WO1999036561A1

WO1999036561A1 - Use of a proline-rich sequence to improve the fusogenic character of retroviral envelopes

Info

Publication number: WO1999036561A1
Application number: PCT/FR1999/000016
Authority: WO
Inventors: François-Loîc COSSET; Dimitri Lavillette
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1998-01-15
Filing date: 1999-01-07
Publication date: 1999-07-22
Also published as: CA2317026A1; EP1047791A1; FR2773561A1; JP2002508975A

Abstract

The invention relates to the use of a proline-rich sequence to improve the fusogenic character of a protein comprising a retroviral envelope glycoprotein of an amphotropic MLV or a retroviral envelope glycoprotein of an MLV 10A1 or a retroviral envelope glycoprotein of a GALV or a retroviral envelope glycoprotein of a xenotropic MLV or a retroviral envelope glycoprotein of a MLV MCF or a retroviral envelope glycoprotein of a FeLV. Said proline-rich sequence corresponds either to the proline-rich sequence of the retroviral envelope glycoprotein of an amphotropic MLV, the retroviral envelope glycoprotein of an MLV MCF, the retroviral envelope glycoprotein of a GALV, the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, and contains at least one mutation which suppresses at least one β-turn in the polyproline helix of the proline-rich sequence, or to the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV.

Description

USE OF A PROLINE RICH SEQUENCE TO INCREASE THE FUSOGENICITY OF RETROVIRUS ENVELOPES

The subject of the invention is the use of a proline-rich sequence to increase the fusogenic character of retrovirus envelopes.

The entry of a retrovirus into a target cell depends on the recognition of a specific surface protein, called a receptor, by the surface subunit (SU) of the viral envelope, followed by the fusion of membranes which is mediated. by the fusion peptide located at the amino terminal end of the transmembrane sub-unit (TM) (29). Both a pH-dependent pathway and an independent pathway have been described for retroviruses (13), but the determinants and process involved in activation of fusion following recognition of the receiver remain unknown. The identification of these stages is essential for understanding the mechanism which modulates infection as well as gene transfer by retroviruses.

The retroviral envelope glycoproteins which assemble in trimer, are a complex comprising a surface subunit (SU) and a transmembrane component (TM) (10). For all enveloped viruses, the initial interactions of this glycoprotein with the cellular receptor (s) lead to conformational rearrangements of the envelope necessary for the exposure of the fusion peptide (30). The determinants of the envelope and the sequence of events causing these conformational changes are well detailed for orthomyxoviruses that require the acidic environment of endocytosis vesicles for their entry (24).

For retroviruses, for which a path independent of pH has also been described, the precise determinants and steps, leading from recognition of the receptor to activation of the fusion, remain unclear. The amphotropic envelope receptor Pit-2 for murine leukemia viruses (14, 28) is present in most species including humans, while the functional receptors for ecotropic envelopes are limited to rat and mouse cells ( 19). The recognition of one or the other of these receptors influences the fusion efficiency, but the ecotropic envelope induces cell-cell fusion and syncytia formation more easily, as tested with XC rat cells, than amphotropic envelopes (17).

The structural domains shared by the Moloney ecotropic envelope (MoMLV) and the amphotropic envelope (MLV-4070A) include: (i) an amino-terminal receptor binding domain (denoted BD) of approximately 200 amino acids (aa ) (3, 7); (ii) the proline-rich region from 45 to 59 a.a long, identified by PRO in the chimeric constructions described below (27); (iii) a carboxy-terminal sequence of SU (denoted C) of 160 a.a. involved in the interaction with TM; (iv) an ectodomain of the 134 a.a. TM designated herein by TM, carrying a potential amino-terminal fusion peptide identified by sequence analogy to the fusion peptides of other envelope proteins of enveloped viruses (11); (v) a membrane anchoring peptide of 28 a.a. and (vi) a cytoplasmic tail containing the small carboxy-terminal peptide R whose late cleavage in virions, increases the fusogenicity of the envelope (20). While the amino-terminal BD and PRR domains of the ecotropic and amphotropic envelope share only 33% and 43% a.a. of homology, respectively, all the other domains have more than 80% similarity (Fig. 1A).

One of the objects of the invention is to provide proline-rich sequences which increase the fusogenic character of retrovirus envelopes. One of the objects of the invention is to provide chimeric or mutated proteins whose fusogenic character is improved.

The subject of the invention is the use of a proline-rich sequence for improving the fusogenic character of a protein comprising a retroviral envelope glycoprotein of an amphotropic MLV or a retroviral envelope glycoprotein of an MLV 10A1 or a retroviral envelope glycoprotein of a GALV or a retroviral envelope glycoprotein of a xenotropic MLV or a retroviral envelope glycoprotein of an MLV MCF or a retroviral envelope glycoprotein of a FeLV, which sequence is rich in proline corresponds to: - either the proline-rich sequence of the retroviral envelope glycoprotein of an amphotropic MLV, or . of the retroviral envelope glycoprotein of an MLV MCF,. of the retroviral envelope glycoprotein of an MLV 1OA1, of the retroviral envelope glycoprotein of a GALV, or of the retroviral envelope glycoprotein of a xenotropic MLV, or

. retroviral envelope glycoprotein of an FeLV, and comprises at least one mutation such that there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence,

- or to the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV.

It has been unexpectedly found that there is an increase in the fusogenic character of a retroviral envelope glycoprotein of an amphotropic MLV or of an MLV MCF or of an MLV 1OA1 or of a GALV or of a Xenotropic MLV or of an FeLV in the following cases: - either by replacing their proline-rich sequence with the proline-rich sequence homologous to an ecotropic MLV,

- Or by point mutation of the proline rich sequence of said amphotropic MLV or of said MLV MCF or of said MLV 1OA1 or of said GALV or of said xenotropic MLV or of said FeLV such that there is suppression of at least one β-turn.

The proline-rich region is probably organized into a poly-proline helix, a secondary structure made up of "beta-turns" which are folds of the peptide chain by 180 °, most often including a proline. These curvatures are arranged differently between the PRO regions (proline-rich region) of the ecotropic and amphotropic envelopes. The latter contains 11 regularly arranged "beta-turns", including 4 in its N-terminal end and 7 in its C-terminal end. The PRO region of the ecotropic envelope contains only 7 beta-turns, of which only 2 are at its N-terminal end.

The expression “mutation such that there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence” means:

- deletion of the 4 amino acids forming the β-turn, or - point mutations of one or more of these 4 amino acids, in particular mutation of the proline responsible for the 180 ° folding of the polypeptide chain, such that the probability of having a β-turn is less than 0.8 according to the algorithm by Chou and Fassman. For example, proline is advantageously replaced by isoleucine, valine or alanine.

As suitable strains of ecotropic MLN, mention may be made of the Moloney MLV strain, Friend MLV (23).

As suitable strains of amphotropic MLV, mention may be made of strain 4070A (16).

As suitable strains of MLV MCF, mention may be made of the strain MLF MCF 247 (9).

As suitable strains of GALV, mention may be made of the Seato strain (Delassus et al, 1989, Virology 173: 205-213). As suitable strains of xenotropic MLV, mention may be made of the strain

ΝZB.IU.6 (15).

As suitable strains of FeLV, there may be mentioned FeLV-C-SARMA FSC (21).

As suitable strains of MLV 10A1, there may be mentioned (Ott et al., 1990 (16)).

The expression "improving the fusogenic character" means that the "improved" retroviral envelope of the invention has a measurable capacity, relative to the parental envelope, to better accomplish the post-binding stages of the viral entry process and consisting ultimately of the molecular fusion of viral and cellular membranes.

The improvement in fusogenic character can be measured according to one or other of the following two tests:

- test of syncytia formation after co-culture between target cells, expressing the retroviral receptor recognized by the binding domain of the mutated or chimeric glycoprotein, and cell lines previously transfected with an expression vector for one of the above protein, - test for infection of target cells, expressing the retroviral receptor recognized by the binding domain of the mutated or chimeric glycoprotein, under limiting conditions where the density of these receptors (that is to say the number of receptors available to the cell surface allowing the attachment of the viral particle) decreases by at least 100 times the infectious titre of the virus comprising the non-mutated parental envelope and by less than 100 times the infectious titre of the virus comprising the chimeric or mutated parental envelope; these values being given in comparison with the infectious titer of the viruses comprising the parental and parental chimera or mutated envelopes on XC cells under conditions defined in Table IB of the example.

The proline-rich sequence of a retroviral envelope glycoprotein of an ecotropic MLV is that shown in FIG. 7.

The proline-rich sequence of a glycoprotein of the retroviral envelope of an amphotropic MLV is that represented in FIG. 8. The proline-rich sequence of a glycoprotein of the retroviral envelope of an MLV MCF is that represented in FIG. Figure 9.

The proline-rich sequence of a glycoprotein of the retroviral envelope of a GALV is that shown in FIG. 10.

The proline-rich sequence of a glycovote from the retroviral envelope of an MLV 10A1 is that shown in FIG. 11.

The proline-rich sequence of a glycoprotein of the retroviral envelope of a xenotropic MLV is that shown in FIG. 12.

The proline-rich sequence of a glycoprotein of the retroviral envelope of an FeLV is that represented in FIG. 13. By proline-rich sequence of a retroviral envelope glycoprotein of an ecotropic MLV, is also meant that comprising mutations, deletion or addition of amino acids such that there is no modification of the sequence of β-burn

According to an advantageous embodiment, the invention relates to the use as defined above of a proline-rich sequence in association with a functional domain of the retroviral envelope glycoprotein of an MLV ecotrope chosen from: the binding domain, the C domain, or the domain of the transmembrane subunit.

The term “functional domain” means any polypeptide sequence that is structurally individualized and capable of performing a determined biological function.

By "binding domain" is defined the functional domain carried by the N-terminal part of the SU subunit of the MLV retroviral envelope and capable of binding to the retroviral receptor. It is shown in Figure 6 as extending from position 31 to 237 of the peptide sequence of the amphotropic envelope glycoprotein MLV.

By “domain C”, we define the functional domain carried by the C-terminal part of the SU subunit of the MLV retroviral envelope and capable of interacting with the TM subunit. It is shown in Figure 6 as extending from position 300 to 458 of the peptide sequence of the amphotropic envelope glycoprotein MLV.

By “TM domain”, we define the domain carried by the ectodomain of the TM subunit of the MLV retroviral envelope and capable of interacting with the SU subunit and of achieving fusion between viral and cellular membranes during of the retroviral entry process. It is shown in Figure 6 as extending from position 459 to 592 of the peptide sequence of the amphotropic envelope glycoprotein MLV.

The binding domain, the C domain, or the domain of the transmembrane subunit can be modified so as to alter their functional capacities with a view to: - interacting with other molecules, such as surface molecules

(antigens, growth factor receptors, etc.), through the insertion of peptides or protein domains with an affinity for these molecules,

- to strengthen the stability of the retroviral envelope glycoprotein, - to increase the fusiogenic capacity of the retroviral envelope glycoprotein. The invention relates in particular to the use as defined above, of a proline-rich sequence corresponding to the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV, in association with a functional domain of the retroviral envelope glycoprotein of an ecotropic MLV chosen from: the binding domain, the C domain, or the domain of the transmembrane subunit.

According to an advantageous embodiment, the invention relates to the use of a proline-rich sequence for improving the fusogenic character of a protein comprising a glycovote of retroviral envelope of an MLV, which proline-rich sequence corresponds to:

- either with the proline-rich sequence

. of the retroviral envelope glycoprotein of an amphotropic MLV, and comprises at least one mutation such that there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence,

The invention also relates, as a new product, to a proline-rich sequence in which the amino acid sequence and the secondary structure correspond to that of the proline-rich sequence of the envelope glycoprotein of an amphotropic MLV or of the MLV 10 A1 retroviral envelope glycoprotein or MLV MCF retroviral envelope glycoprotein or GALV retroviral envelope glycoprotein or xenotropic MLV retroviral envelope glycoprotein or of the retroviral envelope glycoprotein of an FeLV, and comprising at least one mutation on the side of the C-terminal part such that there is suppression of at least 1 β-turn in the polyproline helix of the rich sequence in proline.

The “C-terminal part” defines the amino acid sequence corresponding to the second half of the proline-rich region. The invention also relates to a proline-rich sequence in which the amino acid sequence and the secondary structure correspond to that of the proline-rich sequence of the envelope glycoprotein of an MLV. amphotropic or retroviral envelope glycoprotein of an MLV 10A1 or retroviral envelope glycoprotein of an MLV MCF or retroviral envelope glycoprotein of a GALV or retroviral envelope glycoprotein of a Xenotropic MLV or the retroviral envelope glycoprotein of an FeLV, and comprising at least one mutation on the side of the part

N-terminal such that there is suppression of at least 1 β-turn in the polyproline helix of the proline-rich sequence.

The “N-terminal part” defines the amino acid sequence corresponding to the first half of the proline-rich region. The invention also relates to the mutated proteins containing a proline-rich sequence and in the chain of which the proline-rich sequence is mutated so that there is suppression of at least one β-turn.

More specifically, the invention relates to a mutated protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or to the retroviral envelope glycoprotein of an MLV 10A1 or to the retroviral envelope glycoprotein of an MLV MCF or to the retroviral envelope glycoprotein of a GALV or to the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which the proline-rich domain contains at least one mutation such that 'there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence.

This mutated protein has, with respect to the corresponding non-mutated protein, the property of more easily inducing the formation of syncytia or better infecting the cells expressing a limiting amount of retroviral receptors, relative to the parental envelope from which it is derived.

The invention also relates to the chimeric proteins containing a proline-rich sequence in the chain of which the proline-rich sequence is replaced by the proline-rich sequence homologous to the retroviral glycoprotein of an ecotropic MLV. More specifically, the invention relates to a chimeric protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or to the retroviral envelope glycoprotein of an MLV 10A1 or to the glycoprotein of the retroviral envelope of a MLV MCF or to the retroviral envelope glycoprotein of a GALV or to the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which the domain rich in proline is replaced by the proline rich domain of the retroviral envelope glycoprotein of an ecotropic MLV.

This chimeric protein has, relative to the corresponding protein of origin, the property of more easily inducing the formation of syncytia or better infecting the cells expressing a limiting quantity of retroviral receptors, relative to the parental envelope from which it is derived. In the respective groups of mutated or chimeric proteins as defined above, there can also be replacement of at least one of their functional domains with the homologous functional domain of the retroviral envelope glycoprotein of an ecotropic MLV.

According to an advantageous embodiment, the invention relates to a chimeric protein corresponding to the retroviral envelope glycoprotein of a

Amphotropic MLV or the retroviral envelope glycoprotein of an MLV 10A1 or the retroviral envelope glycoprotein of an MLV MCF or the retroviral envelope glycoprotein of a GALV or the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of an FeLV, in which:

* the proline-rich domain is replaced by the proline-rich domain of the retroviral envelope glycoprotein of an ecotropic MLV,

* and at least one of the functional domains chosen from: the binding domain, the C domain, or the domain of the transmembrane subunit, is replaced by the corresponding domain of the envelope of an ecotropic MLV.

This chimeric protein has the property of inducing more easily the formation of syncytia or of better infecting the cells expressing a limiting quantity of retroviral receptors, relative to the parental envelope from which it is derived.

According to another advantageous embodiment, the invention relates to a chimeric protein corresponding to the retroviral envelope glycoprotein of a Amphotropic MLV or the retroviral envelope glycoprotein of an MLV 10A1 or the retroviral envelope glycoprotein of an MLV MCF or the retroviral envelope glycoprotein of a GALV or the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of an FeLV, in which:

* the proline-rich domain comprises at least one mutation such that there is suppression of at least one β-turn (in the polyproline helix of the proline-rich sequence),

The mutated or chimeric proteins of the invention advantageously have at least one of the following properties:

- there is formation of syncytia after cocultures between target cells expressing a surface molecule serving as a retroviral receptor by interaction with the binding domain carried by a retroviral envelope glycoprotein and cell lines producing or not producing Gag-Pol particles of MLV (or type C mammal retroviruses or lentiviruses) previously transfected with an expression vector for one of the above proteins of the invention,

- there is infection of target cells expressing a retroviral receptor recognized by the binding domain of a mutated or chimeric glycoprotein of the invention, under limiting conditions where the density of this receptor (that is to say the number of receptors available on the cell surface allowing attachment of the viral particle) decreases by at least 100 times the infectious titer of viruses comprising a non-mutated and non-chimeric envelope and by less than 100 times the infectious titer of viruses comprising a chimeric envelope or mutated of the invention, these above values applied to the infectious titre being given by comparison to the infectious titre of viruses comprising an unmutated and non-chimeric envelope or a chimeric or mutated envelope on target cells, such as XC cells, in the infection conditions as defined in the examples and in particular with regard to Table 1B below.

There are therefore in particular two tests making it possible to characterize the mutated or chimeric proteins of the invention.

One of these tests is the formation of syncytia between target cells and cell lines previously transfected with an expression vector for one of the above chimeric or mutated proteins of the invention (and capable of recognizing a retroviral receptor located on said target cells).

As target cells, cells from SCI mice, Mus Duni, human TE671 cells, as well as rat XC cells can be used. The cell lines used may or may not produce particles

Gag-Pol from MLV.

The cell lines may or may not produce Gal-Pol particles of MLV or of other mammalian type C retroviruses, in particular the Moloney MLV virus or of other retroviruses such as lentiviruses, for example HIV-1. Another test for characterizing mutated or chimeric proteins of the invention consists in infecting target cells expressing a retroviral receptor capable of being recognized by a chimeric or mutated protein of the invention, via viral particles containing these proteins chimeras or mutated. As target cells, the XC and XC-A-ST rat cells can be used, the latter being derived from XC cells by transfection of a plasmid expressing the binding domain of the amphotropic retroviral envelope capable of occupying the retroviral receptor amphotropic.

As viral particles, there may be used those produced by MLV retroviruses or other mammalian type C retroviruses, in particular Moloney MLV virus or other retroviruses such as lentiviruses, for example HIV-1. This infection is carried out under limiting conditions, that is to say such that the density of the receptor (that is to say the number of receptors available on the cell surface allowing the attachment of the viral particle) decreases by at least 100 times the infectious titer of virus comprising an envelope mutated or chimeric glycoprotein and by less than 100 times the infectious titer of virus comprising a mutated or chimeric envelope of the invention corresponding to the said envelope neither mutated, nor chimera.

The comparison is made with respect to the infectious titre of viruses comprising a non-chimeric and non-mutated envelope or a chimeric or mutated envelope on target cells such as XC cells.

This infection is comparatively carried out on two cell types, the second being directly derived from the first and has the effect of reducing the number of retroviral receptors available, that is to say such that the decrease in the surface expression of the receptor decreases by at least 100 times the infectious titre of the virus containing the parental envelope, neither mutated nor chimeric.

This value is then compared to that obtained for viruses comprising the chimeric or mutated envelope. A decrease of less than 100 in the infectious titers of viruses comprising the above-mentioned chimera envelope or mutated between the two cell types therefore indicates an improvement in the fusogenic character of the latter.

Advantageous proline-rich sequences according to the invention correspond to one of the following sequences: A ₂

A ₃ A ₃ MB

A ₄ C ₂ C ₃ C ₄ C ₅

Relative to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the A ₂ sequence involves the suppression of the second β-turn which has the effect of being more fusogenic than the parental envelope in syncytia test.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the A ₃ sequence involves the suppression of the third β-turn which has the effect of being more fusogenic than the parental envelope in the syncytia test.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the A ₃ Mo sequence involves the suppression of the third β-turn which has the effect of being more fusogenic than the parental envelope in infection test. as defined above.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the sequence A ₄ involves the suppression of the fourth β-turn which has the effect of being more fusogenic than the parental envelope in infection test such as defined above. Compared to the proline-rich region of the retroviral glycoprotein of a

Amphotropic MLV, the C ₂ sequence includes the deletion of β-turns 9 to 13 which has the effect of being more fusogenic than the parental envelope in infection test as defined above.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the C ₃ sequence involves the deletion of β-turns 11 to 13 which has the effect of being more fusogenic than the parental envelope in test for infection as defined above.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the C ₄ sequence includes the deletion of the last β-turn (13 °) which has the effect of being more fusogenic than the parental envelope under test. infection as defined above.

Compared to the proline-rich region of the retroviral glycoprotein of an amphotropic MLV, the C ₅ sequence includes the deletion of the 11 ° and 12 ° β-turns which has the effect of being more fusogenic than the parental envelope under test. infection as defined above. Advantageous chimeric proteins according to the invention correspond to one of the following sequences:

- PROMO

- PRO FR - BD PRO MO

- PRO C MO

- PRO TM MO

- PRO CTM MO

The PROMO sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV (Moloney strain).

The PRO FR sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV (Friend strain).

The BD PRO MO sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV and in which the binding domain has been replaced by the homologous binding domain of the ecotropic MLV . The PRO CMO sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV and in which the C domain has been replaced by the C domain homologous to the ecotropic MLV.

The PRO TM MO sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV and in which the TM domain has been replaced by the TM domain homologous to the ecotropic MLV.

The PRO CTM MO sequence corresponds to the amphotropic MLV sequence in which the proline-rich region is replaced by the proline-rich region homologous to the ecotropic MLV and in which the respective C and TM domains have been replaced by the C and TM domains respectively. counterparts of the ecotropic MLV. Advantageous chimeric proteins according to the invention comprise one of the proline-rich sequences corresponding to one of the following sequences: A ₂ A ₃ A ₃ Mo

A ₄ C ₂ C ₃ c ₄ C ₅

and are such that at least one of their following functional domains: binding domain, domain C, transmembrane domain, is replaced by the homologous domain of ecotropic MLV. It is shown in the context of the present invention that the proline-rich region (PRR) of the envelope of murine leukemia viruses (MLV), located between the receptor binding domain of the amino terminal part of SU and the domain of interaction with the TM of the carboxy-terminal part of the SU, serves as an intermediary in the changes of conformation of the envelope and in the activation of the fusion. Furthermore, it is shown that the potential beta-tura of the proline-rich region determine the stability of the SU-TM association and the activation threshold for cell-cell fusion and virus-cell fusion.

In order to identify the region (s) responsible for the greatest fusogenicity of the ecotropic envelope of MLVs, a series of chimeric envelopes was generated in which the domains of amphotropic envelopes

BD, PRO, C and TM are exchanged with their counterpart of the ecotropic envelope (Fig 1B). The resulting envelopes are then identified according to the substituted ecotropic domain (s). Cell-to-cell fusion is evaluated by the formation of syncytia after a 24-hour coculture between different target cells (indicator cells) and cell lines, whether or not producing Gag-Pol particles of MLV (5), previously transfected with one either of the envelopes. Although the level of expression on the cell surface of the parental envelopes and chimeras are similar (Fig. 4A), two different cell-cell fusion phenotypes are observed. The CMO and TMMO envelopes, like the wild MLV -4070 A envelope, weakly induce syncytia. However, the BDMO, PROMO and PROFR envelopes strongly induce the formation of syncytia, similar to those induced by the ecotropic envelope. (Fig

2). Syncytia formation is unaffected by the presence or lack of Gag-Pol particles (data not shown), showing that the fusion measured in this test occurs by direct cell-cell contact rather than cell contact mediated by viral particles. Previous reports indicate that the high fusogenicity of the BDMO envelope is mainly due to its ability to interact with the ecotropic receptor (18). However, significant fusogenicity is also observed with the chimeras carrying the amphotropic BD domain (FIGS. 1 and 2) with all the cell types used, including the target cells of rat, mouse and man, with or without the ecotropic receiver (data not shown). Thus, the differences observed, during the formation of syncytia, between the parental and chimeric amphotropic envelopes are due to new properties, different from the interaction of BD with the receptor.

As described for feline leukemia viruses (8) and as suggested by the close cognate with MLV, regular repetition of proline in PRR may cause it to fold into a poly-proline "beta-turn" helix (27 ), a secondary structure which exerts "elastomeric" forces, which can fulfill the function of

"molecular spring" (26). Because of the critical localization of PRR, between the BD and C domains which both influence fusion, its role in fusion has been directly evaluated. Differences in the number and arrangement of "beta-turn" predicted in the PRR of 4070A envelopes, compared to that of MoMLV, suggest a more reactive spring for the latter which could explain the increased fusogenicity of PROMO. The point or deletion mutants, C2, C3, C4 and C5, designed to suppress some of the seven carboxy-terminal "beta-turn" of amphotropic PRR, were tested in the cell-cell XC fusion test. They were found to be weakly fusogenic like the amphotropic parental envelope (Fig. 3B). The

Proline proline-induced "beta-turn" is less entangled in the part amino-terminal than in the carboxy-terminal end. So each of the four

The potential "beta-turn" of the 4070A envelope could be removed individually by substitution of a proline with a valine, an alanine or an isoleucine, giving the mutants Al, A2, A3, A3MO and A4 (Fig. 3C). A second amino acid was substituted in the mutants A2 and A3 in order to avoid the formation of a potential alpha helix or beta sheet, originally absent in the PRR structure predictions of MLV

(not shown). Envelopes A1 and A4, for which the first or fourth amino terminal "beta-turn" respectively have been mutated, retain a regular repeat of three contiguous "beta-turn" and do not induce increased syncytia formation (Table 1). In contrast, the A2 and A3 envelopes in which the continuity of these "beta-turn" is interrupted (Figs. 3C and 3D), are very fusogenic (Table 1), thus demonstrating the critical importance of the adjoining "beta-turn" of PRR in the "mediation" of the fusion following the recognition of the receptor. In order to test whether this increased fusogenicity was due to a lower need for the Pit-2 molecule, cell-cell fusion was compared using XC or XC-A-ST cells. In the latter, the constitutive expression of amphotropic BD reduces the quantity of functional Pit-2 receptor (Table 1).

Interference is effective towards the MLV-4070A envelope while high fusogenicity is always observed with the PROMO, PROFR, A2 and A3 envelopes. However, the fusion is still dependent on Pit-2 since CHO cells not expressing Pit-2 merge only after de novo expression of Pit-2 (not shown). Thus, mutations in the PRR "beta-turn" seem to lower the activation threshold of the fusion taking part after the recognition of the receptor.

After cell-cell fusion, cell-virus fusion was examined by comparing the infectivity of virions carrying the parental or chimeric envelope. The titers are equivalent on the XC rat (Table 1), mouse, human and CHO cells transfected with the Pit-2 receptor expression gene. Surprisingly, the highly fusogenic PROMO and PROFR chimeras, as well as the point mutants A2 and A3 led to undetectable or significantly reduced titers on all the cells tested. The other envelopes confer titers in a range of 10e6-10e7 infectious units per ml (ui / ml) on the XC cells, similar to those obtained with the two parental envelopes 4070 A and MO. Although the infectivity of virions generated with the parental amphotropic envelope is dramatically reduced on XC-A-ST cells, all infectious PRR mutants, except Al, are significantly less sensitive to interference (Table 1 ). The fact that the mutants of the carboxy-terminal part of PRR do not exhibit a modification in cell-cell fusogenicity but nevertheless show an increase in infectivity (virus-cell fusion), demonstrates that the amino motifs, but also carboxy terminals, influence fusogenicity. The receptor binding tests, carried out using an antibody directed against SU, show that the Pit-2 / SU affinity is not significantly altered for these envelopes (not shown). Infection of transfected CHOs expressing Pit-2, as opposed to the failure of parental CHO infection, confirms the need for Pit-2 for virion entry.

Since the low infectivity of virions carrying the fusogenic mutant envelopes in cell-cell tests is due to an increased loss of SU (Fig. 4), PRR seems to play a critical role in the SU / TM association and the conformation of the envelope. The increased release of SU, for the PROMO, PROFR, A2 and A3 mutants, suggests that PRR exerts its function of regulating fusion by interacting with adjacent regions, and that the destruction of these interactions stimulates fusion. The combination of ecotropic PRR with downstream homologous regions, as in the chimeras PROCMO, PROTMMO and PROCTMMO, or with the upstream domain

BD, as for the BDPROMO envelope (Fig. 1C), increases the stability, as estimated with the loss of SU (not shown) (Fig. 1).

Finally, it is shown that after recognition of the receptor, PRR is a key determinant of changes in the conformation of the envelope and of membrane fusion. In addition, the characteristic arrangement of the PRR "beta-turns" in association with the downstream domains C and TM, confers distinct properties between these MLV envelopes.

The potential applications of the invention are as follows:

- ex vivo or in vivo gene therapy for gene transfer into cells expressing a limited number of retroviral receptors, by retroviral vectors derived from type C retroviruses, mammals or lentiviruses comprising: . mutated or chimeric envelope glycoproteins as described in the invention,

. mutated or chimeric envelope glycoproteins as described in the invention, as well as a peptide or a protein domain capable of redirecting the binding of the retroviral vector to a specific molecular target.

I. Legends of the figures

Figs. 1A, 1B, 1C and 1D: Schematic representation of the chimeric envelopes and their properties.

Fig. 1A. The empty boxes are sequences derived from the amphotropic MLV, the filled boxes are sequences from the Moloney ecotropic MLV (black) or the Ecotropic MLV from Friend (gray) and the hatched boxes are sequences common to these two MLVs. BD, receptor binding domain; PRO, proline-rich region;

C, SU terminal carboxy domain; TM, ectodomain and anchoring domain of the TM sub-unit. For each domain, their percentage of identity, as well as the first four N-terminal amino acids, are indicated. A premature stop codon (vertical arrow) is introduced into the cytoplasmic tail of the TM subunit of amphotropic MLV to generate the fusogenic ARless fusogenic envelope glycoprotein.

Fig. 1B. Simple substitution mutants. Fig. 1 C. The substitution mutants combined.

Fig. 1D. The results of cell-cell fusion tests: (-), absence of syncytia; (+), presence of syncytia only in a coculture with XC cells; (+ +), presence of syncytia both in a coculture with rat cells

XC but also with XC A-ST interfering cells. Infection test results: (-), less than 10e2 lacZ u.iJml; (+) and (+ +), more than 10e2 lacZ ui / ml; (+ +); envelopes with a reduced fusion activation threshold, capable of effectively infecting XC-A-ST interfering cells with a titer greater than 10e2 lacZ u.iJml. Fig. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21: Tests for fusion of single substitution mutants with XC cells.

TE671 cells transfected with the expression vectors of the chimeric envelope glycoproteins indicated (FIG. 1) are cocultivated with XC indicator cells for 24 hours. Magnification x250.

Figs. 3A, 3B, 3C and 3D: Mutagenesis of the region rich in amphotropic proline 4070A.

Fig. 3 A: Alignment of the PRR of the MLV of Moloney (Mo), of Friend (Fr) and 4070A (A).

Fig. 3B: Point mutants (amino acids in small bold type) or deletion (dashes) of the C-terminal end of the PRR of MLV 4070A.

Fig. 3C: Point mutants of the C-terminal end of the PRR of MLV 4070A. Amino acids changed or inserted are highlighted in small bold type.

Fig. 3D, 3E, 3F, 3G: Probability profiles of the beta-turn of the PRRs of MLV 4070 A (4070A), Moloney (Moloney) and point mutants of the PRR of MLV 4070A including the second (A2) or third ( A3) beta turn has been removed. The y-axis values represent the probability of beta-turn p (turn) * 10e-4 for each residue of the peptide (on the x-axis). The beta-mrn analysis was carried out using PC / Gene software (Version 6.26, IntelliGenetics, Belgium).

Fig. 4: Immunoblots of amphotropic envelope glycoproteins whose fusogenicity is increased. The cells were transfected with the envelopes indicated in FIGS. 1 and 3 and the expression of their SU and of their envelope precursor (PR) is estimated (cell lysates). The instability of the envelope is shown by the decreased surface expression of the SU relative to the PR (membrane preparations), its release of the SU (shedding) increased in the culture medium (supernatant) and in the virions. Protein transfers (western blots) were revealed with the antibodies indicated. Fig. 5: it represents the sequence of the retroviral envelope glycoprotein of ecotropic MLV (BD, PRO, C and TM domain) (Moloney strain).

Fig. 6: it represents the sequence of the amphotropic MLV retroviral envelope glycoprotein (BD, PRO, C and TM domain) (strain 4070 A).

Fig. 7: it represents the proline-rich region of the glycovote of the retroviral envelope of Mo MLV (Moloney MLV).

Fig. 8: it represents the proline-rich region of the glycovote of the retroviral envelope of MLV-4070A.

Fig. 9: it represents the proline-rich region of the glycoprotein of the retroviral envelope of MLV-MCF (strain 247).

Fig. 10: it represents the proline-rich region of the glycoprotein of the retroviral envelope of GALV, strain Seato.

Fig. 11: it represents the proline-rich region of the glycovote of the retroviral envelope of MLV 10A1.

Fig. 12: it represents the proline-rich region of the glycoprotein of the retroviral envelope of xenotropic MLV, strain NZB.1.V6.

Fig. 13: it represents the proline-rich region of the glycoprotein of the retroviral envelope of FeLV A.

Fig. 14: it represents the sequence of A ₂ .

Fig. 15: it represents the sequence of A ₃ .

Fig. 16: it represents the sequence of A ₃ Mo. Fig. 17: it represents the sequence of A ₄ .

Fig. 18: it represents the sequence of C ₂ .

Fig. 19: it represents the sequence of C ₃ .

Fig. 20: it represents the sequence of C ₄ .

Fig. 21: it represents the sequence of C ₅ .

Fig. 22: it represents the sequence of PROMO.

Fig. 23: it represents the sequence of PRO FR.

Fig. 24: it represents the BD PRO CMO sequence.

Fig. 25: it represents the sequence of PRO CMO.

Fig. 26: it represents the sequence of PRO CTM MO.

Fig. 27: it represents the sequence of PRO TM MO.

Table 1:

A. Results of cell-cell fusion tests.

envelope melt index ^was expressed on the surface XC XC-A-ST

_b 0.02 + 0.01 0.02 + 0

MO ^c 15 ± 2 14 ± 1

A ^d 0.2 + 0.1 0.05 + 0.02

ARLess ^e 74 ± 4 0.85 + 0.03

PROMO ^c 17 + 3 16 + 3

PROFR ^c 18 ± 3 23 ± 6

A2 ^C 13 ± 1 17 ± 1

A3 ^C 14 ± 1 17 + 3

a: merger index calculated according to the formula:

(NS) / T (N, number of nuclei in syncytia; S, number of syncytia; T total number of nuclei) b: untransfected cells. c: syncytia containing more than 20 nuclei d: syncytia containing less than 4 nuclei e: syncytia containing more than 40 nuclei with XC cells and less than 10 nuclei with XC A-ST cells.

B. Results of infection tests.

envelope Titles infectieuj, expressed following the interference XCXC-A-ST virions

MO 7.1xl0 ⁶ 6.1x10 ° 1

A lxlO ⁷ 6.4xl0 ³ 1.369

PROMO 2.2x10 '1x10' na

PROFR 'lxlO ¹ <1x10' na

Al 8.4xl0 ⁶ 2.3xl0 ³ 3.138

A2 <lxlθ '<1x10' na

A3 7.8x10 '3x10' na

A3MO 1.2xl0 ⁷ 3.3xl0 ⁴ 302

A4 5.7xl0 ⁶ 8.1xl0 ³ 605

C2 7.6xl0 ⁶ 5.7X10 ⁵ 11

C3 5 4xl0 ⁶ 4.7xl0 ⁵ 10

C4 2.4xl0 ⁶ 1.7X10 ⁵ 12

C5 9.4xl0 ⁶ 2.7x10 "299

a: lacZ infectious units per ml (u.iJml) b: interference values are calculated according to the formula (title _(XC] / title _[XC . _A. _s - _π ) X (title MO _(XC - _A ^{title M0)} ιχc]) - na: not applicable

II. MATERIAL AND METHODS

II.1. Cell lines.

TELacZ cells contain the retroviral vector MFGnlsLacZ (25) responsible for the expression of a nuclear beta-galactosidase. The cell line

TELCeBό (5) is derived from the TELacZ line and was obtained by transfection of a plasmid expressing the gag and pol proteins of MoMLV. These cells therefore produce non-infectious retroviral particles, since they lack envelopes, carrying the marker gene nlsLacZ. TELCEB6, XC, RD and CEAR13 cells (12) are cultured in medium

DMEM (Life-Technologies) supplemented with 10% fetal calf serum (FCS, Life-Technologies). The CEAR13 culture medium is additionally supplemented with proline (Sigma) at 10 μg / μl. The NIH3T3 line (ATCC CRL 1658) is cultivated in DMEM medium (Life-Technologies) supplemented with 10% newborn calf serum (Life-Technologies).

The XC-A-ST cells were obtained by stable transfection into the XC cells of the plasmid pA-ST (5) expressing the N-terminal fragment of the SU of the MLV-4070A virus capable of binding and blocking the amphotropic receptor.

II.2. Plasmids and construction of mutants.

Plasmids encoding amphotropic envelopes 4070A (FBASALF)

(5), Moloney ecotrope (FBMOSALF) and Friend C57 ecotrope (FB3) allow the expression of the envelope. The envelope genes are under the control of the LTR (5) of the Friend FB29 MLV. In addition, these plasmids contain the phleo marker gene, allowing selection by phleomycin.

To generate the various constructs of mutant envelopes, a PCR mutagenesis technique was used.

For the Rless construct (20), a DNA fragment was obtained by PCR

(amplification by polymerase chain reaction) on the MOMLV envelope (23) using the oligonucleotides OURLess 5 '-TTC TAT GCG GAC CAC ACA GGA and OLRLess 5'-

ATC TCA GTA GTC CAG GCT TTA TGA TAG TCG ACA TGC AT. After digestion with the enzymes Spel and AccI, this fragment was subcloned between the Spel and ClaI sites of the plasmid FBMOSALF. An NdeI / ClaI fragment was then removed from this latter plasmid, and was replaced by NdeI / ClaI originating from the vector FBASALF. The result is an expression vector for an amphotropic envelope containing a premature stop codon a few nucleotides upstream of the normal stop codon. This modification reduces the length of the cytoplasmic part of the amphotropic glycoprotein and is sufficient to make it very fusogenic and induces syncytia in cell culture (20).

For the construction of the PROMO and PROFR chimeric envelopes, a PCR fragment corresponding to the proline-rich region of the virus envelopes

Moloney-MLV (MO) and Friend-MLV (FR), with an Apal site and a Kpnl site created at the beginning and at the end of this region were generated thanks to the nucleotides: Up MO Apal 5 '-CC A ATA GGG CCC AAC CCC GTT CTG and Low MO Kpnl 5'-AAC GTG GTA CCC GCC GGT GGA AGT TGG G for PCR on the plasmid FBMOSALF; Up FRA pal GTC CCG ATA GGG CCC AAC CCC GTC CTG and Low FR Kpnl GAA

TGC GGT ACC TGC TGG CGG GGG CTG AGT GGG for PCR on plasmid FB3. The digested Apal / Kpnl fragments are cloned between the Apal (position 653) and BamHI (position 802) sites of the 4070A envelope using a Kpnl / BamHI adapter fragment generated by PCR on the plasmid FBASALF using oligonucleotides Up A Kpnl 5 '-T ATGCGGTACCGG AGATAGACTACT AGCTC and Low A BamHI

5 '-GGTAGTAGG ATCCTGAGCCGG. The other chimeric envelopes were generated using the envelopes described above by subcloning of different fragments. The plasmid FBASALF was opened in Xhol-Clal and this DNA was used to clone the fragments: (i) XhoI-Draϋl of the PROMO envelope and the Draïl-Clal fragment of the MO envelope, generating the PROCTMMO envelope; (ii) Ncol-ClaI of the MO envelope, co-ligated with an Xhol-Ncol adapter fragment of the A envelope, generating the TM MO envelope; (iii) Xhol-Ncol from the MO envelope thanks to an Ncol-ClaI fragment of the envelope A, generating the PROCMO chimera. The PROTMMO construct was obtained by subcloning the XhoI-BamHI fragment of the PROMO envelope into the vector carrying the TMMO envelope digested with the same enzymes.

Finally, the BDPROMO chimera was made by co-ligating the DralII-Clal fragment of the PROMO envelope in the plasmid FBMOSALF, digested with the BamHI-ClaI enzymes, with a BamHI-DralII fragment of the MO envelope.

Mutants of the amphotropic proline-rich region were generated by the PCR mutagenesis method. An oligonucleotide coding or "sense" ("upper") 805 Fc (5 '-TCC AAT TCC TTC CAA GGG GC), located just upstream of the Xho site

I of envelope 4070A (at position 594) (16) was used in combination with oligonucleotides providing various restriction sites (specified in parenthesis and whose site is underlined in the sequence of the oligonucleotide) and inserting the mutation desired (nucleotides that do not match are indicated by a lowercase letter) A2 UpA2 5'-CGA GTC CCC ATA GGG ATC CAA CCG GTA TTA CCC GAC C

(Agel), A3MO LowA3MO 5'- GCCCAACCCAGTGCTAGCCGACCAAAGAC (Nhel), A3 P249I / Q251V 5'-CCA GTA TTA atC GAC ptc AGA CTC CCT TC (Aat II); A4 P254I 5'-GAC CAA AGA CTg ata TCC TCA CCA A (EcoRV); C5 P286I / P289L 5 '-CTC AAC CTC Cat TAC tAG TCt AAG TGT CCC AC (Spel). Oligonucleotides complementary to these primers (LowA2 GGT CGG GTA ATA

CCG GTT GGA TCC CTA TGG GGA CTC G; Low A3 GAA GGG AGT CTg acG TCG atT AAT ACT GG; LowA3MO GTC TTT GGT CGG CTA GCA CTG GGT TGG GC; Low A4 TTG GTG AGG Ata tcA GTC TTT GGT C; Low C5 GGC TGT GGG ACA CTT aGA CTa GTA atG GAG GTT GAG GGT G) were used with the Low ABamHI primer hybridizing at the BamHI site (position 802) of the 4070 A envelope. The two fragments generated for each mutant is digested with the appropriate enzymes and they are ligated in the plasmid FBASALF, digested in XhoI-BamHI.

The same strategy was used for the C2, C3 and C4 deletion mutants. The oligonucleotide 805Fc, hybridizing upstream of the Xhol site, was used with the oligonucleotides LowA-C4 Kpnl: ATC TCC GGT ACC GAC ACT TGG ACT TGT AGG GGA GGT TGA GGG, LowA-C3 Kpnl: ATC TCC GGT ACC TGG GGG AGG GGA GGT TGA GGG TGT ACT GGT AGT GGA AGG, and LowA-C2 Kpnl: ATC TCC GGT ACC TGG GGG GGG TGT ACT GGT AGT GGA AGG GGG GTA ACT GGT generating, after digestion, Xhol-Kpnl fragments. Each fragment is co-ligated with a KpnI-BamHI fragment, originating from the PROMO envelope, in the vector FBASALF digested into XhoI-BamHI. The plasmids expressing the envelopes are transfected by the calcium phosphate precipitate method (22) in the TELCeBό line. The transfected cells were selected with phleomycin (50 μg / ml) and the resistant clones were mass trypsinized. The confluence cells were used on the one hand to recover viral supernatants after an overnight incubation in normal medium, and on the other hand to make cell lysates. The viral supernatants are used for infection tests, receptor binding tests, and the rest is subjected to ultracentrifigation to obtain virus pellets analyzed by immunoblot.

II.3. Immunoblot.

The virus-producing cells are lysed for 10 minutes at 4 ° C. in a Tris-HCl buffer (pH 7.5), containing 1% TritonXIOO, 0.05% SDS, deoxycholate 5 mg / ml, 150 mM NaCl and a cocktail. protease inhibitor (1 mM PMSF, 6 mM Leupeptin, 0.3 mM Aprotinin). These lysates are centrifuged for 10 minutes at

10,000 g in order to pellet all the organelles, and the supernatants are frozen at -80 ° C. until analysis. Viral samples are obtained by ultracentrifugation of viral supernatants (6 ml) in a Beckman SW41 rotor (30,000 rpm, 1 hour, 4 ° C). The pellets are resuspended in 80 μl of cold PBS (Phosphate Buffered Saline) (life phosphate buffer), Life-Technologies) and frozen at -80 ° C. The samples

(30 μg of cell lysates or 20 μl of purified virus) are mixed in a ratio of 5: 1 (v / v) to 375 mM Tris-HCl buffer (pH 6.8) containing 6% SDS (sodium dodecyl sulfate) , 30% beta-mercaptoethanol, 10% glycerol and 0.06% bromophenol blue, then denatured for 3 minutes at 95 ° C and analyzed on denaturing 10% acrylamide / SDS gel. After transfer of the proteins onto the nitrocellulose membrane, the immunological labeling is carried out in TBS (Tris Base Saline (Tris buffer), pH 7.4) in the presence of skimmed milk (5%) and Tween 0.1%. Antibodies (Quality Biotech Inc., USA) from goat antiserum directed against Rausher Leukemia Virus (RLV) gp70-SU or RLV p30-CA were used at dilutions of 1: 2000 and 1: 10000 respectively . An antibody from rabbit antiserum against pl5E-TM was used at a dilution of 1: 1000. The "blots" were revealed by the use of a conjugated antibody, coupled to peroxidase, of rabbit or goat origin directed against the immunoglobulins of goat or rabbit, respectively (Dako, UK), using a chemiluminescence kit (Amersham Life Science).

II.4. Receptor binding tests.

The target cells are rinsed with PBS and peeled off by a 10-minute incubation at 37 ° C. in 0.02% versene in PBS. The cells are rinsed in PBA (PBS containing 2% of FCS (fetal calf serum) and 0.1% of sodium azide which makes it possible to block the membrane endocytosis). 10 ⁶ cells are then incubated for 1 hour at 37 ° C. with the various viral supernatants (2 ml) containing the soluble SU. These supernatants are discharged from the precipitated viral particles by ultracentrifigation or not. After two rinses with PBA, the cells are incubated in the presence of monoclonal antibody, rat or mouse, directed against SU (83A25) (6) or against TM (MAb2), respectively, for 45 minutes at 4 ° vs. The cells are washed twice in PBA and then incubated in the presence of fluorescent conjugate antibodies directed against rat (Cayla, F) or mouse (Dako, UK) immunoglobulins, for 45 minutes at 4 ° C. The dead cells are then counter-stained with propidium iodide (20 μg / ml) for 5 minutes at 4 ° C. After two rinses in PBA, the fluorescence of the living cells is analyzed by fluorometry, FACSCalibur, Beckton Dickinson).

II.5. Cell labeling test.

The cells carrying the envelopes are washed, detached and rinsed in the same manner as the target cells of the binding test. 10 ° cells are then incubated directly with the anti-SU rat antibody (83A25) for 1 hour at 4 ° C.

The washing, incubation with secondary antibody coupled to FITC and FACS analysis steps are identical to the binding test.

II.6. Infection tests. NIH3T3 target cells are seeded the day before in plates,

24 wells at a density of 5.10 ⁴ cells per well. Cells are incubated with 1 ml different dilutions of the viral stock in the presence of polybrene at 8 μg / ml (a polycation, inhibitor of electrostatic repulsions between viral particles and cells). After 3 to 5 hours of incubation at 37 ° C, the supernatants are replaced with fresh medium and the cells are incubated for 2 days at 37 ° C. Once the cells have been fixed with a 0.5% solution of glutaraldhéhyde bridging agent in PBS, they are stained using a substrate, 5-bromo-4-chloro-3-indolyl-Beta- D- galactopyranoside or X-Gal, for 12 hours. The viral titers are reported in number of positive colonies per ml of viral supernatant (LacZ i.uJml).

II.7. Immunoprecipitation.

A box of confluent cells with a diameter of 35 mm is rinsed once with DMEM medium (Eagle's medium modified by Dubelcco) without metionine or cysteine, supplemented with 10% of fetal calf serum dialyzed in order to remove the amino acids, then incubated for 1 h 30 at 37 ° C in this same medium. After labeling for 45 minutes with methionine and cysteine S35 (100 mCi / ml; TranS35-Label,

ICN Pharmaceuticals), the cells are washed and then incubated at 37 ° C in culture medium (hunting). The cells are lysed at different times in a 0.5% NP40 solution, 150 mM NaCl, 20 mM HEPES supplemented with 1 mM PMSF for 20 minutes at 4 ° C. The lysate is centrifuged at 10,000 g and the supernatant frozen at -80 ° C. These supernatants are clarified using non-immune goat serum (1/100) and

Protein A-Sepharose (6MB, Pharmacia) at 4 ° C for 2 hours or overnight. After centrifugation, the supernatants are incubated with a goat antiserum directed against gp70-SU of Rausher Leukemia Virus (RLV) at 1/100 th for 1 hour at 4 ° C. Protein A-Sepharose 40% in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% Deoxycholate, 1% TritonlOOX, 0.1% SDS) is added for 1 hour at 4 ° C and the complexes are precipitated by centrifugation at 10,000 g for 5 minutes. After three washes in RIPA buffer, the immunoprecipitates are separated on 12% acrylamide / SDS gel. The proteins are fixed in a 25% solution of isopropanol and 10% acetic acid in water and the revelation is done using a P32 Phospholmager screen (Biorad). II.8. Fusion tests.

Two days after their transfection, the effector cells carrying the envelope are seeded at 20% confluence. Three to four hours later, three times more target cells are added. The coculture is incubated for 24 hours at 37 ° C. and then fixed with a 0.5% solution of glutaraldhéhyde in PBS. The nuclei are firstly colored using a May-Griinwald solution (Sigma diagnostics, USA) and then the cytoplasms with Giemsa (Merk, D) diluted to 1/20. The number of syncytia on a square centimeter is reported or relativized between the envelopes.

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Claims

1. Use of a proline-rich sequence to improve the fusogenic character of retrovirus envelopes.

2. Use according to claim 1 of a proline rich sequence for improving the fusogenic character of a protein comprising a retroviral envelope glycoprotein of an amphotropic MLV or a retroviral envelope glycoprotein of an MLV 10A1 or a glycoprotein retroviral envelope of a GALV or a retroviral envelope glycoprotein of a xenotropic MLV or a retroviral envelope glycoprotein of an MLV MCF or a retroviral envelope glycoprotein of a FeLV, which proline-rich sequence corresponds :

- either with the proline-rich sequence

. retroviral envelope glycoprotein from an amphotropic MLV, or

. of the retroviral envelope glycoprotein of an MLV MCF,. of the retroviral envelope glycoprotein of an MLV 10A1,

. of the retroviral envelope glycoprotein of a GALV, or. of the retroviral envelope glycoprotein of a xenotropic MLV, or of the retroviral envelope glycoprotein of a FeLV, and comprises at least one mutation such that there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence,

- either to the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV

3. Use according to claim 1 of a proline-rich sequence in association with a functional domain of the envelope glycoprotein retroviral of an ecotropic MLV chosen from the binding domain, domain C, or the domain of the transmembrane subunit

4. Use according to claim 1 of a proline-rich sequence corresponding to the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV, in association with a functional domain of the retroviral envelope glycoprotein of a Ecotropic MLV chosen from: the binding domain, the C domain, or the domain of the transmembrane subunit.

5. Use according to claim 1 of a proline-rich sequence for improving the fusogenic character of a protein comprising a retroviral envelope glycoprotein of an MLV, which proline-rich sequence corresponds: - either to the proline-rich sequence

. of the retroviral envelope glycoprotein of an amphotropic MLV, and comprises at least one mutation such that there is suppression of at least one β-turn in the polyproline helix of the proline-rich sequence, - either at the proline-rich sequence of the retroviral envelope glycoprotein of an ecotropic MLV.

6. Proline-rich sequence of which the amino acid sequence and the secondary structure correspond to that of the proline-rich sequence of the envelope glycoprotein of an amphotropic MLV or of the retroviral envelope glycoprotein of an MLV 10A1 or of the retroviral envelope glycoprotein of an MLV MCF or of the retroviral envelope glycoprotein of a GALV or of the retroviral envelope glycoprotein of a xenotropic MLV or of the retroviral envelope glycoprotein of a FeLV, and comprising at least one mutation on the C-terminal side such that there is suppression of at least 1 β-turn in the polyproline helix of the proline-rich sequence

7. Proline-rich sequence whose amino acid sequence and secondary structure correspond to that of the proline-rich sequence of the envelope glycoprotein of an amphotropic MLV or of the retroviral envelope glycoprotein of an MLV 10A1 or retroviral envelope glycoprotein of an MCV MLV or retroviral envelope glycoprotein of a GALV or retroviral envelope glycoprotein of a xenotropic MLV or retroviral envelope glycoprotein of a FeLV, and comprising at least one mutation on the side of the N-terminal part such that there is suppression of at least 1 β-turn in the polyproline helix of the proline-rich sequence.

8. Mutated protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or the retroviral envelope glycoprotein of an MLV 10A1 or to the retroviral envelope glycoprotein of an MLV MCF or the envelope glycoprotein retroviral of a GALV or the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which the proline-rich domain comprises at least one mutation such that there is suppression of '' at least one β-turn in the polyproline helix of the proline-rich sequence.

9. Chimeric protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or to the retroviral envelope glycoprotein of an MLV 10A1 or to the retroviral envelope glycoprotein of an MLV MCF or to the envelope glycoprotein retroviral of a GALV or the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which the proline-rich domain is replaced by the proline-rich domain of the glycoprotein retroviral envelope of an ecotropic MLV.

10. Chimeric protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or to the retroviral envelope glycoprotein of an MLV 10A1 or to the retroviral envelope glycoprotein of an MLV MCF or to the retroviral envelope glycoprotein of a GALV or to the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which:

11. Chimeric protein corresponding to the retroviral envelope glycoprotein of an amphotropic MLV or to the retroviral envelope glycoprotein of an MLV 10A1 or to the retroviral envelope glycoprotein of an MLV MCF or to the envelope glycoprotein retroviral of a GALV or the retroviral envelope glycoprotein of a xenotropic MLV or the retroviral envelope glycoprotein of a FeLV, in which:

* the proline-rich domain comprises at least one mutation such that there is suppression of at least one β-turn (in the polyproline helix of the proline-rich sequence), * and at least one of the functional domains chosen from: the binding domain, the C domain, or the domain of the transmembrane subunit, is replaced by the corresponding domain of the envelope of an ecotropic MLV.

12. Mutated or chimeric protein according to one of claims 8 to 11, having at least one of the following properties:

- there is formation of syncytia after cocultures between target cells expressing a surface molecule serving as a retroviral receptor by interaction with the binding domain carried by a retroviral envelope glycoprotein and cell lines producing or not producing Gag-Pol particles of

MLV (or mammalian type C retrovirus or lentivirus) previously transfected with an expression vector for one of the above proteins,

- there is infection of target cells expressing a retroviral receptor recognized by the binding domain of a mutated or chimeric glycoprotein, under limiting conditions where the density of this receptor decreases by at least 100 times the infectious titre of viruses comprising an envelope not mutated or non-chimeric and by less than 100 times the infectious titer of virus comprising a chimeric or mutated envelope, these above values applied to the infectious titer being given by comparison with the infectious titer of virus comprising a neither mutated, nor chimeric or an envelope chimeric or mutated on target cells, such as XC cells.

13. A proline-rich sequence according to claim 6 or 7, corresponding to one of the following sequences: A ₂

At ₃

A ₃ MB A ₄ C ₂ C ₃

C ₅

14. Proteins mutated into a chimera according to one of claims 8, 9, or 10, corresponding to one of the following sequences:

- PROMO

- PRO FR

- BD PRO MO

- PRO C MO - PRO TM MO

- PRO CTM MO