WO1992003578A1 - Analyse d'integration retrovirale in vitro - Google Patents
Analyse d'integration retrovirale in vitro Download PDFInfo
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- WO1992003578A1 WO1992003578A1 PCT/US1991/005828 US9105828W WO9203578A1 WO 1992003578 A1 WO1992003578 A1 WO 1992003578A1 US 9105828 W US9105828 W US 9105828W WO 9203578 A1 WO9203578 A1 WO 9203578A1
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- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a new assay system which screens for anti-retroviral (anti-HIV) drugs.
- the present invention relates to a simple and rapid in vitro integration assay requiring integration or IN protein as the only viral protein necessary for integration. Such an assay may be useful for determining whether viral integration into a host or target DNA is achieved or alternatively, whether integration is inhibited by a test drug.
- the present invention also relates to active MoMLV and HIV recombinant integrase (IN) proteins, each protein has the combined in vitro activities of (1) LT -specific nucleolytic cleavage to expose recessed 3' ends and (2) the integration of LTR recessed 3' ends into DNA targets.
- the retroviral RNA genome is reverse transcribed to make a DNA copy. Integration of this viral DNA into a chromosome of the host cell is necessary for normal viral replication. Transcription of the integrated DNA produces viral RNA species that function as the template for translation of viral proteins or as the genome of progeny virions. Assembled virions bud from the cell membrane and their entry into another cell completes the viral replication cycle.
- retroviral replication see Varmus, H., and Brown, P., In: Mobile DNA. eds. Berg, D.E., and Howe, M.M. , 1989. Genetic studies have identified two classes of mutations that directly affect integration.
- MoMLV Moloney murine leukemia virus
- the endogenous viral DNA made by reverse transcription exists as part of a large nucleoprotein complex, derived from the viral core (Brown et al., Cell 49: 347-356, 1987; Bower an et al.. Genes Dev. 3: 469-478, 1989).
- the ends of the viral DNA isolated from these complexes are two forms: some are blunt and correspond to the expected full-length product of reverse transcription, others are recessed by 2 bases at the 3 1 end (Fujiwara and Mizuuchi, Cell 54: 497-504, 1988; Brown et al., Proc. Natl. Acad. Sci. USA 86: 2525-2529, 1989).
- the MoMLV IN function is implicated in generating these recessed 3' ends because several mutant viruses defective in IN make only the blunt ended viral DNA (Roth et al.. Cell 58: 47-54, 1989; Brown et al., 1989) .
- the endogenous viral DNA can be integrated into a target DNA in vitro and the necessary protein factors copurify with the complex (Brown et al., 1987; Bowerman et al., 1989).
- Analysis of the structure of the MoMLV integration intermediate, made in this cell-free system, has demonstrated that the viral DNA species with recessed 3* ends is the precursor for integration (Fujiwara and Mizuuchi, Cell 54: 497-504, 1988; Brown et al., 1989).
- these recessed 3' ends of the viral DNA are joined to the 5* ends of a double strand cut made in the target DNA; 4 bp staggered cleavage of the target DNA, with a 5* protrusion, is inferred from the characteristic 4 bp duplication of target DNA sequence at the site of MoMLV integration.
- the 5' ends of the viral DNA remain unjoined in the intermediate and a DNA repair step is necessary to complete the integration process.
- mini-MoMLV DNA also carried antibiotic resistance markers, enabling the integration products with phage lambda target DNA to be detected by selection in IL. coli.
- Detergent-disrupted MoMLV virions provided the necessary viral proteins. However, these assays are time consuming and impracticable for drug screening.
- HIV core particle assay An unattractive feature of the HIV core particle assay is that HIV core particles are potentially infectuous.
- the present invention relates to active recombinantly produced MoMLV and HIV retroviral IN protein which has the activities of specific cleavage and integration of MoMLV and HIV long terminal repeat (LTR) ends into a target DNA.
- the present invention relates to a general retroviral DNA in vitro integration assay method. The method comprises the steps of contacting active retroviral IN protein purified from an overexpressed recombinant source with labeled synthetic duplex oligonucleotides encoding at least one LTR terminal end of the same retrovirus under conditions such that integration is effected and detecting the integration products which indicate integration has occurred or the absence thereof indicative that integration has been inhibited or did not occur.
- Integration products may be separated by eletrophoretic methods, by binding to avidin coupled solid supports, or by direct coupling of the oligonucleotide to a solid support. Products may be detected, depending on the label, by autoradiography scintillation counting, treatment with an enzyme substrate or treatment with the proper wave length of light.
- the present invention relates to a modification of the retroviral DNA in vitro integration assay that comprises the steps of contacting active recombinantly produced retroviral IN protein with labeled synthetic duplex oligonucleotides encoding at least one LTR termini end of the same retrovirus and a second heterologous duplex DNA as target under conditions such that integration is effected and detecting the presence or absence of integration products.
- the present invention relates to general retroviral drug screening methods that provides a means of identifying inhibitors of retroviral integration comprising the steps of contacting one or more drug inhibitors with active recombinant retroviral IN protein and labeled duplex oligonucleotides encoding at least one LTR terminal end of same retrovirus under conditions such that inhibition of integration can be effected and detecting the presence or absence of integration products.
- Another embodiment of the present invention relates to a modification for the method for identifying inhibitors of retroviral integration comprising the steps of contacting one or more of said inhibitors with active recombinant retroviral
- duplex oligonucleotides encoding at least one LTR end of same retrovirus and second heterologous duplex target duplex DNA under conditions such that inhibition of integration is effected, and detecting the presence or absence of integration products.
- the present invention relates to a simple in vitro HIV drug screening method that provides a means for identifying inhibitors of growth of HIV.
- the present invention in particular relates to the addition of the sample drug to a test reaction prior to the initiation of the reaction.
- the reaction contains purified HIV IN protein produced in making use of expression systems and synthetic duplex oligonucleotide corresponding to a terminal end of unintegrated linear HIV DNA.
- a second duplex DNA as target DNA can be added as exogenous source of target DNA for HIV integration.
- FIG. 1 shows SDS-polyacrylamide gel analyses of partially purified MoMLV IN protein expressed by Sf9 cells.
- SDS-polyacrylamide gel electrophoresis shows the insoluble protein fraction from uninfected Sf9 cells (lane a) , Sf9 cells infected with wildtype AcMNPV (lane b) , and Sf9 cells infected with recombinant AcMNPV expressing MoMLV IN protein (lane c) .
- the insoluble protein fraction shown in lane c was solubilized in the presence of urea and applied to a Superose 12 column. Peak fractions containing IN protein were pooled (lane d) . The migration positions of molecular weight standards are indicated.
- the gel was stained with Coomassie Blue.
- Figure 2 demonstrates the cleavage activity of MoMLV DNA termini by cloned and partially purified IN protein.
- A DNA substrates for cleavage reactions with IN protein.
- LTRl corresponds to the 5' edge of the MoMLV U3 sequence. Bases that differ from this wildtype sequence in LTR2 and LTR3 are indicated by bold letters. Filled circles indicate the position of the 32 P label.
- B Cleavage of LTRl by IN protein. The reaction products were electrophoresed in a 20% denaturing polyacrylamide gel and visualized by autoradiography. Lanes a and b, chemical cleavage standards; A+G, T+C, respectively.
- the mass of DNA included in the reaction with single stranded DNA substrate (lane g) was adjusted to equal that of the standard reaction with duplex DNA.
- the major cleavage product in the presence of Mn 2+ indicated by an arrow, results from removal of 2 nucleotides from the 3' end of the labeled DNA strand in LTRl.
- the labeling of the lanes in (C) and (D) is the same as in (A) .
- Figure 3 shows the expected products of normal integration with LTRl.
- A) A pair of LTR 1 molecules are brought together by IN protein (shown stippled) . 5' ends of DNA are indicated by small circles and the labeled 5' end in LTRl is shown as a filled circle.
- (C) IN protein makes a 4 bp staggered cut in another LTRl molecule, which acts as the target DNA and integrates the recessed 3' ends (shown in B) to the protruding 5* ends of this cut.
- Figure 4 demonstrates the integration of MoMLV DNA ends by IN protein.
- A DNA substrates for reactions with IN protein. Labeling is the same as in Figure 2A.
- B Integration of LTRl by IN protein. The reactions are the same as in Figure 2B, but a longer exposure of the autoradiogram is shown. Lanes a and b,- chemical cleavage standards; A+G, T+C, respectively. Reaction omitting IN protein (lane c) , standard reaction (lane d) . Other labeling is as in Figure 2B.
- C Reactions with LTR4.
- D Reactions with LTR5.
- E Reactions with LTR6.
- FIG. 5 shows the integration products made with the labeled strand of LTRl.
- a population of integration products made with LTRl was purified from a denaturing polyacrylamide gel and subjected to base-specific chemical cleavage reactions. The products were then electrophoresed in a second sequencing gel Lanes a - d, chemical cleavage reactions; G, A+G, T+C, and C, respectively.
- IP The integration product that remains uncleaved after the chemical cleavage reactions is labeled IP.
- the sequence is unique from the 5' end and matches that of LTRl, but becomes heterogeneous after the CA position that is expected to joined to the target DNA, indicated by the arrow.
- Figure 6 shows the expected product of normal integration of LTRl ends into a circular target DNA.
- the reaction is as shown in Figure 3, except that a circular DNA molecule serves as the target DNA for integration.
- A Synapsis of a pair of LTRl molecules.
- B Two nucleotides are cleaved from the 3 1 end of each LTRl molecule.
- C The recessed 3' ends of LTRl are joined to the 5' ends of a staggered cut made in the target DNA.
- D Melting of the 4 bp between the site of target DNA cleavage generates a linear product with 4 bp single strand gaps and 2 based overhangs at the junctions between LTRl and the target DNA. Labeling is the same as in Figure 3.
- Figure 7 shows the products made in an integration reaction was carried out with labeled LTRl and a circular target DNA.
- a reaction was carried out with LTRl and IN protein in the presence of an unlabeled 400 bp circular target DNA.
- the reaction products were deproteinized and analyzed in a native 5% polyacrylamide gel.
- Lane a pBR322 DNA digested with Mspl and labeled with 32 P;
- lane b linearized target DNA labeled with 32 P;
- lane c products of the complete integration reaction;
- lane d reaction containing LTR5 instead of LTRl;
- lane e the circular target DNA was omitted from the reaction;
- lane f IN protein was omitted from the reaction.
- the arrow indicates the position of the linear integration product.
- the sizes of the pBR322 Mspl fragments are shown.
- Figure 8 demonstrates the presence of a 4 bp target sequence duplication in integration products.
- the linear product of an integration reaction with LTR7, IN protein and the 400 bp circular target DNA was amplified by PCR and cloned into pUC19 (LTR7 is identical to LTRl, except for addition of a BamHl site after position 18 from the LTR end to facilitate cloning) .
- LTR7 is identical to LTRl, except for addition of a BamHl site after position 18 from the LTR end to facilitate cloning
- Nine cloned products were analyzed. The structure of each corresponded to that shown in Figure 6D after repair of the single strand gaps and overhangs.
- Each line shows the target DNA sequence flanking a pair of integrated LTR7 ends. In all cases the terminal two T nucleotides are missing from the integrated LTR7 ends.
- Figure 9 shows the SDS-PAGE analysis of the preparation of HIV IN protein and its substrate miniHIV.
- A SDS-PAGE analysis of extracts of insect cells (Sf9) expressing HIV IN protein. Lane 1) Extract of insoluble proteins from uninfected Sf9 cells. Lane 2) Extract of insoluble proteins from Sf9 cells infected with 561-3, the baculovirus that contains the HIV IN coding region. The arrow marks the position of HIV IN protein. Dashes with numbers indicate the mobilities of marker proteins with the indicated sizes (X10 "3 ) .
- B Structure of the miniHIV DNA.
- the sequence of the oligonucleotide duplex matching the HIV LTR ends (the 5' end of the left LTR and the 3' end of the right LTR of pNL4-3) (Adachi et al., SL. Virology 59: 284-291, 1986) is shown at the top. Digestion of a plasmid containing this duplex with Nde I yields the linear miniHIV DNA. MiniHIV DNA contains the predicted ends of the linear HIV DNA precursor for integration except for the 5'-TA-3' extension at the 5' ends, which is inferred to be 5'-AC-3' in the precursor in vivo.
- Figure 10 represents the sequences of integration products at junctions between miniHIV and lambda DNA.
- A Summary of sequences. The locations of target and miniHIV DNA sequences are indicated at the top of the figure. Arrows over the sequences mark the positions of directly repeated duplications in the target DNA, and duplicated sequences are shown in bold letters.
- Each miniHIV contains the expected 5•-TG and CA-3' sequences at each border with target DNA (not shown) .
- Integration events a- c contain 5 bp target duplications characteristic of HIV integration in vivo.
- Figure 11 illustrates the proposed role of HIV IN protein in integration.
- Linear viral DNA is shown as the darker “ladder,” target DNA is shown as the lighter ladder; “rungs” in the ladders indicate individual base pairs.
- IN protein is shown stippled. DNA 5' ends are indicated by dark dots.
- the integration- competent complex composed of IN and linear viral DNA is shown in the top of part a. Integration proceeds by joining of the recessed 3' ends of viral DNA to protruding 5* ends of a staggered break in a DNA target (part a, middle) .
- LTR B is the same as LTR A except for an A to G change (shown in bold letters) .
- An A to G change in the analogous position in MoMLV disrupts the cleavage and joining reactions.
- the arrow indicates the position of IN-dependent cleavage of LTR A.
- B) Cleavage and joining of oligonucleotide substrates in the presence of HIV IN protein. Lane a) Unreacted oligonucleotide substrate. Lane b) The indicated LTR substrate was incubated for 1 hour in the presence of a suitable buffer containing Mn and partially purified IN protein. The IN protein used in this reaction differed from that of Figure 10 in that IN was solubilized in the presence of 4M urea and then dialyzed. The major cleavage product is indicated with an arrow. Integration products are indicated with a bracket; such molecules have been shown in both the MoMLV and HIV case to be the products of IN-directed DNA integration.
- Figure 13 illustrates the same reaction scheme as depicted in Figure 3 with the following modifications.
- the 32 P (represented as a solid circle in Figure 3) is replaced by the symbol "L" because labels other than 32 P can potentially be used.
- the small open circles now represent the 5' end of the DNA strand that remains unjoined in the reaction product.
- the 3* end of this strand is now shown labeled with biotin (BIO) .
- Figure 14 illustrates the separation and detection of labeled integration products.
- the reaction products, together with unreacted substrate, are bound to a solid support to which avidin (which has a very high affinity for biotin) is covalently coupled (shown in A) .
- the bound DNA is washed with alkali to denature the hydrogen bonded DNA strands.
- the only labeled (with L) DNA strands remaining after the wash are the products of integration (shown in B) .
- the efficiency of integration is quantitated by measuring the amount of label (L) that remains bound to the solid support.
- the present invention relates, in part, to recombinantly produced active retroviral integrase (IN) proteins. Proteins of the present invention have been isolated from cells that over-express the retroviral IN gene and thus provide a means of producing large quantities of the protein.
- a principle embodiment of this aspect of the present invention relates to in vitro retroviral integration assay methods wherein the active retroviral IN protein is the only retroviral protein necessary to generate both the recessed 3' ends of the viral DNA and carry out its integration into target DNA in vitro.
- the present invention also provides a means for identifying inhibitors of retroviral integration. By utilizing the purified retroviral IN protein isolated from insect or £.
- an in vitro retroviral DNA integration system that does not involve potentially infectious derivatives that are found in virus particles or virus-infected cell extracts.
- the present invention is particularly suited for identifying inhibitors of growth of HIV i vitro.
- the present invention relates to a recombinant DNA construct encoding the entire coding sequence of MoMLV and HIV integrase (IN) , and also relates to the recombinant host cells transformed therewith that expresses active IN protein.
- the host cells suitable for use in the present invention can be either eukaryotic or prokaryotic.
- the DNA construct suitable for IN protein expression comprises the coding sequence of MoMLV or HIV IN protein and suitable vector.
- the construct may consist of a baculovirus expression vector containing a promoter element that is operatively linked to the IN coding sequence.
- the present invention further relates to active MoMLV and HIV recombinant IN proteins that are purified from infected host cells using well known methods.
- an active form of the proteins of the present invention can be obtained by one skilled in the art using standard methodologies for protein purification without undue experimentation.
- Sf9 cells infected with recombinant baculovirus overexpressing IN proteins are harvested and lysed in non-ionic detergent.
- the pellet of the centrifuged lysates are resuspended in buffer and further purified by gel filtration. Pooled fractions containing IN protein are identified by SDS-PAGE and monitored for biological integration activity in the assay method described below.
- active protein can be expressed in £• coli and purified from this source.
- the recombinant purified MoMLV and HIV IN protein of the present invention also relates to the two distinct biochemical activities found in recombinant purified MoMLV and HIV IN.
- the dual i vitro activities of proteins are: 1) cleaving and processing MoMLV and HIV LTR termini to expose the correct recessed 3* ends and 2) integrating the processed retroviral LTR ends into target DNA at 5 1 ends of cuts which are also made by IN protein.
- the present invention has determined that the retroviral IN protein is the only viral protein required to cut and process retroviral DNA and target DNA and accomplish integration in vitro.
- the present invention also relates to a method for measuring retroviral integration in vitro requiring only the active recombinant purified retroviral IN protein containing both nuclease and DNA integration activities.
- the method for in vitro retroviral integration is schematically presented in Figures 3 and 6.
- MoMLV retroviral integration is discussed.
- Figure 3 (A) a pair of 5' end labeled duplex DNA oligonucleotides corresponding to the authentic LTRs of unintegrated MoMLV LTR termini are brought together by MoMLV IN obtained from a recombinant source.
- the IN protein correctly cleaves two nucleotides from the 3* ends of the 32 P labeled strand of each LTR molecule to make recessed precursor ends for integration (Figure 3(B)) .
- the IN protein makes a 4 base pair staggered cut in another LTR molecules which acts as the target DNA for the recessed 3' ends ( Figure 3(C)).
- cleavage products as shown in Figure 3 (D) , are detected by electrophoresis in polyacrylamide gels followed by autoradiography.
- integration products are visualized as labeled bands migrating slower on the gel then unit length oligonucleotides.
- Suitable labels for use in the present invention include, but are not limited to 32 P.
- Other labels include florescent labels, enzyme linked labels, or biotin labels.
- the 32 P is replaced by the symbol "L" representing other labels that can be used.
- the small open circle represents the 5' end of the DNA strand that remains unjoined in the reaction product. The 3' end of this strand is labeled with biotin.
- the reaction products are contacted with Avidin-coupled solid supports prepared by commercially available methods.
- Suitable solid supports for use in the present invention include, but are not limited to icrotiter plates. The efficiency of integration is quantitated by measuring the amount of "L" label that remains bound to the solid support after washing.
- the retroviral integration system may be further modified as schematically presented in Figure 6.
- MoMLV retroviral integration system is used as an example of the general method.
- the integration reaction is the same as previously depicted in Figure 3, except that a second heterologous DNA molecule serves as the target DNA for integration.
- the processed recessed 3' ends of the MoMLV LTR are joined to the 5* ends of a staggered cut made in the target duplex DNA molecule instead of another MoMLV LTR oligonucleotide which previously acted as both substrate and target DNA in the schematic of Figure 3.
- the detection means for determining reaction products are analogous to those described above, except that label L is joined to the LTR and biotin is joined to target DNA or vice versa.
- the present invention in particular, is suitable for providing a means for identifying inhibitors of growth of retroviruses such as HIV.
- the in vitro retroviral reaction assay models the normal integration of a DNA copy of the retroviral genome into the genome of infected cells.
- a test drug inhibitor is added to the reaction mixture prior to initiation of the reaction.
- the reaction contains labeled duplex oligonucleotides corresponding to the predicted termini sequence of unintegrated linear retroviral DNA and recombinant purified IN protein. After the appropriate incubation, incubation products are measured by direct physical detection methods such as those described previously. A decrease in integration reaction products of test samples in comparison with controls indicates interference of integration or inhibition of retroviral integration.
- Polynucleotide kinase was purchased from Phar icia, restriction enzymes and DNA ligase from New England Biolabs, and Taq polymerase from Promega. Molecular weight protein standards were purchased from BRL. Standard methods involving DNA manipulation are found in Sambrook, et al., 2nd ed., Cold Soring Harbor Laboratory. 1989) .
- the linker bridging the BAMH1 site of pAc373 and the Xmnl site within the IN coding region has the sequence 5*-GATCCTATAAATATG GAACA-3 1 , where the dashes represent the IN coding sequence between the translation initiation codon and the Xmnl site.
- the Seal restriction site to the 3* side of the pol termination codon was bridged to pAc373 with a BamHl linker.
- the resulting plasmid (pMK556) was recombined with wildtype Autograoha californica nuclear polyhedrosis virus (AcMNPV) by cotransformation of Spodoptera frugiperda (Sf9) cells and recombinants were plaque purified by standard procedures (Summers et al, 1987).
- AcMNPV Autograoha californica nuclear polyhedrosis virus
- Sf9 Spodoptera frugiperda
- recombinants were plaque purified by standard procedures (Summers et al, 1987).
- One purified recombinant baculovirus, 556- 3 was used for expression of MoMLV IN protein. Partial purification of MoMLV IN protein
- Sf9 cells as monolayers and infection with recombinant virus was carried out essentially as described by Summers et al., 1987.
- a 150 cm 2 flask of Sf9 cells was infected with clone 556-3. After incubation for 3 days at 28°C, the cells were harvested. All procedures were carried out between 0°C and 4 ⁇ C. The cells were first chilled, the medium removed, and the monolayer of cells was gently washed with 20 ml of 20 mM Hepes pH 7.6, 150 mM K gluta ate.
- This buffer was removed and the cells were lysed by addition of 5 ml 100 mM K glutamate, 20 mM Hepes pH 7.6, 5 mM MgAc 2 , 1 mM DTT, 0.5% Nonidet P-40, with gentle pipetting. After incubation on ice for 5 minutes, the lysate was centrifuged at 13,000g for 10 min. The pellet was retained and resuspended in 5 ml 0.4 M K glutamate, 20 mM Hepes pH 7.6, 1 mM EDTA, ImM DTT, 0.5% (w/v) Nonidet P-40 by disruption with a glass-glass homogenizer.
- the sample was thawed, centrifuged at 13,000g for 10 minutes, and the pellet was resuspended in 400 ⁇ l HEDG buffer containing 4 M urea, 0.4 M K glutamate, and 0.1 % (w/v) Nonidet P-40.
- the protein was incubated on ice for 15 minutes and then centrifuged at 13,000g for 10 minutes. 250 ⁇ l of the supernatant was then loaded onto a Superose 12 column (HR 10/30, Pharmacia) equilibrated with the same solubilization buffer. The column was run at a flow rate of 0.1 ml/min.
- Substrate LTR 1 corresponding to the U3 end of MoMLV DNA, was made by annealing the synthetic oligonucleotide sequence 5'- AATGAAAGACCCCACCTG-3' with its complement.
- LTR2 through LTR6 differ from LTRl as shown in Figures 2A and 4A. The location of the 32 P label is also noted in these figures.
- LTR7 was formed by annealing the oligonucleotide sequence 5 1 -
- Oligonucleotides were purified by electrophoresis in a DNA sequencing gel before labeling and annealing. 100 ng of the oligonucleotide strand to be labeled with 32 P at its 5' end was phosphorylated by T4 polynucleotide kinase in the presence of 100 ⁇ Ci 32 P ATP (specific activity 3000 Ci/mmol) in a reaction volume of 15 ⁇ l. EDTA was added to a final concentration of 25 mM and the polynucleotide kinase was inactivated by heating at 85 ⁇ C for 15 minutes.
- NaCL NaCL was added to a final concentration of 0.1 M, together with an excess (400 ng) of the unlabeled complementary strand, in a total volume of 40 ⁇ l.
- the mixture was heated to 80 °C and the DNA was annealed by slow cooling. Unincorporated nucleotide was then removed by passage through a G25 Sephadex Quick Spin Column (Boehringer) .
- Oligonucleotide substrate corresponding to the ends of HIV DNA were prepared by the same method•
- Circular target DNA was prepared by ligation of the 0.4 kb EcoRl fragment of pLMF124 (Fisher, et al. EMBO £. 5: 1411-1418, 1986) This DNA is the Alul restriction fragment of pBR322 (position 686-1089) with EcoRl cohesive ends. The EcoRl fragment was circularized by ligation in the presence of HU protein, which greatly increases the yield of monomer circle product (Hodges-Garcia, et al. 2. Biol. Chem. 264: 14621-14623, 1989). The ligated DNA was then extracted twice with phenol, once with chloroform, and precipitated with ethanol.
- reaction conditions for cleavage of the 3' ends MoMLV DNA and integration are identical.
- Reactions (15 ⁇ l) contained 85 mM KCl, 20 mM MOPS pH 7.2, 3 mM MmCl 2 , 10 mM DTT, 20% (w/v) glycerol, 100 ⁇ g/ml BSA, 0.5 pmol LTR substrate (excluding the excess unlabeled strand) , and 1 pmol of MoMLV IN protein (these conditions exclude 13 mM K glutamate and 0.003% Nonidet P-40 continued by the IN protein storage buffer) . Reactions were incubated at 30 °C for 1 hour.
- reaction product in DNA sequencing gels were stopped by addition of 15 ⁇ l 95% formamide, 20 mM EDTA, 0.05% Bromophenol Blue and 0.05% Xylene Cyanol. 1.5 ⁇ l of the sample was loaded after first heating at 95 °C for 2 minutes. Preparative reactions for isolation of the integration products from sequencing gels were scaled up to 75 ⁇ l; the products of four such reactions were pooled, precipitated with ethanol, and the whole sample was loaded.
- Reactions including the 0.4 kb circular target DNA were performed exactly as described above, except the reactions (15 ⁇ l) included 1 ng of the circular target DNA. Reactions were stopped by addition of 5 ⁇ l 5 mg/ml Pronase in 100 mM EDTA, 5 ⁇ l 1 M NaCl, 1 ⁇ l 10% SDS, and 24 ⁇ l H 2 0. After incubation at 30 ⁇ C for 1 hour, 5 ⁇ l of 3 M NaAc was added and the mixture was extracted once with phenol and precipitated with ethanol. The entire sample was loaded onto a polyacrylamide gel.
- the products of reactions with oligonucleotide viral ends were analyzed by electrophoresis in 20% polyacrylamide (19:1, aerylamide:bis) sequencing gels in TBE buffer; chemical cleavage standards were generated by the method of Maxam, and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564, 1977).
- the products of reactions with the circular target DNA were electrophoresed in a 5% polyacrylamide (37.5:1, acrylamide:bis) gel, in TBE buffer, for 90 minutes at 10 V/cm. Protein samples were electrophoresed in 10% SDS- polyacrylamide gels (Laemmli, 1970) , and stained with Coomassie Blue. Amplification and seouencino of the integration products with a circular target DNA.
- the products of an integration reaction with substrate LTR7 and the 0.4 kb circular target DNA were electrophoresed in a 5% polyacrylamide gel, as described above.
- LTR7 was labeled with 32 P, as indicated for LTRl in Figure 2A, in order to facilitate identification of the reaction product.
- the linear product was excised from the gel, the DNA was isolated by the crush/soak method, and then treated with DNA Polymerase 1 to repair the putative single strand gaps in the integration product (see Figure 6) .
- the DNA was then subjected to 30 cycles of PCR amplification using a Perkin Elmer Cetus Thermal Cycler with Taq polymerase (Promega) .
- the primer sequence was 5*-GTTATGGATCCAGGTGGGGTCT-3' .
- the amplified product was digested with Bam HI and ligated into the Bam HI site of pUC19 using standard procedures.
- the DNA sequences of the junctions between LTR7 and target DNA in the cloned products were determined by sequencing double stranded plasmid DNA by the dideoxy method with the Sequenase DNA polymerase (USB) , as recommended by the supplier.
- the pUC sequencing primers were 5'- GTAAAACGACCGCCAGT-3' (forward) and 5'- CAGGAAACAGCTATGAC-3' (reverse)
- the recombinant baculovirus expressing HIV IN protein was made as follows: the 0.7 kb Pvu II to Bsp Ml fragment of pNY5' (Adachi, A., et al. £.
- duplex oligonucleotide linkers were ligated to both ends.
- One synthetic duplex DNA restored the 5' part of the IN coding region and added i) the AUG necessary for translation since the viral IN protein is derived by proteolytic cleavage of a polyprotein precursor, ii) a 9 bp region matching the 9 bp just 5' of the initiation codon of the baculovirus polyhedrin gene, and iii) a Bam HI cohesive end.
- base 4313 was changed from A to G to generate a Bsp Ml site.
- a second synthetic duplex DNA restored the 3' sequence of the gene and added a Bam HI cohesive end.
- This BamHl fragment was then ligated into the BamHl site of pAc373 (Summers et al., 1987) to make pMK561.
- the recombinant baculovirus expressing IN was generated by recombination in vivo between pMK561 and Autographa californica nuclear polyhedrosis virus DNA after contransfection as described (Summers et al., 1987). After plaque purification one isolate, 561-3, was used for expression of IN protein.
- Active HIV IN protein has also been expressed in £. coli.
- the HIV IN coding sequence was cloned using a standard expression system (Rosenberg et al. Gene 56, 125-135, 1987) so as to place the IN coding sequence under the control of T7 promoter.
- HIV IN protein was prepared as follows: Sf9 cells were infected with 561-3 as described (Summers et al., 1987) and HIV IN protein was isolated from infected cells after 3 days of incubation in 150 cm 2 flasks. All the purification steps were carried out at 4 °C. Cells adhered to the flask after removal of the medium, allowing cells to be washed with 5 ml of 150 mM potassium glutamate, 20 mM Tris pH 8, and then lysed by addition of 5 ml of Buffer A containing 100 mM potassium glutamate (Buffer A is 30 mM Tris pH 8, 10 mM MgAc 2 , 1% Nonident P40, and 10% glycerol) .
- Lysates were centrifuged 10 minutes, 13,000 g. The pellet was washed in 2.5 ml of Buffer A containing 400 mM potassium glutamate by homogenizing with a Kimax manual homogenizer. This material was centrifuged again for 10 minutes at 13,000 g, and the insoluble pellet resuspended in 1 ml per flask of starting cells of Buffer A containing 100 mM potassium glutamate. HIV IN protein has been further purified by solubilization in buffer A with 4M urea, followed by gel filtration on a Superose 12 column equilibrated with buffer A containing urea. Control extracts of uninfected or AcMNPV-infected Sf9 cells were prepared in exactly the same way.
- HIV IN protein purified by SDS-PAGE, was sequenced from the amino-terminus in an Applied Biosystems Model 471A Protein Sequencer as described by Matsudaira, P., £. Biol. Chem. 262, 10035-10038 1987.
- the first amino-acid was found to be methionine, and the next 19 a ino-acids matched the sequence of the amino-terminus of HIV IN protein isolated from infected cells, as determined by
- Active HIV IN protein was also purified from induced JL. coli cells containing the HIV IN T7 expression plasmid. Cells were lysed by sonication and an insoluble precipitate containing HIV IN protein and purified by hydrophobic interaction chromatography followed by differential precipitation of HIV IN protein by dialysis against a low ionic strength buffer. HIV IN protein was then resolubilized in a buffer containing 1 M NaCl.
- the synthetic oligonucleotide duplex pictured in Fig. 9(b) was synthesized with Eco RI cohesive ends and inserted into the Eco RI site of pMK468, a pBR322 derivative lacking the Nde I recognition site (Fujiwara and Craigie, 1989), to make pMK564. Digestion of pMK564 with Nde I yields the linear miniHIV DNA.
- mini-MoMLV DNA As a functional assay for putative integration activity of the expressed protein, the previously described in vitro assay for integration of mini-MoMLV DNA was initially employed (Fujiwara and Craigie, 1989) .
- the DNA sequences at the ends of the linear mini-MoMLV DNA mimic the ends of unintegrated MoMLV DNA isolated from the cytoplasm of MoMLV-infected cells and the 3' ends are recessed by two nucleotides as in the authentic precursor for integration. Integration of mini-MoMLV DNA into phage lambda target DNA is detected by in vitro packaging of the reaction products, infecting JL. coli. and selecting for the antibiotic resistance markers present on the mini-MoMLV DNA.
- Integration activity was indeed detected in extracts made from Sf9 cells infected with the recombinant baculovirus; sequencing of the junctions between mini-MoMLV and target DNA of several integration products revealed the expected 4 bp duplication of target DNA sequence at the site of integration, confirming the fidelity of the reaction.
- Preliminary experiments also revealed that most of the IN protein was present in the insoluble fraction of cell lysates, although some integration activity was found in the soluble as well as in the insoluble fraction. A considerable increase in integration activity was obtained by solubilizing the initially insoluble protein fraction in the presence of urea; the protein remained soluble and active after subsequent removal of the urea by dialysis.
- the MoMLV IN function is known to be essential for formation of the recessed ends of the MoMLV DNA precursor for integration in vivo (Roth et al., 1989). Therefore it was tested whether IN protein is able to process blunt-ended MoMLV DNA termini, corresponding to the product of reverse transcription, to generate the correct recessed 3' ends.
- a double stranded DNA LTRl was synthesized corresponding to the first 18 base pairs of one end of unintegrated MoMLV DNA ( Figure 2A) ; the first 13 base pairs of the other end of authentic unintegrated MoMLV DNA are identical and mutations beyond position 13 from the LTR ends do not affect integration in vivo (Roth et al., 1989).
- the DNA strand of LT1 that would be recessed by two nucleotides at its 3• end in the integration precursor was labeled with 32 P at its 5" end.
- oligonucleotides were tested as substrates for cleavage by MoMLV IN protein which contained altered bases (Figure 2C and 2D) .
- LTR2 was processed identically to the wildtype end, with predominant cleavage at position 2 (2C, lane d) .
- LTR 3 exhibited cleavage at positions l and 3 from the 3' end of the labeled strand, with weaker cleavage at position 2 (2D, lane d) .
- the wildtype substrate gives a series of higher molecular weight oligonucleotides which abruptly terminates at a mobility corresponding to about 30 nucleotides ( Figure 4B, lane d) .
- the irregular spacing of the bands indicates that they do not correspond to a unique sequence terminating at different nucleotide positions.
- a similar pattern of bands is also observed with a substrate (LTR4) with recessed 3' ends corresponding to the cleavage product of the full-length substrate (LTRl) by IN protein ( Figure 4C, lane d) .
- this series of bands is absent when the substrate carries an A/T to G/C mutation at position 3(LTR5, Figure 4D) , or when the other strand of the wildtype substrate is labeled at its 5' end (LTR6, Figure
- the labeled products of integration with substrate LTR6 should all be shorter than the initial length (see Figure 3) , as is observed.
- the inventors determined the nucleotide sequence of a population of these products made by incubation of the wildtype substrate LTRl with MoMLV IN protein.
- the longer DNA strands in the integration products should all have a unique sequence from their labeled 5* end up to the CA-3' position that becomes joined to target DNA sequence in the normal reaction, the terminal pair of T nucleotides having been lost.
- the sequence that follows the CA should be heterogeneous because integration can occur at multiple locations in the target DNA.
- MoMLV DNA ends joins each 3' end of the MoMLV DNA to the protruding 5' ends of a 4 bp staggered cut made in the target DNA. Repair of the resulting intermediate generates the characteristic 4 bp target sequence duplication at the site of integration.
- the linear product was cloned and the junctions of several isolates were sequenced.
- the reaction was carried out with LTR7, which differs from LTRl in that a BamHl restriction site is located after position 18 of the MoMLV LTR sequence.
- the linear product was purified from a polyacrylamide gel and amplified by the polymerase chain reaction after treatment with DNA polymerase I to repair the putative single strand gaps in the structure (see Figure 6) .
- the amplified product was cut with Bam HI and cloned into pUC19. As expected, the cloned fragments consisted of linearized target DNA flanked by MoMLV DNA ends.
- MiniHIV consists of a linear DNA molecule bearing ends that resemble the predicted ends of the unintegrated HIV DNA.
- the synthetic oligonucleotide duplex pictured in Fig. 9(b) was synthesized with Eco RI cohesive ends and inserted into the Eco RI site of pMK468, a pBR322 derivative lacking the Nde I recognition site to make pMK564. Digestion of pMK564 with Nde I yields the linear miniHIV DNA.
- miniHIV The 3' ends of miniHIV are recessed, thereby bypassing the need for cleavage by the LTR-specific nuclease activity of IN protein.
- the insertion of miniHIV into lambda DNA is scored by packaging the reaction products into phage heads in vitro and infecting an £. coli strain containing a lambda prophage with the resulting phage particles.
- Lambda cannot grow on the lysogenic strain, but lambda DNA containing integrated miniHIV can persist as a plasmid because miniHIV contains the pB322 origin of replication. Since miniHIV also contains genes conferring resistance to ampicillin and tetracycline, integration events may be detected by plating infected cells on selective plates and scoring the number of colonies which arise.
- Line 1 presents the results of 21 independent reactions
- line 2 presents the results of 6 independent reactions.
- Integration reactions contained 10 ⁇ l of partially purified HIV IN or the same volume of the corresponding protein preparation made from uninfected cells, 1 ug miniHIV DNA, 1.5 ug lambda DNA, 0.1 mg/ml BSA, 0.1 mg/ml RNAse A, 0.1 mg/ml RNAse Tl, 20 ug/ml Xenopus histone HI, 2 ug/ml HU protein (Craigie et al., Proc. Natl. Acad. Sci.
- Example 7 In vitro oligonucleotide assay system for HIV DNA integration.
- the in vitro DNA integration reaction used in this example depends on partially purified integration protein (IN) of HIV, which has been cloned and over-expressed (see materials section entitled Expression of HIV IN protein) .
- the HIV IN protein produced by expression systems in insect cells or £. coli cell can be purified in several ways as described in the methods section and Example 5.
- HIV DNA integration assays were carried out essentially as described in Example 3 for DNA integration assays of the MoMLV retroviral system.
- DNA substrates for HIV IN protein were provided by duplex oligonucleotides matching the predicted termini of the unintegrated linear HIV DNA.
- lpmol of partially purified HIV IN is mixed with 0.5 pmol end-labeled duplex oligonucleotide in a reaction containing 25 mM KCl, 25 mM K Glutamate, 30 mM MES pH 6.2, 7.5 mM MnCl 2 , 10 mM BME, 20% glycerol, and 100 ug/ml BSA.
- the in vitro integration assay described in Examples 3, 4 and 7 can be further adapted for large scale drug screening by replacing the electrophoresis method of detecting the reaction products with a method that is partly automated.
- Figures 13 and 14 schematically depict the integration reaction, and separation and detection of labeled integration products when the assay is scaled up for drug screening.
- the integration of HIV-DNA oligonucleotide into the oligonucleotides using cloned HIV viral IN protein was chosen to exemplify the retroviral integration assay.
- HIV integration protein was cloned and expressed in insect cells and purified as described in the materials section of the Examples.
- Oligonucleotides were synthesized on an Applied Biosystems Model 380B DNA synthesizer and subsequently purified by gel electrophoresis. Biotin was incorporated at the 3' end of one DNA strand ( Figure 13) using a 3'-Amino Modifier (Glen Research) and a biotin-XX-ester 5"-labelling kit (Glen Research) . The DNA strand that becomes joined to a target DNA upon integration was labeled at its 5' end with 32 P as described in Material Section- entitled Oligonucleotide LTR Substrates.
- Avidin was covalently coupled to microtiter cells (CovaLink Modules, Nunc) with the cross-linker Bis (sulfosuccinimidyl) Suberate (Pierce) using a protocol recommended by Nunc Technical Service.
- the HIV integration reactions are described in Example 7. After incubation, the reaction products and unreacted substrate were bound to Avidin-coupled wells in 0.2 M NaCl, 20 mM Tris pH 8.0, 1 mM EDTA for between 2 and 18 hours. The supernatant was then removed and the wells were washed 3 times (5 minutes each wash) with 200 ⁇ l 0.2 M NaCl, 30 mM NaOH, 1 mM EDTA. Residual alkali was removed by a quick wash with 10 mM Tris pH 8.0, 1 mM EDTA. 32 P-labeled nucleotide was then liberated from the wells by treatment with DNAase I (Bethesda Research Laboratories) or formamide and the radioactivity was measured with a Liquid Scintillation Counter.
- DNAase I Bethesda Research Laboratories
- formamide the radioactivity was measured with a Liquid Scintillation Counter.
- the following semi- automated methodology can now be used for large- scale screening of potential inhibitors of HIV DNA integration without further technological development.
- the reactions are carried out as described in Example 7, except that mixtures are assembled in the wells of microtiter plates (standard microtiter plates without avidin) using commercially available automatic liquid handling equipment designed for use with microtiter plates. Control, reactions contain no inhibitor and test reactions contain the potential inhibitor that is to be assayed. After incubation at 30°C for 1 hour, the reactions are stopped by addition of EDTA and the contents of each well are transferred to another microtiter plate that has avidin covalently coupled to the wells.
- the wells are washed with 0.2 M NaCl, 30 mM NaOH, 1 mM EDTA, and with 10 mM Tris pH 8, ImM EDTA, as described in Example 8, except by means of an automatic microtiter plate washer.
- the remaining bound 32 P in each well which is a measure of the quantity of integration product made during the reaction, is released from the wells by addition of formamide.
- the 32 P present in each well can then read using a commercially available detector designed for measuring radioactivity in microtiter well plates. Alternatively, the contents of the wells are then automatically transferred to DEAE membrane (Schleicher & Schuell) . Inhibition of integration is assessed by decrease in detected label relative to control reactions that do not include any inhibitor.
- Example 9 In order to determine the inhibiting effect on HIV DNA integration into target DNA using the in vitro DNA integration assay system, recombinant IN protein is isolated as described in the materials section of the Examples. Briefly, recombinant insect cells expressing HIV IN protein were extracted and IN protein was purified by gel filtration. To confirm that the purified material is HIV IN, the preparation was subjected to SDS-gel electrophoresis and amino-acid sequencing. Radiolabeled duplex oligonucleotide substrate matching the predicted termini of the unintegrated linear HIV DNA are made as described in the materials section of the Examples. Also, in this example, S P is utilized as the detection label and gel electrophoresis and autoradiography as the methods of detecting reaction products.
- test drug is added to the reaction prior to initiating the reaction.
- the reaction contains labeled duplex oligonucleotide and recombinant IN protein.
- the decrease in integration products compared to control reaction mixtures that do not contain test drug are analyzed by electrophoresis as visualized in Figure 12. Because this system safely detects inhibitors of HIV integration in host target DNA in vitro. it is of particular interest that the assay may also be
- the reaction products can be separated on solid supports to which avidin is coupled and the efficiency of integration can be monitored by autoradiography or scintillation counting.
- 32 P label may be substituted with enzyme-linked label or fluorescent label, thus providing the option of detecting the bound label directly on solid supports using enzyme linked or fluorescent detection assays.
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Abstract
L'invention concerne des protéines d'intégrase (IN) actives recombinantes du virus de la leucémie murine de Molony (VLMMo) et du virus d'immunodéficience humaine (VIH) ayant les activités combinées in vitro de (1) clivage nucléolitique spécifique LTR et (2) intégration des extrémités LTR clivées dans une cible ADN. Des procédés utilisant ds protéines IN actives recombinantes sont également décrits. Tant dans le système d'analyse d'intégration rétrovirale in vitro que dans le système d'inhibition de médicaments rétrovirale in vitro, la protéine IN active recombinante est la seule protéine nécessaire pour détecter si une intégration virale dans l'ADN cible a été obtenue ou, alternativement, si l'inhibition de l'intégration par un médicament a été obtenue.
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WO1992020813A1 (fr) * | 1991-05-17 | 1992-11-26 | Dana Farber Cancer Institute | Techniques de determination de facteurs affectant la circularisation de l'adn, techniques de determination de facteurs affectant l'integration de l'adn, facteurs, et utilisations de ceux-ci |
WO1995023875A1 (fr) * | 1994-03-02 | 1995-09-08 | The Johns Hopkins University | Transposition in vitro de transposons artificiels |
WO1996004386A1 (fr) * | 1994-08-05 | 1996-02-15 | Warner-Lambert Company | Procede d'utilisation d'une integrase retrovirale negative transdominante dans le traitement d'infection retrovirale |
US6316261B1 (en) * | 1994-05-20 | 2001-11-13 | Saint Louis University | Method for concerted integration of donor DNA molecules using retroviral integrase proteins |
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JPH02265481A (ja) * | 1988-12-07 | 1990-10-30 | Handai Biseibutsubiyou Kenkyukai | レトロウイルスのプロテアーゼ、逆転写酵素及びエンドヌクレアーゼ、並びにこれ等の酵素の製法 |
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Title |
---|
HIGHLIGHTS OF MODERN BIOCHEMISTRY, Vol. 1, issued 1989, KOTYK (ED), "The Expression of Human Immunodeficiency Virus (HIV) and moloney murine leukemia virus reserve Transcriptase and Integration Proteins in E. Coli", pages 603-14. See abstract No. 1150 42767. * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 88, No. 4, issued 1991, BUSHMAN et al., "Activation of Human Immunodeficiency Virus (HIV) Integration Protein in vitro specific cleavage and Integration of HIV DNA", pages 1339-1393, see abstract No. 91095288. * |
SCIENCE (United States), Vol. 249, No. 4976, issued 28 September 1990, BUSHMAN et al., "Retroviral DNA Integration Directed by HIV Integration Protein in vitro", pages 1555-8, see abstract No. 91019413. * |
VIROLOGY, Vol. 179, No. 2, issued 1990, PAUZE, "Two bases are deleted from the Termini of HIV-1 linear DNA during Integrative recombination", pages 886-889, see abstract No. 91034621. * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1992020813A1 (fr) * | 1991-05-17 | 1992-11-26 | Dana Farber Cancer Institute | Techniques de determination de facteurs affectant la circularisation de l'adn, techniques de determination de facteurs affectant l'integration de l'adn, facteurs, et utilisations de ceux-ci |
US5759768A (en) * | 1991-05-17 | 1998-06-02 | Dana Farber Cancer Institute | Assays for factors affecting circularization of DNA, assays for factors affecting DNA integration, factors, and uses thereof |
WO1995023875A1 (fr) * | 1994-03-02 | 1995-09-08 | The Johns Hopkins University | Transposition in vitro de transposons artificiels |
US5728551A (en) * | 1994-03-02 | 1998-03-17 | The Johns Hopkins University | In vitro transposition of artificial transposons for DNA sequencing |
US5843772A (en) * | 1994-03-02 | 1998-12-01 | John Hopkins University | Artificial transposons |
US6316261B1 (en) * | 1994-05-20 | 2001-11-13 | Saint Louis University | Method for concerted integration of donor DNA molecules using retroviral integrase proteins |
WO1996004386A1 (fr) * | 1994-08-05 | 1996-02-15 | Warner-Lambert Company | Procede d'utilisation d'une integrase retrovirale negative transdominante dans le traitement d'infection retrovirale |
US5908923A (en) * | 1994-08-05 | 1999-06-01 | Warner-Lambert Company | Method of using transdominant negative retroviral integrase in the treatment of retroviral infection |
US6303334B1 (en) | 1994-08-05 | 2001-10-16 | Warner Lambert Company | Nucleic acid encoding a transdominant negative retroviral integrase |
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