JP2002508975A - Use of proline-rich sequences to improve the fusogenicity of retroviral envelopes - Google Patents

Abstract

  (57) [Summary] The present invention relates to a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV10A1, a retroviral envelope glycoprotein of GALV, a retroviral envelope glycoprotein of xenotropic MLV, a retroviral envelope glycoprotein of MLV MCF or It relates to the use of proline-rich sequences to improve the fusogenicity of proteins, including the retroviral envelope glycoprotein of FeLV. The proline-rich sequence may be an amphotropic MLV retrovirus envelope glycoprotein, a GALV MLV MCF retrovirus envelope glycoprotein, an amphotropic retrovirus envelope glycoprotein or a FeLV retrovirus envelope glycoprotein, and a proline-rich sequence. Contains at least one mutation that suppresses at least one of the β-turns in the polyproline helix, or corresponds to the proline-rich sequence of the retroviral envelope glycoprotein of ecotropic MLV.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention has for its object the use of a proline-rich sequence to enhance the fusogenicity of a retroviral envelope.

[0002] Retroviral entry into target cells is mediated by a peptide fusion localized at the amino terminus of the surface subunit (SU) internal to the viral envelope, followed by the internal transmembrane subunit (TM). It relies on the recognition of a specific surface protein, called a receptor, by fusion of different membranes (29). Regarding retroviruses, a pH-dependent entry pathway and an independent entry pathway have been described,
The determinants and the processes involved in fusion activation after receptor recognition are still unknown. Identification of these steps is important not only for controlling infection but also for understanding the mechanism of retroviral gene transfer.

[0003] The retroviral envelope glycoprotein recovered with the trimer is a complex that includes a surface subunit (SU) and a transmembrane (TM) component (10).
For all coated viruses, the initial interaction of this glycoprotein with the cellular receptor causes the conformational rearrangement of the envelope necessary to reveal a peptide fusion. The determinants of the envelope, and the sequence of events that result in a conformational change, have been extensively described for orthomyxoviruses that require endocytic vesicles in an acidic environment for their entry (24).

[0004] Although a pH-independent pathway has been described for retroviruses, the exact determinants and timing derived from receptor recognition for fusion activation are still unknown. The Pit-2 receptor (14, 28) of the amphotropic envelope of mouse leukemia virus is present in most species, including humans, but the functional receptor of the ecotropic envelope is limited to rat and mouse cells. (19). Recognition of one or the other of these receptors affects the efficiency of the fusion, but the ecotropic envelope, compared to the amphotropic envelope, cell-cell fusion and the formation of fused cells, as tested in XC rat cells. Induce more easily (17).

[0005] The structural domain consists of the Moloney ecotropic envelope (MoMLV),
i) an amino-terminal domain (indicated as BD) that links to a receptor of about 200 amino acids (aa) (3, 7); (ii) a 4 identified by the following chimeric PRO4
(Iii) a 160 amino acid SU carboxy terminal sequence (designated C) involved in the interaction with TM; (iv) peptide fusion of the enveloped virus envelope protein protein 1 shown here by TM with a potential amino-terminal peptide fusion identified by the sequence similarity of
Includes a 34 amino acid TM ectodomain (11); (v) a 28 amino acid anchoring peptide of the membrane; and (vi) a small R carboxy terminal peptide that is cleaved late in virions and increases the fusogenic potential of the envelope. It is divided into an amphotropic envelope (MLV-4070A) containing a tail (20) in the cytoplasm. The BD and PRR amino terminal domains between the ecotropic and amphotropic envelopes share only 33% and 43% amino acid homology, respectively, while all other domains have more than 80% homology (FIG. 1A)
.

One object of the present invention is to provide a proline-rich sequence that improves the fusogenicity of a retroviral envelope.

[0007] One of the objects of the present invention is to provide chimeric or mutein proteins that improve fusogenicity.

The present invention provides a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV10A1, a retroviral envelope glycoprotein of GALV, a retroviral envelope glycoprotein of xenotropic MLV, and a retroviral envelope of MLV MCF. A proline-rich sequence for improving the fusogenicity of a protein comprising a glycoprotein or a FeLV retroviral envelope glycoprotein, wherein the proline-rich sequence comprises:-the retroviral envelope glycoprotein of amphotropic MLV; The retroviral envelope glycoprotein of MLV MCF; the retroviral envelope glycoprotein of MLV 1OA1; At least one β in the polyproline helix corresponding to the proline-rich sequence of the retrovirus envelope glycoprotein of the retroviral envelope glycoprotein of xenotropic MLV, or of the retrovirus envelope glycoprotein of FeLV, or Turn suppression comprising at least one mutation of such kind, or-corresponding to the proline-rich sequence of the retroviral envelope glycoprotein of ecotropic MLV) for that purpose.

In the following cases:-by replacing the proline-rich sequence with a homologous proline-rich sequence of an ecotropic MLV, or-of an ampho type of such that there is at least one β-turn suppression. Tropic MLV, MLV MCF, MLV10A1, GALV,
The amphotropic MLV, the MLV MCF, the MLV10A1, the GAL by restricted mutation of the proline-rich sequence of xenotropic MLV or of FeLV
V. It was unexpectedly noticed that there was an increase in the fusogenicity of the retroviral envelope glycoprotein of the xenotropic MLV or the FeLV.

[0010] The proline-rich region is organized in a secondary structure that is presumably a polyproline helix, a "beta-turn" that is a helix of a 180-degree peptide chain that contains more proline. These curves are separately located between the PRO region (proline rich region) of the ecotropic and amphotropic envelopes.
The latter contains 11 regularly arranged "beta turns", 4 of which are at the N-terminus and 7 are at the C-terminus. The PRO region of the ecotropic envelope contains only seven "beta turns", only two of which are at its N-terminus.

The expression “a type of mutation in which there is at least one β-turn suppression in the polyproline helix of the proline-rich sequence” refers to: a deletion of the four amino acids forming the β-turn; One or several selective mutations, in particular, the probability of having a β-turn is less than 0.8 according to the Chow-Fassman algorithm, indicating that Mutation is meant.

By way of example, proline is advantageously substituted with isoleucine, valine or alanine.

The Moloney MLV, Friend MLV (23) strain can be mentioned as a suitable MLV ecotropic strain.

[0014] The 4070A (16) strain may be referred to as a suitable amphotropic MLV strain.

[0015] The MLF MCF 247 (9) strain can be mentioned as a suitable MLV MCF strain.

The Seato strain is a suitable GALV strain (Delassus et al., 1989, Virology 173: 205-2).
13).

The strain NZB. IU. 6 (15) can be mentioned as a suitable xenotropic MLV strain.

[0018] FeLV-C-SARMA FSC (21) can be mentioned as a suitable FeLV strain.

(Ott et al., 1990 (16)) can be mentioned as a suitable MLV 10A1 strain.

The expression “improved fusogenicity” means that the “improved” retroviral envelope of the present invention has the important ability to effectively achieve the post-linking stage of the virus entry process relative to the original envelope. And, in essence, match the molecular fusion of the viral and cell membranes.

[0021] The improvement of fusogenicity is determined by two tests:-a target cell that expresses a retroviral receptor recognized by the linking domain of a chimeric or mutated glycoprotein, and the expression of one of the above proteins Fusion cell formation test after co-culture with cell derivatives pre-transfected with the vector,-Infection of target cells expressing, under limited conditions, retroviral receptors recognized by the linking domain of chimeric or mutated glycoproteins test〔
Here, the density of these receptors (ie the number of cell surface effective receptors that allow the adsorption of virus particles) reduces the infectivity of the virus, including the unmutated original envelope, by at least 100 times, And reduces the infectivity of the virus containing the chimeric or mutated original envelope by less than 100-fold; these values are based on the original XC cell and chimeric mutated sources under the conditions described in Example Table 1B. Given in comparison with the infectivity of the virus containing the envelope].

The proline-rich sequence of the retroviral envelope glycoprotein of ecotropic MLV is shown in FIG.

The proline-rich sequence of the retroviral envelope glycoprotein of the amphotropic MLV is shown in FIG.

The proline-rich sequence of the retroviral envelope glycoprotein of MLV MCF is shown in FIG.

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

The proline-rich sequence of the retroviral envelope of MLV 10A1 is shown in FIG.
Shown in

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

The proline-rich sequence of the retroviral envelope glycoprotein of FeLV is shown in FIG.

The proline-rich sequence of the ecotropic MLV retroviral envelope glycoprotein also refers to those containing mutations, suppressions or additions of amino acids of a type that do not alter the β-turn sequence.

According to an advantageous mode of operation, the present invention provides a proline-rich sequence that binds to a functional domain of an ecotropic MLV retroviral envelope glycoprotein selected from a linking domain, a C domain or a transmembrane subunit. Is intended for use as described above.

A “functional domain” is defined as any polypeptide sequence that is individualized at the structural level and is capable of performing a defined biological function.

A “linking domain” is defined as a functional domain that is carried by the N-terminal portion of the SU subunit of the MLV retroviral envelope and is capable of binding itself to a retroviral receptor. Amphotropic MLV
It is shown in FIG. 6 as extending to positions 31 to 237 of the peptide sequence of the envelope glycoprotein.

A “C domain” is defined as a functional domain that is carried by the C-terminal portion of the SU subunit of the MLV retrovirus envelope and that can interact with the TM subunit. The sequence is shown in FIG. 6 as extending from positions 300 to 458 of the peptide sequence of the amphotropic MLV envelope glycoprotein.

“TM domain” is defined as a domain that is carried by the ectodomain of the TM subunit of the MLV retroviral envelope and is capable of interacting with the SU subunit, and is a virus that is involved in the process of retroviral entry. And achieve fusion between cell membranes. The sequence is shown in FIG. 6 as extending from positions 459 to 592 of the peptide sequence of the amphotropic MLV envelope glycoprotein.

The linking domain, domain C or transmembrane subunit domain comprises:-by the insertion of a peptide or protein domain that exhibits binding to these molecules, other molecules, such as surface molecules (antigen receptor, growth factor receptor, etc.) -Modifying the functional capacity of retroviral envelope glycoproteins with the intention of enhancing their stability;-enhancing their fusogenic potential with retroviral envelope glycoproteins. Can be modified.

The present invention provides a retroviral envelope of an ecotropic MLV that cooperates with a functional domain of an ecotropic MLV retroviral envelope glycoprotein selected from a linking domain, a C domain, or a transmembrane subunit domain. Of particular interest is the use of a proline-rich sequence corresponding to the proline-rich sequence of a glycoprotein as described above.

According to an advantageous method of practice, the present invention relates to a proline-rich sequence for improving the fusogenicity of a protein, including the retroviral envelope glycoprotein of MLV, wherein the proline-rich sequence comprises Comprising at least one mutation of the type corresponding to the proline-rich sequence of the retroviral envelope glycoprotein of the phototropic MLV and being a suppression of at least one β-turn in the polyproline helix of the proline-rich sequence Or a proline-rich sequence corresponding to the proline-rich sequence of the retroviral envelope glycoprotein of ecotropic MLV).

The present invention relates to a novel product whose amino acid sequence and secondary structure are the amphotropic MLV envelope glycoprotein, the MVL 10A1 retrovirus envelope glycoprotein, the MLV MCF retrovirus envelope glycoprotein, and the GALV Corresponds to the proline-rich sequence of the retroviral envelope glycoprotein, the retroviral envelope glycoprotein of xenotropic MLV or the retroviral envelope glycoprotein of FeLV, and suppression of at least one β-turn in the polyproline helix of the proline-rich sequence Also related to proline-rich sequences that contain at least one mutation on the C-terminal part of certain types.

“C-terminal portion” is defined as the sequence of amino acids corresponding to the second half of the proline-rich sequence.

[0040] The present invention provides an amino acid sequence and a secondary structure, wherein the envelope glycoprotein of amphotropic MLV, the retroviral envelope glycoprotein of MVL 10A1, the retroviral envelope glycoprotein of MLV MCF, the retroviral envelope glycoprotein of GALV, N-types of N which correspond to the proline-rich sequence of the retroviral envelope glycoprotein of xenotropic MLV or the retroviral envelope glycoprotein of FeLV and have at least one β-turn suppression in the polyproline helix of the proline-rich sequence The same applies to proline-rich sequences containing at least one mutation on the terminal side.

“N-terminal portion” is defined as a sequence of amino acids corresponding to the first half of a proline-rich sequence.

The invention likewise relates to a mutein comprising a proline-rich sequence in which the proline-rich sequence in the chain has at least one β-turn mutated in such a way that there is suppression.

More precisely, the present invention relates to a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV 10A1, a retroviral envelope glycoprotein of MLV MCF, a retroviral envelope glycoprotein of GALV, xeno. The retroviral envelope glycoprotein of tropic MLV or the retroviral envelope glycoprotein of FeLV (where the proline-rich sequence is at least one of the types such that there is at least one β-turn suppression in the polyproline helix of the proline-rich sequence). Mutated proteins).

This mutein has the property that the envelope from which it was derived can induce fusion cells more easily or effectively infect cells that express limited amounts of retroviral receptors. , For the corresponding non-mutated protein.

The present invention likewise relates to chimeric proteins comprising a proline-rich sequence in the chain from the proline-rich sequence replaced by a proline-rich sequence homologous to the retroviral glycoprotein of ecotropic MLV.

More precisely, the present invention relates to a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV 10A1, a retroviral envelope glycoprotein of MLV MCF, a retroviral envelope glycoprotein of GALV, xeno. To the retroviral envelope glycoprotein of tropic MLV or the retroviral envelope glycoprotein of FeLV, where the proline-rich domain is a proline-rich domain replaced by the proline-rich domain of the ecotropic MLV retroviral envelope glycoprotein For the corresponding chimeric protein.

As compared to the corresponding original protein, the chimeric protein is capable of more easily inducing the formation of fused cells or expressing a limited amount of the retroviral receptor relative to the original envelope from which it was derived. Exhibiting the property of effectively infecting the same.

In each group of mutated or chimeric proteins as described above, a functional domain homologous to the retroviral envelope glycoprotein of ecotropic MLV provides at least one of those functional domains. Can be similarly substituted.

According to a preferred method of practice, the present invention provides a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV 10A1, a retroviral envelope glycoprotein of MLV MCF, a retroviral envelope glycoprotein of GALV. Protein, xenotropic ML
V retrovirus envelope glycoprotein or FeLV retrovirus envelope glycoprotein (where the * proline-rich domain has been replaced by the proline-rich domain of the ecotropic MLV retrovirus envelope glycoprotein and * a linking domain, At least one of the functional domains selected from the C domain or the domain of the transmembrane subunit is ecotropic MLV
Which has been replaced by a domain corresponding to the envelope of

This chimeric protein has the property of more easily inducing the formation of fused cells or of effectively infecting cells expressing a limited amount of the retroviral receptor against the envelope from which it was derived. .

According to another preferred method of implementation, the invention relates to a retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV 10A1, a retroviral envelope glycoprotein of MLV MCF, a retrovirus of GALV. Suppression of the envelope glycoprotein, the retroviral envelope glycoprotein of xenotropic MLV or the retroviral envelope glycoprotein of FeLV, where * the proline-rich domain has at least one β-turn (in the polyproline helix of the proline-rich sequence) There is at least one of a kind
And at least one of the functional domains selected from among linking domains, C domains, or domains of transmembrane subunits comprises ecotropic MLV
(Replaced by the corresponding domain of the envelope).

This chimeric protein has the property of more easily inducing the formation of fused cells or of effectively infecting cells expressing a limited amount of the retroviral receptor with respect to the envelope from which it was derived. .

The mutated or chimeric protein of the present invention has the following characteristics:-a target cell that expresses a surface molecule that serves as a retroviral receptor by interacting with a linking domain carried by a retroviral envelope glycoprotein; Formation of fused cells after co-culture with MLV (or mammalian C-type retrovirus or lentivirus) Gag-Pol particles producing or not producing cell derivatives previously transfected with one expression vector of the above proteins The density of this receptor (ie the number of cell surface effective receptors that allow the attachment of viral particles) reduces the infectivity of viruses, including non-mutated and non-chimeric envelopes, by a factor of 100 and Chimeric or mutant envelopes Under limited conditions that attenuate the infectivity of the containing virus by less than 100-fold, there are infections of target cells that express a mutant virus of the invention or a retroviral receptor that recognizes the linking domain of a chimeric glycoprotein; With target cells, such as X, under infection conditions as defined in the Examples and in particular Table 1B below.
Advantageously, at least one of applying to the infectivity obtained relative to the infectivity of a non-mutated and non-chimeric envelope on C cells, or of a virus comprising a chimeric or mutant envelope.

There are two notable tests that are useful to enable the identification of mutant or chimeric proteins of the present invention.

One of these tests is one of target cells and one of the chimeric or mutated proteins of the invention (and possibly recognizing retroviral receptors located on the target cells). Formation of fused cells with cell derivatives previously infected with an expression vector.

As target cells, SC1 mouse cells, Mus Duni, TE671 human cells, and similarly XC rat cells can be used.

The cell derivative used can or does not produce Gag-Pol particles of MLV.

The cell derivative may be MLV or other mammalian retrovirus type C, especially Moloney M
Can produce or cannot produce Gag-Pol particles of a retrovirus, such as LV or other lentivirus, for example, HIV-1.

Another test for identifying mutated or chimeric proteins of the present invention is by mediating viral particles containing these chimeric or mutated proteins, chimeric or mutated proteins of the present invention. Infecting target cells that express retroviral receptors that may be recognized by the protein.

As target cells, XC and SC-A-ST rat cells can be used, the latter of plasmids expressing the linking domain of the amphotropic retrovirus envelope capable of occupying the amphotropic retrovirus receptor. It is derived from XC cells by transfection.

As virus particles, particles produced by MLV retroviruses or other mammalian retroviruses of type C, in particular other retroviruses such as Moloney MLV virus or lentivirus, for example HIV-1 can be used.

This infection can be effected under limited conditions, that is, when the density of the receptor (ie, the number of receptors available on the cell surface that allows the attachment of the virus particles) can be either mutant or chimeric envelope glycoprotein. Conditions that reduce the infectivity of the virus without the virus by at least 100-fold and reduce the infectivity of the virus containing the mutant or chimeric envelope of the invention that does not correspond to either the above-described mutation or the chimeric envelope by a factor of 100. It is carried out in.

The comparison is carried out on a comparison of the infectivity of non-chimeric and non-mutated envelopes, or viruses containing chimeric or mutated envelopes, on target cells such as XC cells.

This infection was carried out in comparison with two cell types, the second being directly derived from the first and having the effect of reducing the number of effective retroviral receptors, ie the receptor Reducing the surface expression of at least one infectivity of the virus containing the original envelope, which is neither mutated nor chimeric.
Decrease by 00 times.

This utility is then compared with that obtained for viruses containing chimeric or mutated envelopes. A reduction in infectivity of less than 100 of the virus containing the chimeric or mutant envelope between the two cell types thus indicates an improvement in the latter's fusogenic properties.

A preferred proline-rich sequence according to the invention corresponds to one of the following sequences: A ₂ A ₃ A ₃ Mo A ₄ C ₂ C ₃ C ₄ C ₅ .

For the proline-rich region of the retroviral glycoprotein of the amphotropic MLV, the A ₂ sequence has a second β-turn that has the effect of being more fusogenic compared to the original envelope in a fusion cell test. Including suppression of

[0068] Compared to the proline-rich region of the retroviral glycoproteins amphotropic MLV, A ₃ sequences have an effect which is more than the fusion immunogenic than the original envelope in fusion cells tested, third Includes β-turn suppression.

As compared to the proline-rich region of the retroviral glycoprotein of the amphotropic MLV, the A ₃ Mo sequence has the effect of being more fusogenic compared to the original envelope in an infection test as described above. , A third β-turn suppression.

[0070] Compared to the proline-rich region of the retroviral glycoproteins amphotropic MLV, A ₄ sequence has the effect which is a fusion original of the above ratio all the original envelope in infection tests, such as the , A fourth β-turn suppression.

As compared to the proline-rich region of the retroviral glycoprotein of the amphotropic MLV, the C ₂ sequence has the effect of being more fusogenic compared to the original envelope in an infection test as described above. , Β-turns 9-13.

As compared to the proline-rich region of the retroviral glycoprotein of the amphotropic MLV, the C ₃ sequence has the effect of being more fusogenic compared to the original envelope in an infection test as described above. , Β-turns 11-13.

As compared to the proline-rich region of the retroviral glycoprotein of amphotropic MLV, the C ₄ sequence has the effect of being more fusogenic compared to the original envelope in an infection test as described above. , Including the deletion of the last β-turn (13 °).

[0074] Compared to the proline-rich region of the retroviral glycoproteins amphotropic MLV, C ₅ sequence has an effect which is a fusion original of the above ratio all the original envelope in infection tests, such as the , 11 ° and 12 ° β-turn deletions.

A preferred chimeric protein according to the invention corresponds to one of the following sequences: -PROMO-PROFR-BDPROMO-PROCMO-PROTMMO-PROCTMMO.

In the PROMO sequence, the proline-rich region has an ecotropic MLV (Moloney strain)
MLV replaced by a proline-rich region homologous to
Corresponds to an array.

The PRO FR sequence is an amphotropic ML in which the proline-rich region has been replaced by a proline-rich region homologous to ecotropic MLV (Friend strain).
It corresponds to the V array.

[0078] The BD PRO MO sequence corresponds to an amphotropic MLV sequence in which the proline-rich region is homologous to the ecotropic MLV and has been replaced by a linking domain homologous to the ecotropic MLV.

The PRO CMO sequence is an amphotropic MLV in which the proline-rich region has been replaced by a proline-rich region homologous to ecotropic MLV and the C domain has been replaced by a C domain homologous to ecotropic MLV.
Corresponds to an array.

The PRO ™ MO sequence shows that the proline-rich region has been replaced by a proline-rich region homologous to ecotropic MLV, and the TM domain has been replaced by a TM domain homologous to ecotropic MLV. It corresponds to the MLV sequence.

The PRO CTM MO sequence has the proline-rich region replaced by a proline-rich region homologous to ecotropic MLV, and each of the C and TM domains replaced by C and TM domains homologous to ecotropic MLV. Corresponding to an amphotropic MLV array.

A preferred chimeric protein according to the invention is 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 ₄ C ₅ , and at least one of them The following functional domains: linking domain, domain C, including domains in which the transmembrane domain has been replaced by a domain homologous to ecotropic MLV.

The proline-rich region of the mouse leukemia (MLV) envelope, located between the receptor-linking domain of the terminal amino portion of SU and the domain of interaction with TM of the carboxy-terminal portion of SU ( It has been shown in the context of the present invention that PRR) serves as a mediator of the conformation of the envelope. Moreover,
The potential beta-turn of the proline-rich region determines the stability of SU-TM binding and the activation threshold for cell-cell and virus-cell fusion.

To identify the region responsible for the maximal fusion potential of the ecotropic envelope of MLV, the BD, PRO, C and TM amphotropic envelopes were replaced with their homologous ecotropic envelope (FIG. 1B). A series of chimeric envelopes have been created. The resulting envelope is thus identified by the substituting ecotropic domain. Cell-to-cell fusion is performed between various target cells (indicators) and cell derivatives that produce or do not produce Gag-Pol particles of MLV (5) previously transfected with one or the other envelope. Evaluate by the formation of fused cells after co-cultivation for 24 hours.

However, the expression levels on the cell surface of the original envelope and the chimeric envelope are similar (FIG. 4A), and two different cell-cell fusion phenotypes are observed. CM
The O and TMMO envelopes induced cell fusion slightly, as did the wild type envelope MLV-4070A.

However, BDMO, PROMO and PROFR envelopes strongly induce the formation of fused cells similar to those induced by ecotropic envelopes (FIG. 2). The formation of fused cells was not affected by the presence or absence of Gag-Pol particles (obtained but not shown), and the fusion measured in this test was determined by direct cell-cell contact rather than cell contact mediated by virus particles. Is produced by Previous reports have shown that the enhanced fusogenic performance of the BDMO envelope is primarily due to its ability to interact with ecotropic receptors (
18).

However, important fusogenic properties, in addition to, or in a context separate from, ecotropic receptors, are associated with amplifying all cell types used, including rat, mouse and human target cells. Similarly observed for chimeras having a tropic BD domain (FIGS. 1 and 2) (obtained but not shown). Thus, the differences observed during the formation of fused cells between the original and the chimeric amphotropic envelope, unlike the interaction of BD with the receptor, are due to new properties.

As described for the feline leukemia virus (8), and as suggesting a slight homology with MLV, the regular repetition of proline in the PRR is likely to be a “beta turn” (27 ), Ie, its coiling in a polyproline helix in a secondary structure that provides a "elastomer" strength that can achieve the function of a "molecular spring" (26). Because of the important position of the PRR between the BD and C domains that both influence the fusion, its role in the fusion was directly evaluated. Differences in the expected number and arrangement of "beta turns" in the 4070A PRR compared to MoMLV predict the latter more reactive springs which can explain the accumulated fusion potential of PROMO. obtain. C, a selective or deletion mutant that appears to suppress some of the seven carboxy-terminal "beta turns" of the amphotropic PRR
2, C3, C4 and C5 were tested in the XC cell-cell fusion test. They showed a slight fusogenicity, similar to the amphotropic original envelope (FIG. 3B). The "beta turns" induced by PRR proline are less interdigitated at the amino-terminal portion than at the terminal carboxy-terminal. Thus, 407
Each of the four possible "beta turns" of the OA envelope can be separately suppressed by replacement of proline with valine or isoleucine, and A1, A2, A3, A3MO and A4 (FIG. 3C) suddenly Obtain the mutant. M
A2 and A3 mutants have a second amino acid substitution (not shown) to avoid the formation of alpha helices or potential beta sheets, which are not naturally present in the structural predictions of LV PRRs. The A1 and A4 envelopes, in which each of the first or fourth "Beta-turn" amino termini was suppressed, preserved the regular repetition of three adjacent "Beta-turns" and formed fused cells. Does not elicit an increase (Table 1). In contrast, the A2 and A3 envelopes, in which the continuity of these "beta turns" is disrupted (FIGS. 3C and 3D), are highly fusogenic (Table 1), thus "mediating" fusion following receptor recognition. The critical importance of the adjacent "beta turn" of the PRR in "". This cumulative fusion performance is the molecular P
Cell-cell fusion was compared by using XC or XC-A-ST cells to test if it was due to a lower need for it-2. In the latter, constitutive expression of amphotropic BD reduced the amount of functional Pit-2 receptor (
Table 1).

While the interference is effective against the MLV-4070A envelope, increased fusion potential was always observed with the PROMO, PROFR, A2 and A3 envelopes.

Nevertheless, fusion is still dependent on Pit-2, such as CHO cells that do not express Pit-2 but only fused after de novo expression of Pit-2 (
Not shown). Thus, mutations in the "beta turn" of the PRR appear to reduce the activity threshold of fusion that occurs after receptor recognition.

After cell-cell fusion, cell-virus fusion was tested by comparing the infectivity of the original or chimeric enveloped virions. This strength is Pit
XC rats transfected with the gene expressing the -2 receptor (Table 1)
, Mouse, human and CHO. Surprisingly, the intensity of the highly fusogenic chimeras PROMO and PROFR, as well as the selective mutants A2 and A3, was undetectable or clearly reduced in all cells tested.
The other envelope confers strength on XC cells within the range of infectious units per 10 ⁶ to 10 ⁷ mL, similar to that obtained with the original two envelopes, 4070A and MO. However, with the exception of A1, even though the original amphotropic envelope caused a dramatic decrease in virion infectivity in XC-A-ST cells,
All mutants of the infectious PRR are clearly less susceptible to interference (Table 1).
). The fact that, despite showing increased infectivity (virus-cell fusion), mutants of the carboxy-terminal portion of the PRR do not show any improvement in cell-cell fusion performance, the fact that the amino properties, It proves that terminal carboxy also affects fusion performance. Receptor binding tests performed with antibodies to SU show that Pit-2 / SU affinity does not change significantly with these envelopes (data not shown). CHO infection transfected with the expressed Pit-2 confirms the need for Pit-2 for virion invasion, as opposed to lacking the original CHO infection.

PPR is important in SU / TM binding and in the conformation of the envelope, since the weak infectivity of virions with a fusogenic mutant envelope in cell-cell tests is due to a decrease in cumulative SU (FIG. 4). It seems to play an important role. PROMO, PR
It has been suggested that the cumulative expansion of SU for the OFR, A2 and A3 mutants conferred the ability of PRR to control fusion by interacting with adjacent regions and that disruption of these interactions stimulates fusion. Is done. PROCMO, PROTMMO
And regions of low homology, such as PROCTMMO chimeras or BDPROMs
The combination of the BD domain upstream of the O envelope (FIG. 1C) and the ecotropic PRR increases stability (data not shown) as calculated by decreasing SU (FIG. 1).

Finally, following receptor recognition, PRR proved to be a major determinant in conformational changes in envelope and membrane fusion. In addition, the characteristic arrangement of the "beta turns" of the PRR in conjunction with the lower C and TM regions gives different properties between these MLV envelopes.

The possible applications of the present invention are as follows: a mutated or chimeric envelope glycoprotein according to the invention, a mutated or chimeric envelope glycoprotein according to the invention, also specific. Expression of a limited number of retroviral receptors by retroviral vectors derived from C-type, mammalian or lentiviral retroviruses, including peptide or protein domains capable of redirecting the binding of the retroviral vector to molecular targets Ex vivo or in vivo gene therapy for gene transfer into cells.

【Example】

II. Materials and Methods II.1. Cell Derivatives TElacZ cells contain the retroviral vector MFGnlsLacZ (25) that is responsible for the expression of nuclear beta-galactosidase. The cell derivative TELceB6 (5) derived from the TElacZ derivative was obtained by transfection of a plasmid expressing the gag and pol proteins of MoMLV. These cells lack the envelope carrying the marker gene nlsLacZ and therefore produce non-infectious retroviral particles.

TELCEB6, XC, RD and CEAR13 (12) cells were obtained from DMEM (Life-Technologies) supplemented with 10% fetal calf serum (FCS, Life-Technologies).
) The cells were cultured in a culture solution. The CEAR13 culture was further supplemented with 10 μg / μL proline (Sig
ma) was added. The NIH3T3 (ATCC CRL 1658) derivative is a DMEM (Life-Technologies) supplemented with 10% newborn bovine serum (Life-Technologies).
).

The XC-A-ST cells are capable of binding to the amphotropic receptor and blocking the XC of plasmid pA-ST (5), which expresses the N-terminal fragment of SU of the MLV-4070A virus. Obtained by stable transfection into cells.

II.2. Construction of Plasmids and Mutants Plasmids encoding the envelopes of amphotropic 4070A (FBASALF) (5), ecotropic moloney (FBMOSALF), and ecotropic friend C57 (FB3) Was enabled. The envelope gene is under the control of the LTR of the FB29 friend MLV (5)
. In addition, these plasmids allow for phlemycin secretion,
o Includes genetic markers.

[0099] Mutagenesis by PCR procedure was used to produce various constructs of the mutant envelope.

The DNA fragment for the Rless construct (20) was
5'-TTC TAT GCG GAC CAC ACA GGA, and OLR
Less5'-ATC TCA GTA GTC CAG GCT TTA T
MO using GA TAG TCG ACA TGC AT oligonucleotide
Obtained by PCR (amplification of the chain with polymerase) in the MLV (23) envelope. After digestion with the SpeI and AccI enzymes, this fragment was
It was subcloned between the SpeI and ClaI sites of the OSALF plasmid. The NdeI / ClaI fragment was thus removed from the previous plasmid and replaced with the NdeI / ClaI originating from the FBASALF vector. The result was an amphotropic envelope expression vector containing an early stop codon oligonucleotide upstream of the normal stop codon. This modification shortened the length of the cytoplasmic portion of the amphotropic glycoprotein and was sufficient to be strong fusogenicity and to induce fusion cells in cell culture (20).

For the construction of the chimeric envelopes PROMO and PROFR, the Moloney ML using ApaI and KpnI sites created at the start and end of this region.
PCR fragments corresponding to the proline-rich regions of the envelopes of the V (MO) and friend MLV (FR) viruses were cloned using the PCR with plasmid FBMOSALF.
In the following, Up MO ApaI 5′-CCA ATA GGG CCC AAC CCC GTT CTG and Low MO KpnI 5′-AAC GT G GTA CC C GCC GGT GGA AGT TGG G;
In the PCR in B3, nucleotides prepared by using Up FRApal GTC CCG ATA GGG CCC AAC CCC GTC CTG and Low FR KpnI GAA TGC GGT ACC TGC TGG CGG GGG CTG AGT GGG were used.

The digestion fragment ApaI / KpnI was combined with the oligonucleotide Up A Kp
nI 5'-TATGC GGTAC GGAGATAGACTACTAGCTC
And Low A BamHI 5'-GGTAGTAG GGATCC TGAGCC
KpnI generated by PCR on plasmid FBASALF using GG
/ BamHI adapter fragment was used to construct the Ap of envelope 4070A.
It was cloned between the aI (position 653) and BamHI (position 802) sites. Other chimeric envelopes were created using the above envelopes by subcloning different fragments.

The FBASALF plasmid was cleaved with XhoI-ClaI and the DN
A with the fragment: (i) PROCTM producing the PROCTMMO envelope
XhoI-DraIII of MO envelope and DraIII-C of MO envelope
laI, (ii) the NcoI of the MO envelope, co-ligated with the XhoI-NcoI adapter fragment of Envelope A, producing the TM MO envelope
-ClaI, (iii) used to clone the XhoI-NcoI of the MO envelope with the NcoI-ClaI fragment of envelope A, producing the chimeric PROCMO. The PROTMMO construct was digested with the same enzyme and the Xh of the PROMO envelope in a vector carrying the TMMO envelope.
Obtained by subcloning of the oI-BamHI fragment.

Finally, the BDPROMO chimera was constructed using the MO envelope BamHI-DraII.
Created by co-ligation of the I fragment with the DraIII-ClaI fragment of the PROMO envelope in the FBMOS ALF plasmid, digested with the BamHI-ClaI enzyme.

Mutants of the amphotropic proline-rich region were created by mutagenesis by PCR. X of the 4070A envelope (position 594) (16)
The coding oligonucleotide, ie, "sense"("upper") 805Fc (5'-TCC AAT TCC TT, located immediately upstream of the hoI site
C CAA GGG GC) are oligonucleotides provided with various restriction sites (specified in parentheses and which are underlined in the oligonucleotide sequence) and the insertion of the desired mutation (paired). A2 UpA2 5'-CGA GTC CCC ATA GGG ATC CAA CCG GTA TTA CCC GAC C (AgeI), A3
MO LowA3MO 5'-GCCCAACCCAGTGCTAGCCGAC
CAAAGAC (NheI), A3 P249I / Q251V 5'-CCA
GTA TTA atC GAC gtc AGA CTC CCT TC (A
atII); A4 P254I 5'-GAC CAA AGA CT g ata TC C TCA CCA A (EcoRV); C5 P286I / P289L 5'-CTC AAC CTC Cat TAC tAG T Ct AAG
Used in combination with TGT CCC AC (SpeI). Oligonucleotides complementary to these primers (LowA2 GGT CGG GTA ATA CCG GTT GGA TCC CTA TGG GGA CTC G; L
ow A3 GAA GGG AGT C Tg acG TCG atT AA
TACT GG; LowA3MO GTC TTT GGT CGG CTA GCA CTG GGT TGG GC; Low A4 TTG GTG A
GG Ata tc A GTC TTT GGT C; Low C5 GGC
TGT GGG ACA CTT aG A CTa GT A atG GAG
GTT GAG GGT G) at the BamHI site of the 4070 envelope (8
This was used together with a Low ABamHI primer hybridized at position 02). The two fragments generated for each mutant were digested with the appropriate enzymes and then ligated to the XhoI-BamHI digested FBASALF plasmid.

A similar method was used for deletion mutants C2, C3 and C4.

The oligonucleotide 805Fc hybridizing upstream of the XhoI site was
Low-C4 KpnI: ATC producing XhoI-KpnI fragment after digestion
TCC GGT ACC GAC ACT TGG ACT TGT AGG
GGA GGT TGA GGG, LowA-C3 KpnI: ATC TCC GGT ACC TGG GGG AGG GGA GGT TGA GGG TGT ACT GGT AGT GGA AGG, and LowA-C2K
pnI: ATC TCC GGT ACC TGG GGG GGG TGT
Used with ACT GGT AGT GGA AGG GGG GTA ACT GGT oligonucleotide. Each fragment was ligated with KpnI- from the PROMO envelope in an XhoI-BamHI digested FBASALF vector.
Co-bound with the BamHI fragment.

The plasmid expressing the envelope was transfected into the TELCeB6 derivative by the calcium phosphate precipitation method (22). The transfected cells were selected with phleomycin (50 μg / mL), and the resistant clones were combined and trypsinized. The harvested cells were used on the one hand to recover viral supernatants after overnight culture in normal culture medium and on the other hand to make cell lysates. The viral supernatant was used for infection tests, receptor binding tests, and ultracentrifuged to obtain a pellet of the virus, the remainder analyzed by immunoblotting.

II.3 Immunoblotting Virus-producing cells contained Triton X100 1%, SDS 0.05%, deoxycholate 5 mg / mL, NaCl 150 mM and a cocktail of protease inhibitors (PMSF 1 mM, leupeptin 6 mM, aprotinin 0.3 mM). Tris
Dissolved in -HCl (pH 7.5) buffer at 4 ° C for 10 minutes. These lysates were centrifuged at 10,000 g for 10 minutes to pellet all organelles, and the supernatant was then frozen at -80 ° C until analysis. Virus sample, SW
Obtained by ultracentrifugation of virus supernatant (6 mL) in a 41 Beckman rotor (30000 rpm, 1 hour, 4 ° C).

The pellet was resuspended in 80 μL of cold PBS (phosphate buffered saline) (Life-Technologies) and then frozen at −80 ° C. Samples (30 μg cell lysate or 20 μL purified virus) were prepared using 6% SDS (sodium dodecyl sulfate), 30% β-mercaptoethanol, 10% glycerin, and 0.1% bromophenol blue.
It was mixed with 375 mM Tris-HCl (pH 6.8) containing 06% at a ratio of 5: 1 (V / V), then denatured at 95 ° C. for 3 minutes, and then analyzed on SDS denatured 10% acrylamide gel. After transfer of the proteins onto the nitrocellulose membrane, the immunological markings were performed with TB in the presence of skim milk (5%) and Tween 0.1%.
S (Tris base saline (Tris buffer), pH 7.4). Antibodies obtained from sheep antiserum against gp70-SU of Rausher leukemia virus (RLV) or p30-CA of RLV (Quality Biotech Inc.
, USA) diluted 1: 2000 and 1: 10000, respectively. p
Antibodies obtained from rabbit antisera to 15E-TM were used at a dilution of 1: 1000. "Blot" refers to the use of a conjugated antibody conjugated to peroxidase of rabbit or goat origin to goat or rabbit immunoglobulin, respectively (Dako, UK) using a chemiluminescent kit (Amersham Life Science).

II.4. Receptor binding test Target cells were washed with PBS and then washed in a 0.02% versene PBS solution for 37 hours.
Stripped by incubation at 10 ° C for 10 minutes. Cells were washed with PBA (PBS containing 2% FCS (fetal calf serum) and 0.1% sodium azide to allow blockade of membrane endocytosis). 10 ⁶ cells were thus incubated for 1 hour at 37 ° C. with various viral supernatants (2 mL) containing soluble SU 2. Precipitated virus particles were removed from these supernatants, with or without ultracentrifugation. After washing twice with PBA, cells were washed with SU (83A
Incubation was performed at 4 ° C. for 45 minutes in the presence of the monoclonal antibody (6) against 25) or the monoclonal antibody against TM (MAb2), respectively. Cells were washed twice with PBA and then incubated at 4 ° C. for 45 minutes in the presence of conjugated fluorescent antibodies to rat (Cayla, F) or mouse (Dako, UK) immunoglobulin. The dead cells were then counterstained with propidium iodide (20 μg / mL) at 4 ° C. for 5 minutes. After washing twice with PBA, the fluorescence of the living cells was determined by fluorometry (FA
CSCalibur, Beckton Dickinson).

II.5. Cell Marking Test The cells carrying the envelope were washed, detached, and washed like the target cells for the binding test. 10 ⁶ cells were thus immediately incubated with anti-SU (83A25) rat antibody for 1 hour at 4 ° C.

The steps of washing, incubation with the secondary antibody conjugated to FITC and FACS analysis are the same as for the binding test.

II.6. Infection Test Target cells, NIH3T3, were seeded the night before in 24-well plates at a density of 5 × 10 ⁴ cells / well. Cells were incubated with 1 mL of various virus stock dilutions in the presence of 8 μg / mL polybrene (polycation, an inhibitor of electrostatic repulsion between virus particles and cells). After incubation at 37 ° C. for 3-5 hours, the supernatant was replaced with fresh medium, and the cells were incubated at 37 ° C. for 2 days. Cells are shunted with 0.5 shunting agent.
Immediately upon fixation with a solution of 5% glutaraldehyde in PBS, the substrate 5-bromo-
Color was developed using 4-chloro-3-indolyl-β-D-galactopyranoside or X-Gal for 12 hours. The virus intensity ratio was calculated as the number of positive colonies per mL of virus supernatant (LacZ iu / mL).

II.7. Immunoprecipitation A single confluent cell 35 mm in diameter was prepared by adding 10% dialyzed fetal bovine serum to DMEM culture medium without methionine or cysteine (Dulbecco's modification) to remove amino acids. (Eagle's medium) and then incubated with this same medium at 37 ° C for 1 hour 30 minutes. S35 (100 mCi / mL; TransS35-Label
After labeling for 45 minutes with methionine and cysteine (ICN Pharmaceuticals), the cells were washed and then incubated at 37 ° C. in culture (perfusion).
Cells were incubated with 0.5% NP40, 150 mM NaCl and 1 mM PMSF for 20 minutes.
Dissolution was performed at 4 ° C. for 20 minutes at various time points in a mM HEPES solution. 1 lysate
The mixture was centrifuged at 0000 g, and the supernatant was frozen at -80 ° C.

These supernatants were combined with non-immune goat serum (1/100) and ProteinA-Sepharose (
Purification was performed at 4 ° C. for 2 hours or overnight using 6 MB (Pharmacia). After centrifugation, the supernatant was incubated 1/100 with goat antiserum against gp70-SU Rusher leukemia virus (RLV).

RIPA buffer containing 40% A-Sepharose protein (150 mM NaCl, 50 mM Tris, 1% deoxycholate, 1% Triton 100X, 0.1%
% SDS) at 4 ° C. for 1 hour, after which the mixture was precipitated by centrifugation at 10,000 g for 5 minutes. After washing three times with RIPA buffer, immunoprecipitates were fractionated on a 12% SDS acrylamide gel. Protein was added to 25% isopropanol and
Fixation was carried out using an aqueous solution of acetic acid at a concentration of 10% and indicated using a PhosphoImagerP32 screen (Biorad).

II.8. Fusion Test Two days after transfection, active cells carrying the envelope were seeded at 20% confluency. After 3-4 hours, more than 3 times more target cells were added. Co-cultures were incubated at 37 ° C. for 24 hours and then fixed with 0.5% glutaraldehyde in PBS. The nuclei were first washed with May-Grunwald solution (Sigma diag
nostics, USA), and then cytoplasm was diluted to 1/20 with Giemsa (Me
rk, D). The number of fused cells per square centimeter was obtained or otherwise relativized between the envelopes.

References 1. Andersen, KB1994.A domain of murine retrovirus surface protein gp
70 5 mediates cell fusion, as shown in a novel SC-1 cell fusion system.
J. Virol. 68: 3175-3182. 2. Battini, JL, O. Danos, and JM Heard. 1995. Receptor-binding domain
69.713-719. 3.Battini, JL, JMHeard, and O.Danos. 1992.Receptor choice determin
ants in the envelope glycoproteins of amphotropic, xenotropic, and polyt
ropic murine leukemia viruses. J. Virol. 66: 1468-1475. 4. Chou, PY, and GDFasman. 1978. Prediction of the secondary structu
re of proteins from their amino acid sequence.Annu.Rev.Biochem. 47: 45-1
48. 5. Cosset, F.-L., Y. Takeuchi, JLBattini, RAWeiss, and MKLCollins.
1995b.High titer packaging systems producing recombinant retroviruses
J. Virol. 69: 7430-7436. 6. Evans, LH, RPMorrison, FGMalik, J. Portis, and W. Britt. 1990.A
neutralizable epitope common to the envelope glycoproteins of ecotropic,
polytropic, xenotropic and amphotropic murine leukemia viruses.J. Virol
64: 6176-6183. 7. Fass, D., R. Davey, C. Hamson, P. Kim, J. Cunningham, and J. Berger. 1997. Structure of a murine leukemia virus receptor-binding glycoprotein at.
2.0 angstrom resolution. Science. 277: 1662-1666. 8. Fontenot, JD, N. Tjandra, C. Ho, PCAndrews, and RCMontelaro. 1994
.Structure and self assembly of a retrovirus (FeLV) proline rich neutra
lization domain. Journal of Biomolecular Structure and Dynamics. 11: 821-
837. 9. Holland, C., J. Wozney, and N. Hopkins. 1983. Nucleotide sequence of th
e gp70 gene of murine retrovirus MCF 247. J. Virol. 47: 413-420. 10. Hunter, E., and R. Swanstrom. 1990. Retrovirus envelope glycoproteins.
Curr.Top.Microbiol.Immun. 157: 187-253. 11. Jones, JS, and R. Risser. 1993. Cell fusion induced by the murine l.
eukaemia virus envelope glycoprotein. J. Virol. 67: 67-74. 12. Kozak, SL, DCSiess, MPKavanaugh, ADMiller, and D. Kabat. 1995.
The envelope glycoprotein of an amphotropic murine retrovirus binds spe
cifically to the cellular receptor / phosphate transporter of susceptible
species. J. Virol. 69: 3433-3440. 13. McClure, MO, MASommerfelt, M. Marsh, and RAWeiss. 1990. The pH
independence of mammalian retrovirus infection. J. Gen. Virol. 71: 767-773. 14. Miller, DG, RHEdwards, and ADMiller. 1994a. Cloning of the cel.
lular receptor for amphotropic murine retroviruses reveals homology to t
hat for gibbon ape leukemia virus.Proc.Natl.Acad.Sci.USA. 91: 78-82. . E
nvelope and long terminal repeat sequences of a cloned infectious NZB xe
notropic murine leukemia virus. J. Virol. 53: 100-106. 16. Ott, D., R. Friedrich, and A. Rein. 1990. Sequence analysis of amphotro.
pic and 10A1 murine leukemia virus: close relationship to mink cell focu
s forming viruses.J.Virol. 64: 757-766. 17.Pinter, A., T.-E.Chen, A.Lowy, NGCortez, and S.Siligari. 1986. Eco
tropic murine leukemia virus-induced fusion of murine cells.J. Virol. 57
: 1048-1054. 18. Ragheb, JA, H. Yu, T. Hofmann, and WFAnderson. 1995. The amphotrop
ic and ecotropic murine leukemia virus envelope TM subunits are equivale
nt mediators of direct membrane fusion: implications for the role of the
ecotropic envelope and receptor in syncytium formation and viral entry.
J. Virol. 69: 7205-7215. 19. Rein, A. 1982.Interference grouping of murine leukemia viruses: ad
istinct receptor for the MCF-recombinant viruses in mouse cells.Virolog
y. 120: 251-257. 20. Rein, A., J. Mirro, J. Haynes, S. Ernst, and K. Nagashima. 1994. Functio
n of the cytoplasmic domain of a retrovial transmembrane protein: p15E-p
2E cleavage activates the membrane fusion cability of the murine of the
murine leukemia virus env protein.J. Virol. 68: 1773-1781. 21. Riedel, N., E. Hoover, P. Gasper, M. Nicholson, and J. Mullins.
lecular analysis and pathogenesis of the feline aplastic anemia retrovir
us, feline leukemia virus C-Sarma.J.Virol. 60: 242-250.22.Sambrook, J., EFFritsch, and T.Maniatis.1989., 2nd ed. Cold Sprin
g Harbor Laboratory Press, New York. 23. Shinnick, TM, RALerner, and JGSutcliffe. 1981.Nucleotide sequ.
ence of Moloney murine leukemia virus.Nature (London). 293: 543-548.24.Skehel, JJ, PMBayley, EBBrown, SRMartin, MDWaterfield, JM.
.White, LAWilson, and DCWiley. 1982.Changes in the conformation of
influenza virus hemagglutinin at the pH optimum of virus-mediated membra
ne fusion.Proc.Acad.Natl.Sci.USA.79: 968-972.25.Takeuchi, Y., FLCosset, PJLachmann, H. Okada, RAWeiss, and MK.
L. Collins. 1994. Type C retrovirus inactivation by human complement is d
etermined by both the viral genome and producer cell.J.Virol. 68: 8001-8
007. 26. Urry, DW 1988. Entropic elastic processes in protein mechanisms. I
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7.1-34. 27.Valsesia-Wittmann, S., FJMorling, T. Hatziioannou, SJrussell, and ity due to internal chain dynamics.
F.-L.Cosset. 1997.Receptor co-operation in retrovirus entry: recruitme
nt of an auxilliary entry mechanism after retargeted binding.The Embo J
16: 1214-1223. 28. VanZeiji, M., SVJohann, E. Cross, J. Cunningham, R. Eddy, TBShows,
and B.O'Hara. 1994.An amphotropic virus receptor is a second member of
the gibbon ape leukemia virus receptor family.Proc.Natl.Acad.Sci.USA. 9
1: 1168-1172. 29. Weiss, RA, and CSTailor. 1995. Retrovirus receptors. Cell. 82:53
1-533. 30. White, JM 1992. Membrane Fusion. Science. 258: 917-924.

A. Cell-cell fusion test results

[0121]

[Table 1]

A: The fusion index was calculated according to the following formula: (N−S) / T (N, number of nuclei in fused cells; S, number of fused cells; T, total number of nuclei). b: uninfected cells c: fusion cells with more than 20 nuclei d: fusion cells with less than 4 nuclei e: fusion cells with more than 40 nuclei in XC cells and 10 in XCA-ST cells
Fusion cells containing less than nuclei.

B. Infection test results.

[0124]

[Table 2]

A: lacZ infectious units per mL (ui / mL) b: The level of interference was calculated by the following formula (intensity [ _XC ] / intensity [ _XC-A-ST ]) X (intensity MO [ _{XC-A -ST} ] / MO intensity [ _XC ])
It calculated according to. na: Not applicable

[Brief description of the drawings]

1A, 1B, 1C and 1D: Schematic representation of chimeric envelopes and their properties. FIG. 1A: Open squares are sequences from amphotropic MLVs, filled squares are ecotropic Moloney MLV (black) or ecotropic friend M
The sequence obtained from the LV (ash) and the shaded squares are the sequences common to these two MLVBDs, the binding domain for the receptor, PRO is the proline-rich region, C is the carboxy of SU. Terminal domain, TM is T
The ectodomain and the anchor domain of the M subunit. For each domain,
The first 4 amino acids at the N-terminus and their percent identity are shown. An early stop codon (vertical arrow) is introduced into the cytoplasmic tail of the TM subunit of the amphotropic MLV, which produces the Arless fusogenic mutant envelope glycoprotein.

1A, 1B, 1C and 1D: Schematic representation of chimeric envelopes and their properties. FIG. 1B: Simple substitution mutant.

1A, 1B, 1C and 1D: Schematic representation of chimeric envelopes and their properties. FIG. 1C: Combination substitution mutant.

1A, 1B, 1C and 1D: Schematic representations of chimeric envelopes and their properties. FIG. 1D: Cell-cell fusion test results. (-) Absence of fusion cells, (+) XC
Presence of fused cells only in co-culture with E. coli, (++) cells, ie, presence of fused cells in co-culture with XC A-ST interfering cells as well as XC rat cells. Infection test results: (-) less than 10 ² lacZ ui / mL; (+) and (++) more than 10 ² lacZ ui / mL, (++) XC-A-ST with a strength of more than 10 ² lacZ ui / mL. An envelope with a lower fusion activity that the interfering cells can effectively intercept.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

FIG. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I: Fusion test of simple substitution mutants of XC cells. TE671 cells transfected with the indicated expression vector of the chimeric envelope glycoprotein (FIG. 1) were co-cultured with XC indicator cells for 24 hours. Proliferation, 250-fold.

3A, 3B, 3C and 3D: amphotropic proline rich region 4070
Mutagenicity of A. Figure 3A: Moloney MLV (Mo), Friend (Fr) and 4070A (A) P
FIG.

3A, 3B, 3C and 3D: amphotropic proline-rich region 4070
Mutagenicity of A. Figure 3B: Selective mutant (lowercase bold amino acid) or deletion (dash) at the C-terminus of the MLV4070A PRR.

3A, 3B, 3C and 3D: amphotropic proline-rich region 4070
Mutagenicity of A. FIG. 3C: Selective mutant at the C-terminus of MLV4070A PRR. Modified or inserted amino acids are highlighted in lower case bold.

3A, 3B, 3C and 3D: amphotropic proline-rich region 4070
Mutagenicity of A. 3D, 3E, 3F, 3G: MLV 4070A (4070A), Moloney (Mo
loney) and MLR4070A PRR selective mutants, the possible profiles of PRR "beta turns", the second (A2) or third (A3) beta turns have been deleted. The y-axis value represents the probability of beta turn p (turn) * 10 ^-4 for each peptide residue (on the x-axis). Beta turn analysis was performed using PC / Gene software (version 6.26. IntelliGenetics, Belgium).

FIGS. 3A, 3B, 3C and 3D: amphotropic proline-rich region 4070.
Mutagenicity of A. 3D, 3E, 3F, 3G: MLV 4070A (4070A), Moloney (Mo
loney) and MLR4070A PRR selective mutants, the possible profiles of PRR "beta turns", the second (A2) or third (A3) beta turns have been deleted. The y-axis value represents the probability of beta turn p (turn) * 10 ^-4 for each peptide residue (on the x-axis). Beta turn analysis was performed using PC / Gene software (version 6.26. IntelliGenetics, Belgium).

3A, 3B, 3C and 3D: amphotropic proline-rich region 4070
Mutagenicity of A. 3D, 3E, 3F, 3G: MLV 4070A (4070A), Moloney (Mo
loney) and the possible profile of the PRR “beta turn” of the PRR selective mutant of MLV4070A, the second (A2) or third (A3) beta turn has been deleted. The y-axis value represents the probability of beta turn p (turn) * 10 ^-4 for each peptide residue (on the x-axis). Beta turn analysis was performed using PC / Gene software (version 6.26. IntelliGenetics, Belgium).

FIGS. 3A, 3B, 3C and 3D: amphotropic proline rich region 4070
Mutagenicity of A. 3D, 3E, 3F, 3G: MLV 4070A (4070A), Moloney (Mo
loney) and MLR4070A PRR selective mutants, the possible profiles of PRR "beta turns", the second (A2) or third (A3) beta turns have been deleted. The y-axis value represents the probability of beta turn p (turn) * 10 ^-4 for each peptide residue (on the x-axis). Beta turn analysis was performed using PC / Gene software (version 6.26. IntelliGenetics, Belgium).

FIG. 4 is an immunoblot of an amphotropic envelope glycoprotein with improved fusogenic performance. Cells were transfected with the envelopes shown in FIGS. 1 and 3, and the expression of their SU and their envelope precursor (PR) was calculated (lysed cells). Envelope instability was demonstrated by expression of SU-reduced surfaces relative to PR, expansion of SU (split) accumulated in culture (supernatant), and virions. Protein transfer (Western blot) was indicated with the indicated antibodies.

FIG. 5 Ecotropic MLV (BD, PRO, C and TM domains) (Moloney strain)
1 shows the sequence of the retrovirus envelope glycoprotein of FIG.

FIG. 6: Amphotropic MLV (BD, PRO, C and TM domains) (strain 407)
2A shows the sequence of the retroviral envelope glycoprotein of 0A).

FIG. 7 shows the proline-rich region of the retroviral envelope glycoprotein of Moloney MLV (Moloney MLV).

FIG. 8 shows the proline-rich region of the retroviral envelope glycoprotein of MLV-4070A.

FIG. 9 shows the proline-rich region of the retroviral envelope glycoprotein of MLV-MCF (strain 247).

FIG. 10 shows the proline-rich region of the Seato strain GALV retrovirus envelope glycoprotein.

FIG. 11 shows the proline-rich region of the retroviral envelope glycoprotein of MLV 10A1.

FIG. 12 shows the strain NZB. 1. 3 shows the proline-rich region of the retroviral envelope glycoprotein of the V6 xenotropic MLV.

FIG. 13 shows the proline-rich region of the retroviral envelope glycoprotein of FeLVA.

14 shows an A ₂ sequence.

15 shows an A ₃ sequence.

FIG. 16 shows the A ₃ Mo sequence.

Figure 17 shows the A ₄ sequence.

FIG. 18 shows the C ₂ sequence.

Figure 19 shows the C ₃ sequence.

Figure 20 shows the C ₄ sequence.

21 shows a C ₅ sequence.

FIG. 22 shows a PROMO arrangement.

FIG. 23 shows the PRO FR sequence.

FIG. 24 shows a BD PRO CMO sequence.

FIG. 25 shows the PRO CMO sequence.

FIG. 26 shows the PRO CTM MO sequence.

FIG. 27 shows the PRO ™ MO sequence.

[Procedure amendment]

[Submission date] October 16, 2000 (2000.10.16)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Fig. 1A

[Correction method] Change

[Correction contents]

1A, 1B and 1C: Schematic representation of chimeric envelopes and their properties. FIG. 1A: Open squares are sequences from amphotropic MLVs, filled squares are ecotropic Moloney MLV (black) or ecotropic friend M
The sequence obtained from the LV (ash) and the shaded squares are the sequences common to these two MLVBDs, the binding domain for the receptor, PRO is the proline-rich region, C is the carboxy of SU. Terminal domain, TM is T
The ectodomain and the anchor domain of the M subunit. For each domain,
The first 4 amino acids at the N-terminus and their percent identity are shown. An early stop codon (vertical arrow) is introduced into the cytoplasmic tail of the TM subunit of the amphotropic MLV, which produces the Arless fusogenic mutant envelope glycoprotein. It is a cell-cell fusion test result. (-) Absence of fusion cells, presence of fusion cells only in coculture with (+) XC, (++) cells, ie, fusion cells in coculture with XCA-ST interfering cells as well as XC rat cells Existence. Infection test results: (
−) Less than 10 ² lacZ ui / mL; (+) and (++) 10 ² lacZ ui /
mL ^{exceeded, (++) 10 2 lacZ ui} / mL exceeded XC-A-ST interfering cells having strength can be effectively interfere fusion activity lower envelope.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Fig. 1B

[Correction method] Change

[Correction contents]

1A, 1B and 1C: Schematic representations of chimeric envelopes and their properties. FIG. 1B: Simple substitution mutant. It is a cell-cell fusion test result. (-) Absence of fused cells, presence of fused cells only in co-culture with (+) XC, (++) cells, ie, XC rat cells as well as X
Presence of fused cells in co-culture with CA-ST interfering cells. Infection test results: (-
) Less than 10 ² lacZ ui / mL; (+) and (++) 10 ² lacZ ui / mL
Exceed, envelope of lower fusion activity that XC-A-ST interfering cells with a strength of more than (++) 10 ² lacZ ui / mL can effectively interfere.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] Fig. 1C

[Correction method] Change

[Correction contents]

1A, 1B and 1C: Schematic representations of chimeric envelopes and their properties. FIG. 1C: Combination substitution mutant. It is a cell-cell fusion test result. (-) Absence of fused cells, presence of fused cells only in co-culture with (+) XC, (++) cells, ie, XC rat cells as well as X
Presence of fused cells in co-culture with CA-ST interfering cells. Infection test results: (-
) Less than 10 ² lacZ ui / mL; (+) and (++) 10 ² lacZ ui / mL
Exceed, envelope of lower fusion activity that XC-A-ST interfering cells with a strength of more than (++) 10 ² lacZ ui / mL can effectively interfere.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] Fig. 1D

[Correction method] Deleted

[Procedure amendment 5]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1D

[Correction method] Deleted

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor LaVillette, Dimitri F-10290, France Bersnay-le-Aye, Rue de Bushbourg 8 F-term (reference) 4B024 AA20 BA80 CA04 DA02 EA04 GA11 HA01 4H045 AA10 AA20 BA10 BA53 CA01

Claims (14)

[Claims] 1. Use of a proline-rich sequence to improve the fusogenicity of a retroviral envelope. 2. A retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV10A1, GA
A proline-rich sequence for improving the fusogenicity of a protein comprising the retroviral envelope glycoprotein of LV, the retroviral envelope glycoprotein of xenotropic MLV, the retroviral envelope glycoprotein of MLV MCF or the retroviral envelope glycoprotein of FeLV ( Wherein the proline-rich sequence is:-a retroviral envelope glycoprotein of amphotropic MLV,-a retroviral envelope glycoprotein of MLV MCF,-a retroviral envelope glycoprotein of MLV 10A1, or a GALV retrovirus. Envelope glycoprotein, xenotropic MLV retrovirus envelope glycoprotein, or FeLV retrovirus d Contains at least one mutation of the kind corresponding to the proline-rich sequence of the envelope glycoprotein and having at least one β-turn suppression in the polyproline helix of the proline-rich sequence; or 3. The use of claim 1, wherein the proline-rich sequence of a viral envelope glycoprotein corresponds to the proline-rich sequence of a viral envelope glycoprotein. 3. The use according to claim 1, wherein the proline-rich sequence cooperates with a functional domain of an ecotropic MLV retroviral envelope glycoprotein selected from a linking domain, a C domain or a transmembrane subunit. 4. An ecotropic MLV retroviral envelope glycoprotein that cooperates with a functional domain of an ecotropic MLV retroviral envelope glycoprotein selected from a linking domain, a C domain, or a transmembrane subunit domain. Use according to claim 1, of a proline-rich sequence corresponding to the proline-rich sequence of 5. A proline-rich sequence for improving the fusogenicity of a protein comprising a retroviral envelope glycoprotein of MLV, wherein the proline-rich sequence comprises:-a proline of an amphotropic MLV retroviral envelope glycoprotein. Contains at least one mutation of the kind that corresponds to the abundant sequence and is a suppression of at least one β-turn in the polyproline helix of the proline-rich sequence; or-a retroviral envelope sugar of ecotropic MLV The proline-rich sequence of the protein or the proline-rich sequence of the protein). 6. The amino acid sequence and the secondary structure of the envelope glycoprotein of amphotropic MLV, the retrovirus envelope glycoprotein of MLV 10A1, the retrovirus envelope glycoprotein of MLV MCF, GA
At least one of the proline-rich sequences of the retroviral envelope glycoprotein of LV, the xenotropic MLV retroviral envelope glycoprotein or the retroviral envelope glycoprotein of FeLV, and A proline-rich sequence comprising at least one mutation on the side of the C-terminal part of the kind such that there is β-turn suppression. 7. The amino acid sequence and secondary structure of the envelope glycoprotein of amphotropic MLV, the retrovirus envelope glycoprotein of MLV 10A1, the retrovirus envelope glycoprotein of MLV MCF, and GA
At least one of the proline-rich sequences of the retroviral envelope glycoprotein of LV, the xenotropic MLV retroviral envelope glycoprotein or the retroviral envelope glycoprotein of FeLV, and in the polyproline helix of the proline-rich sequence A proline-rich sequence comprising at least one mutation on the N-terminal part of the type such that there is β-turn suppression. 8. A retroviral envelope glycoprotein of amphotropic MLV, a retroviral envelope glycoprotein of MLV 10A1, M
Retroviral envelope glycoprotein of LV MCF, retroviral envelope glycoprotein of GALV, retroviral envelope glycoprotein of xenotropic MLV or retroviral envelope glycoprotein of FeLV (where the proline-rich domain is a polyproline helix with a proline-rich sequence) Mutein protein comprising at least one mutation of the type in which there is at least one β-turn suppression. 9. The envelope glycoprotein of the amphotropic MLV, M
Retroviral envelope glycoprotein of LV 10A1, retroviral envelope glycoprotein of MLV MCF, retroviral envelope glycoprotein of GALV, retroviral envelope glycoprotein of xenotropic MLV or retroviral envelope glycoprotein of FeLV (where the proline-rich domain Is replaced by the proline-rich domain of the retroviral envelope glycoprotein of ecotropic MLV). 10. An envelope glycoprotein of an amphotropic MLV,
Retroviral envelope glycoprotein of MLV 10A1, MLV MCF
Retrovirus envelope glycoprotein of GALV, retrovirus envelope glycoprotein of xenotropic MLV, or retrovirus envelope glycoprotein of FeLV (where * the proline-rich domain is the retrovirus envelope glycoprotein of ecotropic MLV (A linking domain, C domain, or transmembrane subunit has been replaced by the corresponding domain of the ecotropic MLV envelope). 11. An envelope glycoprotein of an amphotropic MLV,
Retroviral envelope glycoprotein of MLV 10A1, MLV MCF
Retroviral envelope glycoprotein of GALV, retroviral envelope glycoprotein of xenotropic MLV or retroviral envelope glycoprotein of FeLV (where * the proline-rich domain has at least one β-turn ( A linking domain, C domain, or transmembrane that contains at least one mutation of some type (in the polyproline helix of the proline-rich sequence) and is replaced by the corresponding domain of the ecotropic MLV envelope Comprising at least one functional domain selected from subunits). 12. The following characteristics:-a target cell expressing a surface molecule acting as a retrovirus receptor by the interaction of a linking domain carried by a retrovirus envelope glycoprotein, which has been previously transfected with an expression vector of one of said proteins. MLV (or C-type mammalian retrovirus or lentivirus)
Forming a fusion cell after co-cultivation with a cell derivative that produces or does not produce Gag-Pol particles, the infectivity of a virus containing an envelope containing a non-mutated or non-chimeric envelope of this receptor. In a limiting condition, which reduces the infectivity of a virus comprising a chimeric or mutated envelope by at least 100-fold, and reduces the infectivity of a virus comprising a chimeric or mutated envelope by less than 100-fold. There is an infection of target cells that express the viral receptor (where the above values applied to these infectivity values are not mutated or chimeric envelopes, or chimeric or mutated envelopes on target cells, eg, XC cells). Given by comparison with the infectivity of viruses, including envelopes) Both have a 1, set forth in any one mutation or chimeric protein of claim 8 to 11. 13. The proline-rich sequence according to claim 6, which corresponds to one of the following sequences: A ₂ A ₃ A ₃ Mo A ₄ C ₂ C ₃ C ₄ C ₅ . 14. The method according to claim 8, which corresponds to one of the following sequences: PROMO-PROFR-BDPROMO-PROCMO-PROTMMO-PROCTMMO. Chimeric muteins.