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WO2000061749A1 - Proteine cellulaire p60 interagissant avec des proteines regulatrices du virus de l'herpes - Google Patents

Proteine cellulaire p60 interagissant avec des proteines regulatrices du virus de l'herpes Download PDF

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Publication number
WO2000061749A1
WO2000061749A1 PCT/US2000/009214 US0009214W WO0061749A1 WO 2000061749 A1 WO2000061749 A1 WO 2000061749A1 US 0009214 W US0009214 W US 0009214W WO 0061749 A1 WO0061749 A1 WO 0061749A1
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hsv
gene
composition
polynucleotide
cell
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PCT/US2000/009214
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English (en)
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Renato Bruni
Beatrice Fineschi
William O. Ogle
Bernard Roizman
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Arch Development Corporation
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Priority to AU42079/00A priority Critical patent/AU4207900A/en
Publication of WO2000061749A1 publication Critical patent/WO2000061749A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates generally to the field of virology, molecular biology and cell biology and, more specifically, it relates to interactions of a novel cellular protein with herpesvirus regulatory proteins.
  • the herpes group of viruses is composed of at least eight human viruses including: herpes simplex virus types 1 and 2 ("HSV-1" and "HSV-2”); varicella virus; cytomegalovirus; Epstein-Barr virus; human herpesvirus type 6; and human herpesvirus type 8 (Karposi's sarcoma-associated herpesvirus). All human herpesviruses have a similar morphology and size: a 120-250 nm diameter lipoprotein envelope; a 95-100 nm diameter icosahedral nucelocapsid; and a 30 to 60 nm diameter core containing a double-stranded DNA genome.
  • HSV-1 herpes simplex virus types 1 and 2
  • HSV-2 varicella virus
  • cytomegalovirus Epstein-Barr virus
  • human herpesvirus type 6 human herpesvirus type 8
  • All human herpesviruses have a similar morphology and size: a 120-250 nm diameter
  • Herpes simplex viruses are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. These viruses cause a broad spectrum of disease, which ranges from relatively insignificant to severe and life- threatening. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of anti-viral therapy. Despite some successful efforts in treating HSV infectious, dermal and epidermal lesion often recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality.
  • Herpes simplex virus 1 causes two kinds of infection. The first, exemplified most dramatically after first exposure to the virus, results in productive infection at the portal of entry of the virus into the body. In productive infection, approximately 80 different genes are expressed, viral protein and DNA are made, viral progeny is assembled and, ultimately, the cell is destroyed. The second type of infection, latent infection, takes place only in sensory neurons populated by viruses brought to that sites by retrograde transport along axons from the portal of entry. In latently infected cells, viral DNA is maintained as an episome, and the only products detected to date are transcripts arising from two copies of a 8.5 kb domain of the DNA.
  • the herpes simplex virus 1 (HSV-1) genome encodes at least 84 genes, whose expression is coordinately regulated and sequentially ordered in a cascade fashion (Honess and Roizman, 1974; Honess and Roizman, 1975; Roizman and Sears, 1996).
  • the first genes to be expressed are the ⁇ genes, whose products enable the expression of the ⁇ and ⁇ genes.
  • ICP47 infected-cell protein No. 47
  • all products of the ⁇ genes have been shown to be regulatory proteins (Roizman and Sears, 1996).
  • ICP4 encoded by the ⁇ 4 gene, is an essential viral regulatory protein that binds DNA at specific sites and can both transactivate and repress expression of viral genes (Roizman and Sears, 1996).
  • ICP27 the product of the ⁇ 27 gene mediates the processing and translocation of mRNA (Hardwicke and Sandri-Goldin, 1994; Hardy and Sandri-Goldin, 1994; Sandri-Goldin and Mendoza, 1992).
  • ICP22 is a 420-amino acid protein encoded by the ⁇ 22 gene.
  • a smaller transcript promoted from the amino-terminal domain of the ⁇ .22 gene encodes a polypeptide collinear with the carboxyl-terminal 60% of ICP22 and designated U s 1.5 (Carter and Roizman, 1996a).
  • the ⁇ 22 gene is dispensable in Vero and HEp-2 (permissive) cell lines, but ⁇ 22 " mutant viruses replicate poorly in rodent or rabbit skin (restrictive) cell lines, or in confluent primary human fibroblasts (Meignier et al. , 1988; Post and Roizman, 1981; Sears et al, 1985).
  • the R325 mutant HSV-1 virus lacks the carboxyl-terminal 220 codons of the ⁇ .22 gene (Post and Roizman, 1981). In the restrictive cells, the mutant virus exhibit a reduction of ⁇ O and of U s ll mRNA and proteins (Purves et al, 1993).
  • ICP22 localizes in punctate nuclear structures. At the time of onset of viral DNA synthesis, ICP22 colocalizes in infected nuclei with ICP4, viral DNA, RNA polymerase II and a small cellular protein (EAP) named on the basis of its association with Epstein Barr virus small nuclear RNAs.
  • ICP22 is required for the alternative splicing of the ⁇ O gene and for the accumulation of the viral host shutoff protein in infected cells (Carter and Roizman, 1996b; Ng et al, 1997). ICP22 also has been reported to be responsible for the aberrant phosphorylation of the carboxyl-terminal domain of the large subunit of RNA Polymerase II (Rice et al, 1994; Rice et al, 1995). More recently, it has been reported that ICP22 interacts with a previously unknown cell cycle-regulated cellular protein p78 (Bruni and Roizman, 1998).
  • ICP22 also accumulated in a cell cycle-specific fashion and novel forms of ICP22 could be detected during the cell cycle. These forms differ with respect to their electrophoretic mobility from the previously published isoforms. These results suggest that ICP22 is able to interact with cell cycle-regulated proteins (Bruni and Roizman, 1998).
  • ICPO a protein of 775 amino acids
  • ICPO a protein of 775 amino acids
  • HSV-1 viruses lacking the ⁇ O gene replicate less efficiently than the wild-type parent (Sacks and Schaffer, 1987; Stow and Stow, 1986).
  • ICPO is a multifunctional protein inasmuch as it interacts with several cellular proteins.
  • the protein has been shown to bind and colocalize with the cell cycle regulator cyclin D3 (Kawaguchi et al, 1997b).
  • ICPO also colocalizes with an ubiquitin-specific protease in nuclear dense bodies known as PML or NDlOs (Everett et al, 1997; Maul and Everett, 1994). Later in infection ICPO is translocated into the cytoplasm and interacts with the elongation factor EF-l ⁇ (Kawaguchi et al, 1997a). The possible role of ICPO in the cytoplasm and especially its interactions with EF-l ⁇ are reflected in the finding that EF-l ⁇ is phosphorylated by the viral protein kinase U L 13 (Kawaguchi et al, 1998). ICPO is also phosphorylated by the U L 13 protein kinase (Ogle et al, 1997).
  • ICP4 has been reported to interact with ICPO (Yao and Schaffer, 1994) and, as noted above, ICP4 and ICP22 colocalize late in infection in structures containing viral DNA and RNA polymerase (Leopardi et al, 1997). Interaction with viral regulatory proteins with cellular proteins is less well understood or investigated. Identification of novel cellular proteins interacting with viral regulatory proteins will provide important new tools for those working in this arena and also provides new points in intervention for the development of new anti-viral therapeutic agents.
  • an object of the present invention to provide methods for the treatment of herpesvirus infections by modulating cellular protein interactions with herpesvirus regulatory proteins, and compositions therefor. More specifically, the invention discloses the identification of a hitherto unknown cellular protein designated p60, that binds independently to both ICPO and ICP22. The p60 polypeptide presents a novel point of intervention for new anti-viral therapies.
  • One aspect of the present invention is an isolated polynucleotide, or complement thereof, which encodes a p60 polypeptide.
  • the encoded p60 is a mammalian p60.
  • the p60 is a human p60.
  • the p60 may be a wild-type, an allelic variant, a polymorphic variant, a mutant, or a recombinant p60.
  • the p60 may be a truncated p60.
  • the polynucleotide that encodes p60 comprises the amino acid sequence of SEQ ID NO:2 or a contiguous amino acid sequence thereof.
  • the polynucleotide may have or comprise the sequence represented in SEQ ID NO:l, or the complements thereof.
  • the polynucleotide may comprise a contiguous polynucleotide segment of SEQ ID NO:l, or the complement thereof.
  • the polynucleotide may be DNA or complementary.
  • the polynucleotide may include a promoter operably linked to the coding region, or the complement thereof, encoding the p60-molecule.
  • the polynucleotide may further contain a polyadenylation signal which is operably linked to said coding region.
  • the polynucleotide also may contain an origin of replication.
  • the polynucleotide may be contained in a viral vector.
  • the viral vector may be selected from a number of viruses including, but not limited to, retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.
  • the polynucleotide may be packaged in a virus particle.
  • the term "contiguous polynucleotide segment” will be understood to include a contiguous polynucleotide sequence of abut 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 40, about 45, about 50, about 60, about 80, about 90, about 100, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000,, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800,
  • the polynucleotide that encodes p60 may be, but not limited to, a polynucleotide consisting of about 1200 bases, about 1500 bases, about 2000 bases, about 3500 bases, about 5000 bases, about 10,000 bases, about 15,000 bases, about 20,000 bases, about 25,000 bases, about 30,000 bases, about 35,000 bases, about 40,000 bases, about 45,000 bases, about 50,000 bases, about 75,000 bases or about 100,000 bases.
  • Another aspect of the present invention is an isolated oligonucleotide of between about 10 and about 50 contiguous nucleotides of SEQ ID NO:l .
  • the oligonucleotide may be about 11, about 12 bases, about 14 bases, about 16 bases, about 17 bases, about 18 bases, about 19 bases, about 20 bases, about 25 bases, about 30 bases, about 35 bases, about 40 bases, or about 50 base pairs in length.
  • Another aspect of the invention is a contiguous polynucleotide segment characterized as a sequence having of at least about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 25, that have the same sequence as, or are complementary to, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 25 contiguous polynucleotide segments of SEQ ID NO:l .
  • the contiguous polynucleotide segment may also be characterized as a sequence of from about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 25 to about 1,000,000 nucleotides in length that hybridizes to the polynucleotide segment of SEQ ID NO:l, or the complements thereof, under stringent hybridization conditions.
  • the polynucleotide may be about 550 nucleotides in length or may be about 1.32 kilobasepairs in length.
  • polypeptide may have or comprise the amino acid sequence set forth in SEQ ID NO:2.
  • the p60 polypeptide includes a contiguous amino acid sequence of at least 8 amino acids from SEQ ID NO:2.
  • the term "contiguous amino acid sequence” will be understood to include a contiguous amino acid sequence of at least about 4, about, 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 600 or about 700, about 800, about 900, about 1000, or about 1100 amino acids or so.
  • the isolated gene encodes p60 comprising about 441 amino acids in length that comprises the amino acid sequence of SEQ ID NO:2.
  • Yet another aspect of the invention is a monoclonal antibody that binds immunologically to a protein, polypeptide or peptide molecule designated as p60 or a contiguous amino acid sequence thereof.
  • the anti-p60 monoclonal antibody does not bind immunologically to other human polypeptides.
  • the monoclonal antibody may contain or be attached to a detectable label. This label may be selected from, but not be limited to, a fluorescent label, a chemiluminescent label, a radiolabel, or an enzyme.
  • the invention also encompasses a hybridoma cell that produces an anti-p60 monoclonal antibody.
  • polyclonal antisera antibodies of which bind immunologically to a polypeptide designated as p60.
  • Anti-p60 antibodies derived from polyclonal antisera may contain or be attached to a detectable label. This label may be selected from, but not be limited to, a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme.
  • One aspect of the present invention is a method of modulating herpesvirus replication by providing a p60 polypeptide to a herpesvirus-infected cell.
  • the p60 polypeptide is provided to the infected cell by a polynucleotide encoding a p60 polypeptide.
  • the polynucleotide may be operably linked to a promoter, which may be selected from, but not limited to, the group comprising of CMV IE, SV40 IE, RSV, ⁇ -actin, tetracycline regulatable and ecdysone regulatable promoters.
  • the polynucleotide may be contained in a viral vector.
  • the viral vector may be selected from a number of viruses including, but not limited to, retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.
  • the method of modulating herpesvirus replication by providing a p60 polypeptide to a herpesvirus infected cell further comprises administering to the cell an anti-viral therapeutic agent.
  • the anti-viral agent may be selected, but not limited to, a group comprising of acyclovir, valacyclovir, famciclovir or foscarnet.
  • the anti-viral therapy may be given before, simultaneously, or subsequent to providing a p60 polypeptide to a herpesvirus-infected cell.
  • the herpes virus may be HSV, which may be HSV-1 or HSV-2.
  • Another aspect of the present invention is a method for modulating herpesvirus replication by providing to a herpesvirus infected cell an agent that induces the expression of p60 in said cell.
  • This agent may be a trans-acting agent that increases transcription; an agent that stabilizes p60 mRNA; an agent that decreases p60 degradation; or an agent that otherwise increases the intercellular level of a p60 polypeptide.
  • One aspect of the present invention is a method of modulating herpesvirus replication which comprises administering to a cell an effective amount of an agent that modulates the binding of p60 to one or more HSV gene-encoded proteins.
  • the cell is a human cell.
  • the one or more HSV ⁇ gene-encoded proteins may be one or more HSV-1 ⁇ gene-encoded proteins.
  • the one or more HSV-1 ⁇ gene-encoded proteins may be, but not limited to, either ICP22 or ICPO or both ICP22 and ICPO.
  • the modulating agent may inhibit or enhance the binding of p60 to one or more HSV ⁇ gene-encoded proteins.
  • the modulating agent may bind to p60.
  • Such an agent may be an anti-p60 antibody, which may be a single chain anti-p60 antibody.
  • the modulating agent may bind to one or more HSV ⁇ gene-encoded proteins.
  • the p60 comprises the amino acid sequence of SEQ ID NO: 2, or a contiguous amino acid sequence thereof.
  • the method may also further comprise administering to the cell an anti-viral therapeutic agent.
  • the anti-viral agent may be selected, but not limited to, a group comprising of acyclovir, valacyclovir, famciclovir or foscarnet.
  • the anti-viral therapy may be given before, simultaneously, or subsequent to an agent that modulates the binding of p60 to one or more HSV ⁇ gene- encoded proteins.
  • Another aspect of the invention is a method for identifying a modulator of p60 binding to one or more HSV ⁇ gene-encoded proteins.
  • This screening method may comprise mixing a candidate substance with a p60 composition and a HSV ⁇ gene- encoded protein composition.
  • the bound p60-HSV ⁇ gene-encoded protein complex is quantified and substances that modulate binding identified by comparison to the binding of p60 to the target protein or proteins in the absence of the candidate substance.
  • the HSV ⁇ gene-encoded protein composition is a HSV-1 ⁇ gene-encoded protein composition, which may in turn be a ICP22 composition or a ICPO composition.
  • the p60 composition is a p60 fusion protein.
  • Such a fusion protein may be a GST-p60 fusion protein.
  • the GST- p60 fusion protein may comprise a p60 amino acid sequence encoded by SEQ ID NO: 3, or a contiguous amino acid sequence of SEQ ID NO: 4.
  • compositions that comprises an agent that modulates the binding of p60 to a HSV ⁇ gene-encoded protein.
  • This composition may further comprise a pharmaceutically acceptable carrier.
  • the agent is identified by the foregoing method for identifying substances that modulate p60 binding to one or more HSV ⁇ gene-encoded proteins.
  • Another aspect of the invention is a method for identifying a modulator of p60 translocation.
  • Such a method may comprise exposing a cell population to an infectious herpes virus composition in the presence or absence of a candidate substance.
  • the candidate substance when present, may be added prior or subsequent to exposure to the herpes virus.
  • the intracellular location of p60 is identified by immunohistological methodologies.
  • the results of cells incubated with or without the candidate substance are compared.
  • the cell population is permissive for the ⁇ 22 gene and may be, but not limited to, either HEp-2 cells or Vero cells.
  • the herpes virus composition is a HSV composition, which maybe a HSV-1 composition or a HSV-2 composition
  • One aspect of the invention is a method for screening the ability of candidate substances to modulate herpes virus-dependent p60 posttranslational modifications.
  • the assay may comprise mixing a mammalian cell population with a candidate substance and exposing the cell population to a herpes virus.
  • the candidate substance may be added before or after exposure to a herpes virus.
  • a cell extract is prepared and the relative molecular weights of p60 isoforms are evaluated. Determination of whether a candidate substance modulates p60 posttranslational modification is by comparison to p60 isoforms from a cell population infected with herpes virus in the absence of the candidate substance.
  • the cell population is restrictive for the ⁇ 22 gene.
  • a cell population may be, but not limited to, a primary human cell strain, a primary rabbit cell strain or a primary rodent cell strain.
  • the herpes virus composition may be a HSV composition, which may be a HSV-1 composition or a HSV-2 composition.
  • the candidate substance may be a substance that decreases the activity of a herpes virus protein kinase.
  • the protein kinase may be, but not limited to, U L 13 or U s 3.
  • a final aspect of the present invention is a method of engineering a mammalian cell that does not express p60.
  • Such a method may comprise of providing to the mammalian cell an expression comprising a p60 encoding polynucleotide which is positioned antisense to and operatively linked to a promoter.
  • the result of the expression of the antisense p60 transcript is a cell deficient in p60 production.
  • A Schematic representation of the DNA sequence arrangements of the HSV-1 genome of new recombinant viruses used in this study.
  • Line 1 linear representation of the HSV-1 genome.
  • the open rectangles represent the terminal repeats flanking the unique long (U L ) and the unique short (U s ) sequences. The location of the ⁇ 22/U s 1.5 gene is shown.
  • Line 1 representation of the BamHI N fragment which contains the ⁇ 22/U s 1.5 gene.
  • the transcript is represented by the arrow and the coding domain of the ⁇ 22/U s 1.5 genes is represented by the open rectangle.
  • Line 2 in recombinant R7820 the C-terminal 22 amino acids of the ⁇ 22 open reading frame have been deleted.
  • B is an abbreviation of BamHI.
  • B Polypeptide sequence of the carboxyl terminus of ICP22. Line 1, the 43 amino acids of the carboxyl-terminal domain of ICP22. Line 2, sequence of the 21 amino acids remaining within the carboxyl-terminal domain of R7820. Line 3, the 3 amino acids remaining within the carboxyl-terminal domain of R7810.
  • FIG. 2 Nucleotide and amino acid sequence of the p60 polypeptide isolated from a Hela cell library.
  • the top line represents the nucleotide sequence and the bottom line shows the amino acid sequence. Numbers on the right refer to the nucleotide sequence.
  • the underlined sequence indicates the nucleotides equating to the 550 bp sequence from the B-lymphocyte cell line cDNA clone used to construct the GST-p60 fusion protein.
  • FIGS. 3a AND 3b Schematic representation of (A) the interaction of p60 with ICPO and ICP22 and (B) the distribution of ⁇ 60 in HEP-2 and rabbit skin cells.
  • A although p60 interacts with ICPO and ICP22, there is currently no evidence that it can interact with both proteins simultaneously.
  • B the schematic diagram shows only the distribution of p60 and not those of ICPO or ICP22. Note that at 16 h after infection the small structures containing p60 decrease in number in cells infected with wild-type or ICP22VU S 1.5 ⁇ virus but are present in increasing numbers in cells infected with U L 13- or U s 3 -viruses.
  • Herpes simplex virus 1 encodes two multifunctional regulatory proteins, ICP22 and ICPO.
  • ICPO is a promiscuous transactivator whereas ICP22 is required in vivo for efficient replication and expression of a subset of late ( ⁇ 2 ) genes in restrictive cells, e.g., such as primary human cell strains, but not in permissive cells, e.g., HEp-2 or Vero cells.
  • the present invention identifies a cellular protein, designated p60, that interacts with ICP22. This novel protein (apparent M r of 60,000) has no known motifs. Analyses revealed that p60 bound one isoform of fast migrating, underprocessed wild-type ICP22.
  • the p60 binding site has been mapped to a stretch of approximately 16 amino acids located close to the carboxyl terminus of ICP22. This domain is essential for the processing of ICP22. The interaction of p60 with only one ICP22 isoform indicates a specific function for that isoform. p60 also bound ICPO, the binding of which was independent of that of ICP22. The p60- binding domain has been mapped to the carboxyl-terminal 130 amino acids of exon 2 of ICPO. p60 localized in uninfected rabbit skin cells, restrictive for the ⁇ 22 gene, in both nuclei and cytoplasm.
  • p60 was posttranslationally processed to a higher apparent M r but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and of U L 13 protein kinase.
  • uninfected HEp-2 cells permissive for the ct22 gene, p60 localized primarily in nuclei. After infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICPO. Late in infection, both ICPO and p60 tended to disperse but p60 did not change in apparent M r . The localization of p60 was independent of ICP22.
  • posttranslational modification of p60 is mediated either by ICPO in permissive cells or by ICP22 and U L 13 protein kinase in restrictive cells, and that the restrictive phenotype may be related to the failure to process p60 by mutants lacking the genes encoding U L 13 or ICP22.
  • the binding of the cellular protein to two multifunctional HSV-1 regulatory proteins indicates a role for p60 in modulating the efficiency of HSV-1 replication and thereby presents a new point of intervention for anti-herpesvirus therapeutic agents.
  • Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding p60, and the creation and use of recombinant host cells through the application of DNA technology that express p60.
  • the present invention concerns mammalian DNA segments, isolated away from other mammalian genomic DNA segments or total chromosomes.
  • Representative sources for the p60 are human gene sequences. In cloning a p60 sequence of the invention, one may advantageously choose an established human cell line. But other sources will be equally appropriate, such as cDNA or genomic libraries.
  • the DNA segments of the invention have been found to be isolatable from a Hela cell library and an Epstein-Barr virus-immortalized human peripheral blood B-lymphocyte cell line cDNA cloned in the yeast two-hybrid system vector pACT.
  • the DNA segments of the invention are capable of conferring p60-like activity or properties, such as defined herein below, to a recombinant host cell when incorporated into the recombinant host cell.
  • Important aspects of the present invention concern isolated polynucleotide segments and recombinant vectors encoding p60, and the creation and use of recombinant host cells through the application of DNA technology, that express wild-type p60 comprising the sequence of SEQ ID NO:l and biologically functional equivalents thereof.
  • polynucleotide segment refers to a polynucleotide molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a polynucleotide segment encoding p60 refers to a polynucleotide segment that contains wild-type, polymorphic or mutant p60 coding sequences yet is isolated away from, or purified free from, total mammalian genomic DNA. Included within the term “polynucleotide segment,” are polynucleotide segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • a DNA segment comprising an isolated or purified p60 gene refers to a polynucleotide segment including p60 coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutants.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case the p60 gene, forms the significant part of the coding region of the polynucleotide segment, and that the polynucleotide segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the polynucleotide segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • the invention concerns isolated polynucleotide segments and recombinant vectors incorporating polynucleotide sequences that encode an p60 protein or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:2, corresponding to p60.
  • sequence essentially as set forth in SEQ ID NO:2 means that the sequence substantially corresponds to a portion of SEQ ID NO:2, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO: 2 will be sequences that are "essentially as set forth in SEQ ID NO:2," provided the biological activity of the protein is maintained.
  • the invention concerns isolated polynucleotide segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1.
  • the term "essentially as set forth in SEQ ID NO:l" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l, and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:l.
  • polynucleotide segments that encode proteins exhibiting p60 activity will be most preferred.
  • codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.
  • the codons are shown in preference of use from left to right, in Table 1. The most preferred codon for alanine is thus “GCC,” and the least is “GCG” (see Table 1, below).
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e. introns, which are known to occur within genes.
  • sequences that have between about 70% and about 79%; or more preferably, between about 80% and about 89%; or even more preferably, between about 90% and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO:l . will be sequences that are "essentially as set forth in SEQ ID NO:l".
  • Sequences that are essentially the same as those set forth in SEQ ID NO: 1 may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art, as disclosed herein.
  • Hybridization is understood to mean the forming of a double stranded molecule or a molecule with partial double stranded nature. Stringent conditions are those that allow hybridization between two homologous nucleic acid sequences, but precludes hybridization of random sequences. For example, hybridization at low temperature and/or high ionic strength is termed low stringency. Hybridization at high temperature and/or low ionic strength is termed high stringency. Low stringency is generally performed at 0.15 M to 0.9 M NaCl at a temperature range of 20°C to 50°C. High stringency is generally performed at 0.02 M to 0.15 M NaCl at a temperature range of 50°C to 70°C.
  • the temperature and ionic strength of a desired stringency are determined in part by the length of the particular probe, the length and base content of the target sequences, and to the presence of formamide, tetramethylammomum chloride or other solvents in the hybridization mixture. It is also understood that these ranges are mentioned by way of example only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to positive and negative controls.
  • nucleotide sequences of the disclosure may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues.
  • relatively stringent conditions For applications requiring high selectivity, it is preferred to employ relatively stringent conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific genes or detecting specific mRNA transcripts. It is generally appreciated that conditions may be rendered more stringent by the addition of increasing amounts of formamide.
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:l under relatively stringent conditions such as those described herein.
  • nucleic acid segments of the present invention may be combined with other polynucleotide sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments may be prepared that include a contiguous polynucleotide segment to or complementary to SEQ ID NO:l, such as about 8, about 10 to about 14, or about 15 to about 20 nucleotides, and that are up to about 1,000,000, about 750,000, about 500,000, about 250,000, about 100,000, about 50,000, about 20,000, or about 10,000, or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases.
  • nucleotide segments of a million bases or more, including chromosome sized pieces of DNA are contemplated as being useful.
  • Polynucleotide segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
  • intermediate lengths means any length between the quoted ranges, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500- 1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002, 15,000, 20,000 and the like.
  • the various probes and primers designed around the disclosed nucleotide sequences of the present invention may be of any length.
  • an algorithm defining all primers can be proposed:
  • n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one, where n + y does not exceed the last number of the sequence.
  • the probes correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and so on.
  • the probes correspond to bases 1 to 15, 2 to 16, 3 to 17 ... and so on.
  • the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on.
  • this invention is not limited to the particular nucleic acid and amino acid sequence of SEQ ID NO:l.
  • Recombinant vectors and isolated polynucleotide segments may therefore variously include these coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include such coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
  • DNA segments of the present invention encompass biologically functional equivalent p60 proteins, polypeptides, and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins, polypeptides or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.
  • Changes designed by man may be introduced, for example, through the application of site-directed mutagenesis techniques as discussed herein below, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine p60 activity at the molecular level.
  • fusion proteins polypeptides and peptides, e.g., where the p60 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • An example of such a fusion protein is the exemplified GTS-p60 formed by ligating the 5'-end of SEQ ID NO: 3 in frame with the glutathione S-transferase ("GST”) gene in PGEX4T-3 (Pharmacia).
  • polynucleotide segments encoding relatively small peptides, such as, for example, peptides of from about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; as set forth in SEQ ID NO:l and also larger polypeptides up to and including proteins corresponding to the full-length sequence set forth in SEQ ID NO:l .
  • modified bases are also contemplated for use in particular applications of the present invention.
  • a table of exemplary, but not limiting, modified bases is provided herein below.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest.
  • the present invention involves the manipulation of genetic material to produce expression constructs that encode p60.
  • the present invention also envisages the use of multigene constructs wherein a p60 is administered with another regulatory or therapeutic gene.
  • the isolated nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis.
  • TATA box In some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter may be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
  • Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • a transgene is toxic to the host cells, it may be desirable to prohibit or reduce expression of the transgene.
  • transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes.
  • inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.
  • An inducible promoter may be used to control the expression of P60.
  • the ecdysone system (Invitrogen, Carlsbad, CA) is one such inducible system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene such that high levels of mRNA transcripts are attained.
  • both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid.
  • cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
  • expression of the transgene could be activated with ecdysone or muristeron A.
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-OnTM system gene expression is turned on in the presence of doxycycline
  • Tet-OffTM system gene expression is turned on in the absence of doxycycline.
  • Tet-OffTM system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
  • a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired.
  • the CMV immediate early promoter if often used to provide strong transcriptional activation.
  • Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • viral promoters that may be used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the El A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
  • Enhancers include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the El A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • SAA Human Serum Amyloid A
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human or bovine growth hormone and SV40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • IRES elements are used to create multigene polycistronic messages.
  • IRES elements are able to bypass the ribosome scanning model of 5'-methylated, Cap- dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • a gene bombardment (“gene gun") delivery system is used (Yang et al, 1990).
  • the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites.
  • the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in a cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991a).
  • Lipoplexes have been widely used as gene transfer vectors (Escriou et al 1998a, 1998b; Zelphati et al, 1996, 1998; Dodds et al, 1998).
  • a cationic lipid vector has been used in the Phase I study of direct gene transfer of HLA-B7 in patients with metastatic melanoma (Stopeck et al, 1997).
  • cationic polymer-DNA complexes can be used as delivery vehicles for DNA.
  • Simple cationic polymers will bind to DNA and assemble into discrete complexes (Wolfert et al, 1996).
  • Cationic-hydrophilic block copolymers can be used to increase aqueous solubility (Toncheva et al, 1998).
  • DNA- cationic polymer complexes have been widely used as synthetic vectors for delivery of genes in vitro and in vivo (Wagner et al, 1991 ; Trubetskoy et al, 1992; Pocet et al, 1996; Coll et al, 1997; Goldman et al, 1997; Abdallah et al, 1996; Ferrari et al, 1997).
  • U.S. Patent 5,679,559 discloses the use of a lipoprotein containing cationic polymer system for gene delivery.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • One embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • the expression construct comprises a virus or engineered construct derived form a viral genome. It is contemplated that a variety of viral particles may be employed according to the present invention.
  • herpes simplex virus is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non-dividing neuronal cells without integrating in to the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency makes HSV an attractive vector. And though much attention has focused on the neurotropic applications of HSV, this vector also can be exploited for other tissues given its wide host range.
  • HSV is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings.
  • Avirulent variants of HSV have been developed and are readily available for use in gene therapy contexts (U.S. Patent No. 5,672,344). ii. Adenovirus
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
  • the roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained c. ' s-acting elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal repeats
  • E and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.
  • adenovirus In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included.
  • the large displacement of DNA is possible because the cis elements required for viral DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al, 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication.
  • ITR inverted terminal repeats
  • packaging signal for viral encapsidation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al, 1987).
  • El substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et ⁇ /., 1991).
  • adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants.
  • First generation adenovirus vectors contain deletions in the El region, and the replication of these defective vectors is supported by packaging cell lines such as 293 cells that provide the El region gene products. Similarly, adenovirus vectors with the El and E4 gene deleted, but provided by 293 cells expressing both viral gene products, have been made. It is possible to make even larger deletions on the adenovirus genome and then provide the deleted genes in trans, either by a helper virus or helper cell, or both.
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene, termed ⁇ functions as a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol and env genes but without the LTR and ⁇ components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al, 1975). iv. Adeno-associated Virus
  • AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (NP), designated NP-1, NP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins ( ⁇ S). One or more of these rep gene products is responsible for transactivating AAV transcription.
  • AAV -based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, 1995; Chatterjee et al, 1995; Ferrari et al, 1996; Fisher et al, 1996; Flotte et al, 1993; Goodman et al, 1994; Kaplitt et al, 1994; 1996, Kessler et al, 1996; Koeberl et al, 1997; Mizukami et al, 1996; Xiao et al, 1996).
  • AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995; Flotte et al, 1993).
  • the prospects for treatment of muscular dystrophy by AAV- mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al, 1996; Flotte et al, 1993; Kaplitt et al, 1994; 1996; Koeberl et al, 1997; McCown et al, 1996; Ping et al, 1996; Xiao et al, 1996).
  • Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying D ⁇ A.
  • Vaccinia contains a linear, double-stranded D ⁇ A genome of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal repeats of about 10.5 kb flank the genome. The majority of essential genes appear to map within the central region, which is most highly conserved among poxviruses. Estimated open reading frames in vaccinia virus number from 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common.
  • Prototypical vaccinia vectors contain transgenes inserted into the viral thymidine kinase gene via homologous recombination. Vectors are selected on the basis of a tk-phenotype. Inclusion of the untranslated leader sequence of encephalomyocarditis virus, the level of expression is higher than that of conventional vectors, with the transgenes accumulating at 10% or more of the infected cell's protein in 24 h (Elroy-Stein et al, 1989).
  • simian virus 40 is a double-stranded, circular DNA of about 5000 bases encoding large (708 AA) and small T antigens (174 AA), agnoprotein and the structural proteins VP1, VP2 and VP3. It is contemplated that the present invention will encompass SV40 vectors lacking all coding sequences. The region from about 5165-5243 and about 0-325 contains all of the control elements necessary for replication and packaging of the vector and expression of any included genes. Thus, minimal SV40 vectors are derived from this region and contain at least a complete origin of replication.
  • the promoter driving the heterologous gene be a polyomavirus early promoter or a heterologous promoter.
  • the SV40 promoter and enhancer elements are dispensable.
  • Vectors may also be employed as expression constructs within the scope of the present invention.
  • Vectors may be derived from viruses such as lentivirus, poxvirus, alphavirus and coxsackie virus.
  • nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments.
  • hybridization probe of between 17 and 100 nucleotides in length, or in some aspect of the invention even up to 1-2 Kb or more in length, allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • polynucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues.
  • one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g. , one will select relatively low salt and or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific genes or detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • polynucleotide sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target polynucleotide, source of polynucleotide, size of hybridization probe, etc.).
  • hybridization is detected, or even quantified, by means of the label.
  • a polynucleotide used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989).
  • the polynucleotide may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA and is used directly as the template for amplification.
  • primers that selectively hybridize to polynucleotides corresponding to P60 genes are contacted with the isolated polynucleotide under conditions that permit selective hybridization.
  • the term "primer,” as defined herein, is meant to encompass any polynucleotide that is capable of priming the synthesis of a nascent polynucleotide in a template-dependent process.
  • primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single- stranded form, although the single-stranded form is preferred.
  • the polynucleotide primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product is detected.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
  • PCRTM polymerase chain reaction
  • PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase.
  • a DNA polymerase e.g., Taq polymerase.
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of n polynucleotide which involves multiple rounds of strand displacement and synthesis, i.e. nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • RNA amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Genomerase binding et al, PCT Application WO 88/10315, incorporated herein by reference.
  • the polynucleotides can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • RNA polymerase such as T7 or SP6
  • T7 or SP6 an RNA polymerase
  • ssRNA single- stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a polynucleotide sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence.
  • This scheme is not cyclic, i.e. new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, 1990, incorporated herein by reference).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of polynucleotide having the sequence of the resulting "di-oligonucleotide,” thereby amplifying the di-oligonucleotide may also be used in the amplification step of the present invention.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989).
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
  • Amplification products must be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled, polynucleotide probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the polynucleotide and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
  • kits This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying polynucleotides including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • enzymes suitable for amplifying polynucleotides including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair.
  • Preferred pairs of primers for amplifying polynucleotide are selected to amplify the sequence specified in SEQ ID NO: 1.
  • kits will comprise hybridization probes specific P60 corresponding to the sequences specified in SEQ ID NO:l .
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.
  • DGGE denaturing gradient gel electrophoresis
  • SSCP single-strand conformation polymorphism analysis
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.
  • U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • RNase mismatch cleavage assays including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA probes.
  • Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A.
  • Other investigators have described the use of an E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches.
  • the use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.
  • the RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution.
  • the assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription.
  • the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters.
  • the probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe.
  • the RNase used is specific for single-stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel.
  • the RNase Protection assay was adapted for detection of single base mutations.
  • radiolabeled RNA probes transcribed in vitro from wild-type sequences are hybridized to complementary target regions derived from test samples.
  • the test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCRTM), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch”) can be recognized and cleaved in some cases by single-strand specific ribonuclease.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a polynucleotide sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • the predicted amino acid sequence of human p60 based on the sequence of a 1.5-kb cDNA clone isolated from Hela cells, comprises SEQ ID NO:2. This molecule has 441 amino acids. The clone upon which this predicted sequence is based does not contain a methionine residue. Although SEQ ID No: 2 shows two leucine residues at position 15 and 17 which could potentially act as initiator amino acids, it is likely that the 1.5-kb clone does not encode a full-length p60 and that a portion of the amino terminus is missing.
  • purification and in particular embodiments, the substantial purification, of a p60 protein, polypeptide or peptide.
  • a purified p60 protein therefore also refers to a protein, free from the environment in which it may naturally occur in intact cells.
  • the purified p60 proteins, polypeptides or peptides of the invention will generally possess p60 activity.
  • p60 "activity” or “function” is the ability to bind to herpesvirus regulatory proteins, and in HSV specifically ICP22 and ICPO. That is, they will have the capacity to bind to herpesvirus regulatory proteins in herpesvirus infected cells or cell free preparations.
  • purified will refer to a p60 composition that has been subjected to fractionation to remove various non-p60 components such as other cell components.
  • Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
  • substantially purified will refer to a composition in which p60 forms the major component of the composition, such as constituting about 50% of the proteins in the composition or more. In preferred embodiments, a substantially purified protein will constitute more than 60% of the proteins in the composition.
  • a polypeptide or protein that is "purified to homogeneity," as applied to the present invention, means that the polypeptide or protein has a level of purity where the polypeptide or protein is substantially free from other proteins and biological components.
  • a purified polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.
  • Various methods for quantifying the degree of purification of the p60 protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by gel electrophoresis. Assessing the number of polypeptides within a fraction by SDS/PAGE analysis will often be preferred in the context of the present invention, e.g., in assessing protein purity.
  • a preferred method for assessing the purity of a p60 fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial cell extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number".
  • the actual units used to represent the amount of cell binding activity will, of course, be dependent upon the particular assay technique chosen to follow the purification.
  • the present inventors prefer to use SDS-PAGE and western blotting to examine the relative amounts of p60 proteins.
  • polyclonal antibodies against p60 antibodies that recognize several epitopes of these molecules.
  • the inventors currently have rabbit polyclonal antibodies against a GST- p60 fusion protein.
  • the test samples will be examined for protein concentration, separated by SDS-PAGE, and stained by coomassie blue.
  • An additional SDS-PAGE gel that will be run in parallel will then be examined by western blotting with polyclonal antibodies to identify the putative band for p60.
  • the amounts of p60 proteins will then be calculated by multiplying the total protein concentration with the relative purity that will be determined by densitometric analysis of the coomassie-stained SDS-PAGE gel. For example, if one fraction contains 1 mg/ml protein and contains p60 70% purity, this fraction is calculated to contain 0.7 mg/ml p60 protein.
  • An advantage of this system will be that one can test simultaneously the protein profile of p60, so that one can eliminate contamination problems of degraded p60.
  • the inventors will employ a double sandwich ELISA assay in which ELISA plates were first coated with a monoclonal antibody ("MAb") against p60, incubated with test samples, and finally incubated with polyclonal antibodies against p60.
  • MAb monoclonal antibody
  • the amounts of p60 in the test samples will be determined based on the amounts of polyclonal antibodies binding to the plates.
  • Purified p60 proteins will serve as a standard in these assays
  • Relative protein amounts of p60 may not necessarily represent relative biological activities. This is especially the case when p60 proteins are degraded and/or denatured during purification procedures or if different isoforms of p60 protein exhibit different degrees of biological activity. Therefore, it will be important to measure relative biological activity. Biological activity based on the capacity to bind to HSV proteins as described in Section D.5, infra.
  • the specific activity As is generally known in the art, to determine the specific activity, one would calculate the number of units of activity per milligram of total protein. In the purification procedure, the specific activity of the starting material, i.e. tissue extract, would represent the specific activity of the p60 in its natural state. At each step, one would generally expect the specific activity of the p60 to increase above this value, as it is purified relative to its natural state. In preferred embodiments, it is contemplated that one would assess the degree of purity of a given p60 fraction by comparing its specific activity to the specific activity of the starting material, and representing this as X-fold purification. The use of "fold purification" is advantageous as the purity of an inhibitory fraction can thus be compared to another despite any differences that may exist in the actual units of activity or specific activity.
  • the p60 of the present invention be purified to between about 10-fold and about 30-fold, and preferably, of between about 30-fold and about 100-fold, and even more preferably, to about 300-fold, relative to its natural state.
  • the preferred purification method disclosed herein below contains several steps and represents the best mode presently known by the inventors to prepare a substantially purified p60 protein. This method is currently preferred as it results in the substantial purification of the protein or polypeptide, as assessed by western blotting, in yields sufficient for further .characterization and use.
  • This preferred mode of p60 protein, polypeptide, or peptide purification involves the execution of certain purification steps in the order described herein below. However, as is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified p60 protein, polypeptide, or peptide
  • p60 proteins, polypeptides, or peptides there is no general requirement that the p60 proteins, polypeptides, or peptides always be provided in their most purified state. Indeed, it is contemplated that less substantially purified proteins, polypeptides or peptides, which are nonetheless enriched in p60 activity relative to the natural state, will have utility in certain embodiments. For example, less purified p60 preparations may contain molecules that are associated naturally with p60. If so, this may, ultimately, lead to the identification of unique molecules that associate with p60 on the cell surfaces (e.g., co-receptors) or in the cytoplasum (e.g., signaling components).
  • co-receptors e.g., co-receptors
  • cytoplasum e.g., signaling components
  • Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • Inactive products also have utility in certain embodiments, such as, e.g. , in antibody generation.
  • Partially purified p60 fractions for use in such embodiments may be obtained by subjecting a cell extract to one or a combination of the steps described. Substituting certain steps with improved equivalents is also contemplated to be useful. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater -fold purification than the same technique utilizing a low pressure chromatography system.
  • Modification and changes may be made in the structure of p60 and still obtain a molecule having like or otherwise desirable characteristics.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, ICP22, ICPO and antigen-binding regions of antibodies. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. Equally, the same considerations may be employed to create a protein, polypeptide or peptide with countervailing (e.g., antagonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of p60 proteins, polypeptides or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.
  • Biologically functional equivalent polypeptides are thus defined herein as those polypeptides in which certain, not most or all, of the amino acids may be substituted.
  • a plurality of distinct proteins/polypeptides/peptides with different substitutions may be made and used in accordance with the invention.
  • residues may not generally be exchanged.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
  • Conservative substitutions well known in the art include, for example, the changes of alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycogen to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • Polypeptides corresponding to one or more antigenic determinants, or "epitopic core regions,” of p60 can also be prepared. Such polypeptides should generally be at least five or six amino acid residues in length, and may contain up to about 35-50 residues or so.
  • Synthetic polypeptides will generally be about 35 residues long, which is the approximate upper length limit of automated polypeptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA). Longer polypeptides may also be prepared, e.g., by recombinant means.
  • MacVector IBI, New Haven, CT
  • major antigenic determinants of a polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response.
  • PCRTM can be used to prepare a range of polypeptides lacking successively longer fragments of the C-terminus of the protein. The immunoactivity of each of these polypeptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.
  • polypeptides are prepared that contain at least the essential features of one or more antigenic determinants.
  • the polypeptides are then employed in the generation of antisera against the polypeptide.
  • Minigenes or gene fusions encoding these determinants can also be constructed and inserted into expression vectors by standard methods, for example, using PCRTM cloning methodology.
  • polypeptides for vaccination typically requires conjugation of the polypeptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin. Methods for performing this conjugation are well known in the art.
  • immunogenic carrier protein such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin.
  • peptidyl compounds described herein may be formulated to mimic the key portions of the polypeptide structure.
  • Such compounds which may be termed peptidomimetics, may be used in the same manner as the polypeptides of the invention and hence are also functional equivalents.
  • Certain mimetics that mimic elements of protein secondary structure are described in Johnson et al. (1993).
  • the underlying rationale behind the use of polypeptide mimetics is that the polypeptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a polypeptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
  • ⁇ -turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention (either with or without prior immunotolerizing, depending on the antigen composition and protocol being employed) and collecting antisera from that immunized animal.
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
  • a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, ⁇ -maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
  • Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, ⁇ -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP
  • CGP CGP
  • MTP-PE CGP
  • MTP-PE CGP
  • MTP-PE CGP
  • MTP-PE CGP
  • MTP-PE CGP
  • MPL monophosphoryl lipid A
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/
  • Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • BRM biologic response modifiers
  • BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m 2 ) (Johnson/Mead, NJ) and cytokines such as ⁇ -interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as CD80 (B7-1) and CD86 (B7-2).
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
  • a second, booster injection may also be given.
  • the process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies (MAbs).
  • the animal For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots.
  • the serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a polypeptide bound to a solid matrix, or by using, e.g. , protein A or protein G chromatography.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified p60 protein, polypeptide or peptide (or any p60 composition, if used after tolerization to common antigens).
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • the methods for generating MAbs generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • the animals are injected with antigen, generally as described above.
  • the antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary.
  • the antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant.
  • adjuvant such as Freund's complete or incomplete adjuvant.
  • Booster injections with the same antigen would occur at approximately two-wk intervals.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x IO 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
  • P3-NS-l-Ag4-l NS-1 myeloma cell line
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 x IO "6 to 1 x IO "8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants (after about two to three wk) for the desired reactivity.
  • the assay should be sensitive, rapid and easy to use, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse).
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • a hydrocarbon especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • MAbs will be chimeric MAbs, including "humanized” MAbs.
  • the chimeric MAb is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse anti-p60 producing cell and the constant-region exons from a human antibody gene. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse lg into a human V-domain. This can be followed by the replacement of some human residues in the framework regions of their murine counterparts.
  • CDRs mouse complementary determining regions
  • the antibody encoded by such recombinant genes is a mouse-human chimera.
  • the specificity of the chimeric antibody is determined by the variable region derived from mouse sequences while the isotype, which is determined by the constant region, is derived from human DNA.
  • These humanized anti-p60 antibodies are especially suitable for use in in vivo diagnostic and therapeutic methods.
  • the nucleotide sequence encoding the variable domain of the light and heavy chains of mouse anti-human p60 MAb will be first cloned by PCRTM and then inserted into the expression vector containing the human light and heavy chain constant regions. These expression vectors are used routinely by many investigators (Co et al, 1996; Co et al, 1992).
  • Recombinant proteins may be produced in mammalian cells (e.g., mouse myeloma cell line SI 94) and then purified with protein A sepharose column.
  • mammalian cells e.g., mouse myeloma cell line SI 94
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
  • a molecular cloning approach may be used to generate MAbs.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells e.g., normal-versus-tumor cells.
  • the advantages of this approach over conventional hybridoma techniques are that approximately IO 4 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
  • autoantibodies against p60 proteins, polypeptides or peptides may be generated, under pathological conditions.
  • such autoantibodies may be present in detectable levels in HSV infected human patients.
  • Autoantibodies may be detected by ELISA using relevant antibodies that recognize p60 proteins, polypeptides or peptides.
  • ELISA plates will be first coated with (rabbit) p60 antibodies and then coated with recombinant or native form of p60. These plates will be incubated with test samples (e.g., human serum) and then with antibodies against (human) immunoglobulin.
  • test samples e.g., human serum
  • recombinant or native forms of p60 may be immobilized directly on the ELISA plates.
  • the amounts of autoantibodies will be determined by measuring the amounts of anti-immunoglobulin antibodies that bind to the plates. This and other assays to measure autoantibodies against p60 may be useful for diagnostic purposes.
  • Further aspects of the present invention include the down-regulation or suppression of p60 genes so that p60 production is reduced or even eliminated. It is contemplated that the expression of p60 will be suppressed by the incorporation a DNA segment into the genome of recombinant host cells or animals such that the expression of the p60 gene is disrupted.
  • constructs are designed to homologously recombine into particular endogenous gene locus, i.e. p60 gene locus, rendering the endogenous p60 gene nonfunctional.
  • constructs are designed to randomly integrate throughout the genome, a non-specific effect which may ultimately result in loss of expression of the p60 gene.
  • antisense constructs are designed to introduce nucleic acids complementary to the target endogenous gene. Expression of RNAs corresponding to these complementary nucleic acids will interfere with the transcription and/or translation of the target sequences.
  • constructs are designed to introduce nucleic acids encoding ribozymes - RNA-cleaving enzymes - that will specifically cleave a target mRNA corresponding to the endogenous gene.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementary rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementary to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences that are completely complementary will be sequences that are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990; Sioud et al, 1992).
  • ribozymes elicited genetic changes in some cell lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • homologous recombination relies, like antisense, on the tendency of nucleic acids to base pair with complementary sequences. In this instance, the base pairing serves to facilitate the interaction of two separate nucleic acid molecules so that strand breakage and repair can take place.
  • the "homologous” aspect of the method relies on sequence homology to bring two complementary sequences into close proximity, while the "recombination” aspect provides for one complementary sequence to replace the other by virtue of the breaking of certain bonds and the formation of others.
  • homologous recombination is used as follows. First, a target gene is selected within the host cell. Sequences homologous to the target gene are then included in a genetic construct, along with some mutation that will render the target gene inactive (stop codon, interruption, etc.). The homologous sequences flanking the inactivating mutation are said to "flank" the mutation. Flanking, in this context, simply means that target homologous sequences are located both upstream (5') and downstream (3') of the mutation. These sequences should conespond to some sequences upstream and downstream of the target gene. The construct is then introduced into the cell, thus permitting recombination between the cellular sequences and the construct.
  • the genetic construct will normally act as far more than a vehicle to interrupt the gene.
  • a selectable marker gene This gene permits selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic and biocidal drugs.
  • a heterologous gene that is to be expressed in the cell also may advantageously be included within the construct. The arrangement might be as follows:
  • Another refinement of the homologous recombination approach involves the use of a "negative" selectable marker.
  • This marker unlike the selectable marker, causes death of cells which express the marker. Thus, it is used to identify undesirable recombination events.
  • it is difficult in the initial screening step to identify proper homologous recombinants from recombinants generated from random, non-sequence specific events.
  • These recombinants also may contain the selectable marker gene and may express the heterologous protein of interest, but will, in all likelihood, not have the desired "knock out" phenotype.
  • a target gene within a host cell is selected as the location into which a selected gene is to be transferred. Sequences homologous to the target gene are included in the expression vector, and the selected gene is inserted into the vector such that target gene homologous sequences are interrupted by the selected gene or, put another way, such the target gene homologous sequences "flank" the selected gene.
  • a drug selectable marker gene also is inserted into the target gene homologous sequences.
  • flanking sequences need not directly abut the genes they "flank.”
  • a modification of this procedure is one where no selected gene is included, i.e. only the selectable marker is inserted into the target gene homologous sequences. Use of this kind of construct will result in the "knock-out" of the target gene only. Again, proper recombinants are screened by drug resistance.
  • random integration is like homologous recombination, however, in that a gene construct, often containing a heterologous gene and a selectable marker, integrates into the target cell genomic DNA via strand breakage and reformation.
  • p60-related agents described herein will be useful in many areas, for example in screening assays, immunoassays, immunodetection methods, suppressing herpesvirus replication, and monitoring amounts and qualities of p60 in clinical samples.
  • p60-related agents refers to full length as well as partial polynucleotide segments; full length, isoforms, mutated, truncated or elongated forms of p60 proteins, polypeptides and peptides; other members of the p60 family; isolated and purified native p60 as well as recombinantly produced p60; antibodies raised to any of the above forms; cells and animals engineered to overproduce p60; and cells and animals engineered not to produce p60.
  • the p60-related agents described herein may, of course, additionally be used to search for molecules that modulate the expression and/or function of p60 (e.g., chemicals, synthetic peptides, carbohydrates, lipids, recombinant proteins, cell extracts, and supernatant, etc.). This may, for example, involve the use of p60 transfectants to search for molecules that modulate HSV replication.
  • molecules that modulate the expression and/or function of p60 e.g., chemicals, synthetic peptides, carbohydrates, lipids, recombinant proteins, cell extracts, and supernatant, etc.
  • agents described herein may be used to upregulate as well as downregulate binding of p60 to ICP22 and ICPO.
  • agents against p60 may be used in this manner.
  • Another example is the use of p60 mimetics to competitively bind to p60 ligands and thereby exhibit beneficial effects to prevent or even treat HSV infections.
  • transgenic animals that express "marker genes” (e.g., green fluorescence proteins, ⁇ -galactosidase) under the control of the p60 promoter.
  • markers genes e.g., green fluorescence proteins, ⁇ -galactosidase
  • Such transgenic animals will be useful for studying the replication and virulence of HSV.
  • these animals may serve as useful tool to search for candidate substances that upregulate or downregulate HSV replication.
  • transgenic animals will be treated with candidate substances and then examined for the expression of p60.
  • p60-related reagents a) polynucleotide segments of p60 including the 5'- and 3'-flanking regions, b) RNA segments of sense or anti-sense strands of p60, including the truncated or mutated transcripts, c) p60 peptides, polypeptides or proteins, including truncated or mutated forms and their biological equivalents, d) polyclonal or monoclonal antibodies against p60, e) vectors designed to produce p60 peptides, polypeptides or proteins, f) cell lines that are engineered to express p60, g) cell lines that are engineered to lack the expression of p60, h) animals that are engineered to overproduce p60, i) animals that are engineered to lack the expression of p60, j) other members of the p60 family of genes and their products which can be identified with the above rea
  • p60-related assays a) assays to detect p60 polynucleotides, including Southern blotting, genomic PCRTM, colony and plaque hybridization, and slot blotting; b) assays to detect p60 RNA, including northern blotting, RT-PCRTM, in situ hybridization, primer extension assay, and RNase protection assay; c) assays to detect p60 peptides, polypeptides or proteins, including ELISA, Western blotting, immunoprecipitation, radioimmuno-absorption and -competition assays, and immunofluorescence and immunohistochemical stainings; and d) assays to search for agents that modulate p60 binding to herpes virus regualtory proteins; e) assays to search for agents that modulate herpes virus-dependent p60 post-translational modifications, and f) assays to search for agents that modulate herpes virus-dependent p60 post-translational modifications,
  • Polynucleotides of p60 e.g., SEQ ID NO:l and SEQ ID NO:3 or related polynucleotides that exhibit significant homologies with or that contain portions of p60 will be used as probes to detect members of the p60 family of genes.
  • the p60 family of genes is defined as genes that are detectable with at least one of these probes.
  • standard assays including Southern blotting, PCRTM, colony and plaque hybridization, and slot blot hybridization will be employed under various conditions with different degrees of stringency as described previously. Specimens to be tested include cDNA libraries, genomic DNA, cDNA, and DNA fragments isolated from cells or tissues.
  • these assays may be modified to detect selectively mutated p60 DNA.
  • Southern blotting or PCRTM will be employed to detect or amplify the mutated DNA segments. These segments will then be sequenced to identify the mutated nucleotides.
  • a combination of selected restriction enzymes will be employed to reveal molecular heterogeneity in Southern blotting.
  • these assays may be modified to detect selectively different domains or different portions of the p60 polynucleotide sequences. For this aim, one may employ probes or primers for different portions of the nucleotide sequences. More sophisticated methods may be employed to screen point mutations. For example, it is contemplated that one may choose a PCRTM-single-strand conformation polymorphism (PCRTM-SSCP) analysis (Sarkar et al, 1995).
  • PCRTM-SSCP PCRTM-single-strand conformation polymorphism
  • Polynucleotides of p60 e.g., SEQ ID NO:l and SEQ ID NO:3 or related polynucleotides that exhibit significant homologies with or that contain portions of with p60 will be used as probes to detect transcripts of the p60 family of genes.
  • standard assays including northern blotting, RT-PCRTM, in situ hybridization, primer extension assay and RNase protection assay will be employed under various conditions with different degrees of stringency as described previously.
  • Specimens to be tested include total RNA and mRNA isolated from cells or tissues and cell and tissue samples themselves obtained from living animals or patients. These assays may be modified to detect selectively the transcripts for different domains or different isoforms.
  • the inventors will employ probes or primers for different portions of the nucleotide sequences.
  • the inventors have been able to identify several truncated transcripts of p60 by RT-PCRTM using a panel of different primer sets. These transcripts have been found to be produced by alternative splicing mechanisms. Similar methods using RT-PCRTM may be employed to identify other spliced variants and even other isoforms that are produced by other mechanisms. Alternatively, northern blotting may be used to detect selectively different isoforms.
  • oligonucleotide probes will be constructed, each covering different portions of the nucleotide sequences.
  • RNA isolated from DC will be analyzed by northern blotting or RT-PCRTM.
  • assays may be designed to detect selectively different RNA species.
  • the inventors have been able to identify several truncated transcripts of p60 by RT-PCRTM using a panel of different primer sets. These transcripts have been found to be produced by alternative splicing mechanisms. Similar methods using RT-PCRTM may be employed to identify other spliced variants and even other isoforms that are produced by other mechanisms. Alternatively, northern blotting may be used to detect selectively different isoforms.
  • oligonucleotide probes will be constructed, each covering different portions of the nucleotide sequences. To define the nucleotides that are deleted from the original sequence, RNase protection assays may be employed.
  • Antibodies against p60 will be used to detect p60 proteins or polypeptides.
  • standard assays including ELISA, western blotting, immunoprecipitation, radioimmuno-absorption and radioimmuno-competition assays, and immunofluorescence and immunohistochemical stainings will be employed under various conditions with different degrees of specificity and sensitivity.
  • Specimens to be tested include viable cells, whole cellular extracts, and different subcellular fractions of established cell lines, as well as cells, tissues, and body fluids isolated from living animals or patients.
  • These assays may be modified to detect selectively different epitopes, domains, or isoforms of p60 peptides, polypeptides or proteins.
  • the inventors will develop and employ a panel of MAb against different epitopes or domains.
  • Functionality of p60 may be evaluated by binding of p60 to HSV proteins.
  • Such assays may comprise mixing a p60 composition with a HSV protein composition, and then quantifying the amount of p60 bound to the HSV protein composition.
  • the HSV protein composition may be a HSV-1 protein composition and may comprise ⁇ gene-encoded proteins such as ICP22 and ICPO.
  • a ICP22 composition may include an amino acid sequence of about 16 amino acids from the carboxyl terminus of ICP22.
  • the p60 composition may be a GST-p60 fusion protein.
  • the polynucleotide that encodes a GST-p60 fusion protein may include SEQ ID NO:3. GST fusion proteins may be used to pull down proteins in cell extract as in the exemplified procedure.
  • Such proteins may then be solubilized, electrophoretically separated, and then transferred to a nitrocellulose sheet and proteins reacted with an appropriate antibody.
  • Other methods of quantifying bound p60-HSV protein complexes may be undertaken by attaching a detectable label, such as, but not limited to, a radiolabel, fluorescent label or enzyme, to either the p60 composition or HSV protein composition.
  • the intracellular location of p60 in a cell infected with HSV may be determined.
  • Such assays are preferably undertaken with cells that are permissive in regard to the HSV ⁇ 22 gene and will primarily be an indicator of binding of ICPO.
  • Such an assay may comprise infecting a cell population with a herpes virus and after incubation for 1 to 40 hours, the cells are fixed and the intracellular location of p60 identified by use of a suitably labeled anti-p60 antibody.
  • Herpesvirus-dependent posttranslational modification of p60 also may be determined.
  • Such assays are preferably undertaken with cells that are restrictive in regard to the HSV ⁇ 22 gene and will primarily be an indicator of binding to ICP22 and/or U L 13 and U s 3 protein kinase function.
  • Such an assay may comprise mixing a cell population with a candidate substance and then exposing the cell population to an infectious herpesvirus. After incubation for 1-40 hours, a cell extract is prepared, proteins separated by polyacrylamide gel electrophoresis under denaturing conditions, transferred to nitrocellulose and protein bands identified by an anti-p60 antibody.
  • Methods for Screening of Active Compounds p60-related assays described above also may be used to search for molecules that modulate expression and function of p60. Screening will include the determination as to whether candidate substances may affect the expression of p60 in a specified cell population. For this purpose, cell preparations (e.g., HEp-2, HeLa, or human or rabbit skin cells) will be treated with candidate substances either individually or in combination and then examined for p60 expression at the levels of mRNA, protein, and function. Alternatively, those candidate substances may be tested in vivo by administering into living animals. In this case, cells will be isolated from those animals after treatment and then examined in vitro for p60 expression, once again, at the levels of mRNA, protein, and function.
  • cell preparations e.g., HEp-2, HeLa, or human or rabbit skin cells
  • candidate substances may be tested in vivo by administering into living animals. In this case, cells will be isolated from those animals after treatment and then examined in vitro for p60 expression,
  • Candidate substances that modulate the binding of p60 to HSV proteins may be identified by the above described binding assays. Such assays may comprise mixing a p60 composition with a HSV protein composition, and then quantifying the amount of p60 bound to the HSV protein composition. Identification of a substance that modulates p60 binding to the HSV protein composition is by comparison to a negative control conducted in the absence of the candidate substance. Methods for screening compounds that modulate the interaction between p60 and products of the ⁇ 22 gene are also described in U.S. Patent Application entitled Methods and Compositions Involving Posttranslational Modifications of Herpesvirus Proteins submitted April 9, 1999 by applicants William O. Ogle and Bernard Roizman, which is herein incorporated by reference in its entirety. Candidate substances also may be screened for the ability to alter the intracellular location of p60 in a cell infected with HSV as described above. Such a screening assay will primarily identify modulating the binding of binding of p60 to ICPO.
  • Screening of compounds that modulate herpesvirus-dependent posttranslational modification of p60 will primarily identify compounds modulating p60 binding to ICP22 and or activity of U L 13 and/or U s 3.
  • useful compounds in this regard will in no way be limited to antibodies.
  • the most useful pharmacological compounds for identification through application of the screening assay will be non-peptidyl in nature and serve to inhibit p60 binding, which may be elicited by, but not limited to, a tight binding or other chemical interaction with p60 or target HSV proteins or modulation of the activity of U L 13 and/or U s 3.
  • Molecules may be examined for their capacities to suppress or to enhance the expression of p60 at mRNA or protein levels. For this aim, cells will be incubated with test samples and then examined for p60 expression by northern blotting, RT-PCRTM, in situ hybridization, primer extension assay and RNase protection assay (at RNA levels) or by ELISA, western blotting, immunoprecipitation, radioimmuno-absorption and competition assays, and immunofluorescence and immunohistochemical stainings (at protein levels).
  • Candidate substance that modulate p60 expression or function are considered to posses the ability to modulate the replication of herpes virus and specifically HSV.
  • p60 related agents described herein i.e. p60 proteins, polypeptides or peptides, antibodies raised against such proteins, polypeptides or peptides, mutated, truncated or elongated forms of p60, antibodies raised against such forms, and cells engineered to overproduce or lack p60 may be used to suppress herpesvirus replication. That is, they may be used for the treatment of HSV infection.
  • Diagnostic Application p60-related reagents and assays may be applicable clinically to diagnostic purposes. For example, functional activities of p60 may will provide important information with respect to diagnosis and prognosis patients infected with a herpes virus. Moreover, some infected patients may have p60 mutations, or autoantibodies against p60.
  • the antibodies of the invention will find utility in diagnostic agents, purifying p60 molecules, and in the recombinant cloning of p60-like molecules.
  • One evident utility of the p60-related reagents is in immunoassays for the identification of p60, p60-HSV protein complexes, and different isoforms of p60 as needed in diagnosis and prognostic monitoring.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections also is particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like also may be used.
  • anti-p60 antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the p60 antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound p60 antigen may be detected. Detection is generally achieved by the addition of another anti-p60 antibody that is linked to a detectable label.
  • ELISA is a "sandwich ELISA.” Detection also may be achieved by the addition of a second anti-p60 antibody, followed by the addition of a third antibody that has binding affinity for the second anti-p60 antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the p60 antigen are immobilized onto the well surface and then contacted with the anti-p60 antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-p60 antibodies are detected. Where the initial p60 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-p60 antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the proteins or polypeptides are immobilized involves the use of antibody competition in the detection.
  • labeled anti- p60 antibodies are added to the wells, allowed to bind, and detected by means of their label.
  • the amount of p60 antigen in an unknown sample is then determined by mixing the sample with the labeled anti-p60 antibodies before or during incubation with coated wells.
  • the presence of p60 antigen in the sample acts to reduce the amount of anti-peptide antibody available for binding to the well and thus reduces the ultimate signal.
  • This is also appropriate for detecting anti-p60 antibodies in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described as follows:
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the control protein and/or clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation.
  • Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gammaglobulin
  • PBS phosphate buffered saline
  • suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 h at room temperature in a PBS -containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • the present invention concerns immunodetection methods for binding, purifying, removing, quantifying or otherwise generally detecting biological components.
  • the p60 proteins, polypeptides or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect p60 proteins, polypeptides or peptides.
  • Ligands of p60 including carbohydrate determinants as well as peptide determinants; other members (subunits and isoforms) of the p60 family; molecules that are associated with p60 (e.g., co-receptors, subunits, signaling molecules, etc.); molecules that bind to the 5'- or 3'- flanking regions, as well as the coding sequences of p60 genes; and/or molecules that are found to regulate the expression and/or function of p60.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.
  • the immunobinding methods include obtaining a sample suspected of containing a p60 protein, polypeptide or anti-p60 antibody, and contacting the sample with an antibody or p60 protein, polypeptide or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • an antibody will most likely be used to remove an antigenic component from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the unwanted antigenic component will be applied to the immobilized antibody.
  • a purged or purified sample may be obtained free from the unwanted antigen by collecting the sample from the column and leaving the antigen immunocomplexed to the immobilized antibody.
  • the immunobinding methods also include methods for detecting or quantifying the amount of a reactive component in a sample, which methods require the detection or quantification of any immune complexes formed during the binding process.
  • methods for detecting or quantifying the amount of a reactive component in a sample which methods require the detection or quantification of any immune complexes formed during the binding process.
  • one would obtain a sample suspected of containing a p60 protein, polypeptide, peptide or anti-p60 antibody and contact the sample with an antibody or p60 protein, polypeptide or peptide, as the case may be, and then detect or quantify the amount of immune complexes formed under the specific conditions.
  • p60 in order to monitor the amounts and qualities of p60 in clinical samples (e.g., sera, cells, tissues, etc.), one may use antibodies against p60 (e.g., in ELISA, RIA, immunoblotting as described herein), recombinant p60 (e.g., to provide competition at the level of binding to T cells) or cDNA and/or oligonucleotides (e.g., in RT-PCRTM or northern blotting as described herein).
  • antibodies against p60 e.g., in ELISA, RIA, immunoblotting as described herein
  • recombinant p60 e.g., to provide competition at the level of binding to T cells
  • cDNA and/or oligonucleotides e.g., in RT-PCRTM or northern blotting as described herein.
  • the biological sample analyzed may be any sample that is suspected of containing a p60 protein, polypeptide or peptide.
  • a cryostat skin tissue section or specimen such as a homogenized skin tissue extract, an isolated cell, separated or purified forms of any of the above p60 containing compositions.
  • immune complexes are generally a straightforward matter of adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e. to bind to, any antigens present.
  • the sample- antibody composition such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the p60 protein, polypeptide, peptide or antibody employed in the detection may itself be linked to a detectable label, wherein one would then detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first added component that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the p60 protein, polypeptide, peptide or antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the p60 protein, polypeptide, peptide or antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • the detection of p60 levels or activity, p60-HSV protein complexes, or autoantibodies against p60, or an increase in the levels of such molecule, in comparison to the levels in a corresponding biological sample is indicative of dysfunction of p60-mediated activity.
  • RNA isolated from peripheral blood DC will be examined by northern blotting or RT-PCRTM. If patients exhibit p60 bands of different sizes or no detectable bands, this will indicate the genetic mutation. On the other hand, this screening, which can be routinely and rapidly performed, will not allow one to identify point mutations. Point mutations may be identified by sequencing the cDNA isolated from patients. More sophisticated methods may be employed to screen point mutations. For example, it is contemplated that one may choose a PCRTM-single-strand conformation polymorphism (PCRTM-SSCP) analysis (Sarkar et al, 1995).
  • PCRTM-SSCP PCRTM-single-strand conformation polymorphism
  • p60-related reagents may be used to down regulate herpesivrus replication.
  • the present invention is concerned with a method of inhibiting HSV replication by subjecting the HSV infected cells to an effective concentration of a p60 inhibitor such as one of the family of positive candidate substances discussed above, but not limited to antibodies or with a candidate substance identified in accordance with the candidate screening assay embodiments.
  • a p60 inhibitor such as one of the family of positive candidate substances discussed above, but not limited to antibodies or with a candidate substance identified in accordance with the candidate screening assay embodiments.
  • Aqueous pharmaceutical compositions of the present invention will have an effective amount of a compound that modulates p60 binding to HSV proteins, a p60 expression construct, an antisense p60 expression construct, an expression construct that encodes a therapeutic gene along with an p60 gene, an anti-p60 antibody, or any other suitable composition.
  • Such compositions generally will be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • An "effective amount,” for the purposes of therapy, is defined at that amount that causes a clinically measurable difference in the condition of the subject. This amount will vary depending on the substance, the condition of the patient, the type of treatment, the location of the lesion, etc.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other antiviral agents, can also be incorporated into the compositions.
  • other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
  • the active compounds of the present invention will often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains glycosylceramide synthesis inhibitory compounds alone or in combination with a chemotherapeutic agent as active ingredients will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the active compounds may be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions. These forms may be used for treating skin-associated diseases.
  • the formulation will be geared for administration to the central nervous system, e.g., the brain.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed, will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Persistent infection and latency represent major problems in clinical virology.
  • One goal of current viral research is to find ways to improve the efficacy of anti-viral therapy.
  • One way is by combining such traditional therapies with a therapy that modulates p60 function of activity.
  • therapeutic formulations of the present invention could be used similarly in conjunction with anti-viral agents, such as acyclovir, valacyclovir, famciclovir and foscarnet
  • compositions would be provided in a combined amount effective to inhibit replication and infection by a herpesvirus.
  • This process may involve contacting the cells with the p60 modulating agent and the traditional anti-viral agent(s) or factor(s) at the same time.
  • This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
  • the p60 modulating agent may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and p60 modulating agent are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and p60 modulating agent would still be able to exert an advantageously combined effect on the cell.
  • A/A/B/B A B/A/B A/B/B/A B/B/A A B/A/B/A B/A/A B B/B/B/A
  • both agents are delivered to a cell in a combined amount effective to achieve efficacy.
  • agents or factors suitable for use in a combined therapy are any chemical compound or treatment method with antiviral activity; therefore, the term "antiviral agent” that is used throughout this application refers to an agent with antiviral activity.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents, which are both chemically and physiologically related, may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Example 1 Materials and Methods Cell lines and viruses.
  • HeLa and HEp-2 cell lines were obtained from the American Type Culture Collection. Rabbit skin cells were originally obtained from J. McClaren.
  • HSV- 1(F) is the prototype HSV-1 strain used in this laboratory (Ejercito et al, 1968).
  • R7810 and R7820 are recombinant viruses from which the carboxyl-terminal 40 and 24 codons of ⁇ 22, respectively, had been deleted (FIG. 1). Preparation of the recombinant viruses R7810 and R7820 are described in the U.S. Patent Application entitled Methods and Compositions Involving Posttranslational Modifications of Herpesvirus Proteins submitted April 9, 1999 by applicants Willaim O.
  • Recombinant virus R325 has been described previously (Post and Roizman, 1981). Recombinants lacking the genes encoding the protein kinase U s 3 (R7041) or U L 13 (R7356) have been previously described (Purves et al, 1987; Purves et al, 1993).
  • Plasmids. pBH1025 contained a 1-kb cDNA insert encoding 300 amino acids of p60 in the yeast two-hybrid system vector pACT (Clontech).
  • pBH1026 was constructed by ligating the 550 bp shown in SEQ ID NO: 3 of the 5'-end of the cDNA insert of pBH1025 in frame with the glutathione S-transferase (GST) gene in pGEX4T-3 (Pharmacia, Columbia, MD).
  • GST glutathione S-transferase
  • SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.
  • pRB4965 contained the carboxyl-terminal 370 codons of p78 in pGEX4T-3 and has been described previously (Bruni and Roizman, 1998).
  • pRB5113 contained the entire coding sequence of ⁇ 22/U s 1.5 genes in pGBT9 (Bruni and Roizman, 1998).
  • yeast strain HF7c (Clontech, Palo Alto, CA) was transformed with pRB5113, grown to large-scale and transformed with a cDNA library derived from an Epstein-Barr virus-immortalized human peripheral blood B-lymphocyte cell line (Clontech, Palo Alto, CA) cloned in pACT. Positive clones were selected and isolated as described elsewhere (Bruni and Roizman, 1996). Isolation of cDNA clones containing larger p60 inserts was also done as described elsewhere (Bruni and Roizman, 1996) using the 1-kb insert of pBH1025 as a probe.
  • Recombinant pGEX vectors were grown in BL21 and fusion proteins were isolated as recommended by the manufacturer (Pharmacia, Columbia, MD). Binding reactions containing 300 ⁇ l of cell extract mixed with 3 to 5 ⁇ g of fusion protein were incubated at 4°C for 4 to 5 h. Beads were collected by centrifugation, rinsed three times with PBS* (1 ml) and resuspended with 100 ⁇ l of 2 ⁇ disruption buffer (100 mM Tris-Cl pH 6.8, 200 mM dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol).
  • 2 ⁇ disruption buffer 100 mM Tris-Cl pH 6.8, 200 mM dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol.
  • Electrophoretic separation of proteins Subconfluent rabbit skin cells or HEp-2 cells grown in 25c ⁇ r-flasks were infected with 10 PFU of virus per cell. After 18 h at 37°C, the cells were scraped in PBS, rinsed once with PBS and resuspended with 130 ⁇ l of 2 x disruption buffer (Sambrook et al, 1989). Cells were sonicated for 10 sec and 40 to 60 ⁇ l subjected to electrophoretic separation.
  • Monoclonal antibody to ICPO purchased from the Goodwin Institute (Plantation, Florida) and the polyclonal antiserum R77 to ICP22 have been described elsewhere (Ackerman et al, 1984; Ackermann et al, 1985).
  • the polyclonal serum to p60 was generated as follows: pBH1026 was grown in BL21 and the fusion protein was purified from large-scale culture as recommended by the manufacturer (Pharmacia, Columbia, MD). Two rabbits were injected subcutaneously at Josman Laboratories (Nape, California) with 1 mg of fusion protein each time at 14-day intervals. The serum used in this study was collected 1 week after the fifth immunization.
  • nitrocellulose sheets were rinsed four times in 5% milk (in PBS) and reacted for 1 h at room temperature with the secondary antibody conjugated to either alkaline phosphatase (BioRad, Hercules, CA) or to horseradish peroxidase (Amersham, Harlington Heights, IL). The sheets were then rinsed four times in PBS and enzymatic reactions were done as recommended by the manufacturer.
  • HEp-2 or rabbit skin cells were grown in wells on 1 x 3-in. slides, exposed to 10 PFU of HSV- 1(F) per cell, maintained for 18 h at 37°C and then fixed in methanol at - 20°C for 20 min. The cells were blocked in 1% BSA in PBS containing 20% normal human serum at room temperature for 60 min. rinsed once with PBS and then reacted for 18-24 h at 4°C with the primary antibodies diluted in 1% BSA in PBS containing 10% normal human serum. Dilutions were 1 :2000 for the polyclonal serum to p60 and 1:300 for the ICPO monoclonal antibody.
  • the cells were rinsed three times with PBS and then reacted for 1 h at room temperature with goat anti-rabbit IgG conjugated to Texas red (Molecular Probes) and goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC, Sigma).
  • the cells were rinsed again with PBS and mounted in PBS containing 90% glycerol and 1 mg of p-phenylenediamine per ml of solution.
  • the slides were examined with a Zeiss confocal fluorescence microscope, and digitized images were acquired with software provided by the manufacturer and printed by a Tektronix 440 phaser printer.
  • Single-color images were acquired by excitation using an argon-krypton laser at 488 rim (FITC) or 568 nm (Texas red). Double-stained images were acquired by a split image of both fluorochromes filtered by a 515- to 540-nm band pass (FITC) and a 590-nm long pass (Texas red) filters. Overlays were acquired with the software provided by the manufacturer. Each set of images was acquired with the same set of setting and was not modified.
  • FITC argon-krypton laser at 488 rim
  • Texas red 568 nm
  • Double-stained images were acquired by a split image of both fluorochromes filtered by a 515- to 540-nm band pass (FITC) and a 590-nm long pass (Texas red) filters. Overlays were acquired with the software provided by the manufacturer. Each set of images was acquired with the same set of setting and was not modified.
  • An Epstein-Barr virus-immortalized human peripheral blood B-lymphocyte cell line cDNA cloned in the yeast two-hybrid system vector pACT was screened with the full-length ⁇ 22 gene as a bait.
  • Several positive clones were identified and further analyzed for specificity as described in Example 1, Materials and Methods.
  • One of the clones, designated 22.6 interacted with full-length ICP22 and in a subsequent study, with ICPO amino acids 111-241, but not with a truncated ICP22 protein extending from amino acids 1-267 or with an irrelevant protein (e.g., ORF P, ICPO amino acids 543-775).
  • Clone 22.6 contained a cDNA insert of approximately 1 kb.
  • the sequence of this clone revealed an open reading frame of 300 amino acids.
  • the 550 bp of the 5'-end of this clone used to construct the GST-p60 fusion protein is shown in SEQ ID NO: 3.
  • SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.
  • a larger 1.5-kb cDNA clone was isolated from a Hela cell library (FIG. 2 and SEQ ID NO: 1) and on sequencing was found to contain an open reading frame of 441 amino acids as represented in FIG. 2 and SEQ ID NO: 2.
  • the protein encoded by this open reading frame was designated p60 on the basis of the apparent molecular weight of the protein it encodes as described below.
  • a GST-p60 fusion protein was reacted with a HSV- 1(F) infected HeLa cell extract.
  • the proteins pulled down by the GST-p60 protein were solubilized, electrophoretically separated on a denaturing gel, transferred to a nitrocellulose sheet and reacted with antibody to ICP22 as described in Example 1, Materials and Methods.
  • GST-p60 was reacted with HSV-l(F)-infected extract. Briefly, fusion proteins were grown in BL21 and purified as recommended by the manufacturer (Pharmacia). GST and GST-p60 were mixed with HSV-l(F)-infected HeLa extract, and beads were collected and washed as described in Materials and Methods. Proteins were separated in 7% denaturing polyacrylamide gel, transferred to nitrocellulose, blocked, reacted with R77 and then with a goat anti-rabbit antibody conjugated to horseradish peroxidase and processed as described by the manufacturer (Amersham). GST-p60 bound ICP22, whereas GST alone did not.
  • ICP22 Only the fastest migrating, underprocessed form of ICP22 was able to bind to GST-p60. This is in contrast to another ICP22-binding cellular protein, p78, which the inventors found to bind all forms of ICP22 (Brunt and Roizman, 1998).
  • Proteins were separated in 7% denaturing polyacrylamide gels, transferred to nitrocellulose, blocked, reacted with R77 or with a monoclonal antibody to ICPO and then with a goat anti-rabbit antibody (ICP22) or a goat anti-mouse antibody (ICPO) conjugated to horseradish peroxidase and processed as described by the manufacturer (Amersham). The results indicated the following:
  • GST-p60 bound the full-length ICP22 as well as the ICP22 lacking the carboxyl-terminal 24 amino acids, but not the protein lacking the terminal 40 amino acids.
  • the binding site on ICP22 for p60 maps in the carboxyl-terminal 40 amino acids, approximately between amino acids 24 and 40 from the carboxyl terminus of ICP22.
  • p60 interacts with ICPO independently of ICP22.
  • HEp-2 cell extracts and rabbit skin cell extracts were separated on 10% denaturing polyacrylamide gels, transferred to nitrocellulose, blocked, reacted with a serum to p60 and then with a goat anti-rabbit antibody conjugated to alkaline phosphatase.
  • the immunoblots were developed as described by the manufacturer (BioRad, Hercules, CA).
  • the antiserum reproducibly detected a closely migrating doublet bands with an apparent M r of 60,000 in electrophoretically separated lysates of mock-infected HEp-2.
  • the antibody reacted with well resolved doublet of bands in lysates of mock-infected rabbit skin cells. These bands migrated with a mobility similar mobility to those of mock infected HEp-2 cells.
  • the antibody reacted with a higher molecular weight band in lysates of rabbit skin cells.
  • the electrophoretic mobility of p60 in lysates of rabbit skin cells infected with the R325 ( ⁇ 22 " /U s 1.5 " ) mutant p60 was processed to a higher apparent molecular weight. The results may be summarized as follows:
  • Electrophoretically separated p60 from lysates of rabbit skin cells infected with wild type virus formed several additional bands. These bands were also formed by p60 from lysates of cells infected with the U s 3 " - virus (R7041) - infected cells but were not formed by the p60 present in lysates of mock-infected cells or cells infected with U L 13 " (R7356) or ⁇ 22VU s 1.5 " (R325) viruses. These results indicate that in wild-type virus-infected rabbit skin cells p60 is posttranslationally modified to a higher apparent molecular weight and that this posttranslational modification requires the U L 13 protein kinase and ICP22 and or U s 1.5 protein.
  • Example 5 p60 translocation to the nucleus and co-localization with ICPO in infected HEp-2 cells but not in infected rabbit skin cells
  • HEp-2 cells grown as described in Example 1 Materials and Methods were exposed to 10 PFU of HSV-l(F), R7356 (U L 13 ), R7041 or R325 per cell. The cultures were fixed and reacted with antibody to p60 (FITC) or ICPO (Texas Red). The results were as follows:
  • p60 was distributed mainly in the nucleus.
  • p60 was distributed in some cells in small nuclear bodies and in others throughout the nucleus.
  • p60 colocalized with ICPO in the small nuclear bodies but not in cells in which p60 was dispersed throughout the nucleus.
  • the distribution of p60 in cells infected with R325 ( ⁇ 22 ⁇ U s 1.5 " ) mutant was similar to that observed in wild-type infected cells.
  • ICPO also localized in the cytoplasm of infected cells.
  • a novel cellular protein designated p60, has been identified that interacts with two HSV-1 regulatory proteins, ICP22 and ICPO, in the yeast two-hybrid system and in in vitro biochemical assays.
  • ICP22 and ICPO two HSV-1 regulatory proteins
  • FIG. 3 The salient features of results, summarized in part by FIG. 3, are as follows:
  • p60 binds only one of the multiple electrophoretic isoforms of ICP22.
  • phosphorylation, and concurrent alteration in electrophoretic mobility of ICP22 have been shown to be necessary for the up-regulation of selected HSV-1 genes (Purves et al, 1993), this interaction suggests that each ICP22 isoform performs a specific function.
  • the p60-ICP22 interaction is different from that of the p78-ICP22 interaction in that in the latter case all isoforms of ICP22 interact with p78.
  • the significance of the results presented in the Examples stems from two considerations. First, HSV proteins frequently accumulate in several isoforms differing in electrophoretic mobility.
  • the p60 binding domain was mapped to a stretch of approximately 16 amino acids located close to the carboxyl terminus of ICP22. This domain is essential for the processing of ICP22. It is conceivable that phosphorylation of ICP22 could interfere with binding to p60.
  • p60 binds to ICPO and the domain responsible for binding has been mapped to the carboxyl-terminal 130 amino acids of exon 2 of ICPO.
  • the data indicate that p60 can bind to both proteins as illustrated in FIG. 8 Panel A, the inventors have not rigorously proven that p60 can simultaneously bind to both proteins. Yet similarities in the functions of ICP22 and of ICPO suggest that this is likely to occur at specific stages of viral replicative cycle.
  • ICPO and ICP22 are both multifunctional proteins and recent studies indicate that both interact with cell-cycle dependent cellular proteins.
  • ICPO binds and stabilizes cyclin D3 whereas ICP22 interacts with p78, a protein detected only briefly early in the S phase (Kawaguchi et al, 1997).
  • the accumulation of ICP22 itself was also found to be cell-cycle dependent.
  • Both rabbit skin and HEp-2 cell lines are highly permissive to wild-type virus. They differ in two respects. First, rabbit skin cells restrict the replication of ⁇ 22 " mutants whereas HEp-2 cells are equally permissive to both wild-type and ⁇ 22 " viruses. The evidence presented in this report shows that p60 is similarly dispersed in uninfected rabbit skin and HEp-2 cells but differs quite significantly after infection. In infected HEp-2 cells p60 is translocated into dense nuclear bodies containing ICPO. Late times after infection, p60 is also found in dispersed throughout the nucleus of infected cells.
  • Translocation of p60 to the dense nuclear bodies does not require ICP22/U S 1.5 protein inasmuch as p60 is translocated to these structures in cells infected with virus mutants lacking the carboxyl-terminal domain of ICP22. Dispersal of the p60 throughout the nucleus requires the presence of U L 13 protein kinase and to a slightly lesser extent, of U s 3 protein kinase. In rabbit skin cells p60 is processed to isoforms of higher apparent molecular weight and is not translocated into the dense nuclear structures. Moreover, the modification of p60 to isoforms of higher apparent molecular weight requires the participation of ICP22/U S 1,5 and of U L 13 protein kinase.
  • Nicolas and Rubenstein In: Vectors: A survey of molecular cloning vectors and their uses, Stoneham: Butterworth, 1988. Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982. Ogle et ⁇ /., Virology, 235:406-413, 1997. Paskind et al, Virology, 67:242-248, 1975. PCT/US87/00880. PCT/US89/01025.

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  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne p60, une nouvelle protéine cellulaire qui interagit avec des protéines régulatrices du virus de l'herpès. Des analyses de protéines se liant à HSV-1 ont révélé que p60 se lie à un isoforme d'ICP22 de type sauvage. Le site de liaison de p60 a été cartographié sur une étendue d'environ 16 acides aminés situés à proximité de l'extrémité terminale carboxyle d'ICP22, un domaine essentiel au traitement d'ICP22. L'interaction de p60 avec un seul isoforme d'ICP22 uniquement indique une fonction spécifique de cet isoforme. La protéine p60 se lie également à ICP0, cette liaison étant indépendante de celle d'ICP22.
PCT/US2000/009214 1999-03-09 2000-04-07 Proteine cellulaire p60 interagissant avec des proteines regulatrices du virus de l'herpes WO2000061749A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU42079/00A AU4207900A (en) 1999-03-09 2000-04-07 Cellular protein p60 that interacts with herpesvirus regulatory proteins

Applications Claiming Priority (2)

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US12850199P 1999-03-09 1999-03-09
US60/128,501 1999-04-09

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WO2000061749A1 true WO2000061749A1 (fr) 2000-10-19

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109929014A (zh) * 2019-02-18 2019-06-25 北京市农林科学院 一种抑制鸡先天性免疫反应鸡马立克氏病病毒蛋白及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018127A1 (fr) * 1997-10-02 1999-04-15 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018127A1 (fr) * 1997-10-02 1999-04-15 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRUNI R. & ROIZMAN B.: "Herpes Simplex Virus 1 Reglatory Protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.", J. VIROL., vol. 72, no. 11, November 1998 (1998-11-01), pages 8525 - 8531, XP002146335 *
BRUNI R. ET AL.: "A novel cellular protein, p60, interacting with both Herpes Simplex Virus 1 Regulatory Proteins IPC22 and IPC0 is modified in a cell-type specific manner and is recruited to the nucleus after infection.", J. VIROL., vol. 73, no. 5, May 1999 (1999-05-01), pages 3810 - 3817, XP002146334 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109929014A (zh) * 2019-02-18 2019-06-25 北京市农林科学院 一种抑制鸡先天性免疫反应鸡马立克氏病病毒蛋白及其应用

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