EP0513116A1 - Self-inactivating bird vector - Google Patents

Abstract

The subject of the present invention is a self-inactivating viral vector for integration and expression of at least one heterologous gene in avian cells, characterized in that it is constituted from the proviral genome of an avian retrovirus and which 'it comprises between two avian LTR sequences the said heterologous gene (s) which is (are) under the control of a heterologous internal promoter, deletions or mutations in the LTR 3' having been carried out from so as to inactivate the viral promoters present in the LTRs.

Description

 AVIAN SELF-ACTIVATING VECTOR

The present invention relates to new retroviral vectors, of avian origin, of integration and expression of at least one heterologous gene in avian cells.

 The present invention constitutes a development of the inventions described in European patent applications EP 178 996 and international patent WO 89/03877 in the name of the present applicant. It should be referred to if necessary for the best understanding of the present invention.

 By heterologous gene is meant a gene which is not found in the original avian genome and preferably which is not found in the genome of a virus. It may be a useful gene coding for a protein of industrial interest intended to be obtained by cell culture or else a gene which may be of particular interest for breeding birds, in particular chickens or quails, or a gene coding for an immunogenic protein intended to serve as a vaccine, or it may be a marker gene in particular for resistance to an antibiotic.

 Retroviruses are now widely used for gene transfer because of their high efficiency in both their penetration through the cell membrane and the integration of their genome into the DNA of the host cell. Of particular interest are retroviral vectors in which the inserted foreign sequences are transcribed directly under the control of internal promoters. They allow efficient translation of TARN transcribed as a native protein. However, there are functional interferences between the viral and internal promoters.

This is why a new generation of avian retroviral vectors has been developed according to the present invention. These vectors carry mutations in their terminal "long repeated sequences" (so-called LTR sequences) which inactivate transcription of the integrated provirus without affecting the expression of internal promoters. These vectors are called SIN ("self inactivating"), that is to say self-inactivating.

 The article by Dougherty and Temin 1987, PNAS 84, 1197-1201, describes a self-inactivating vector constructed from the SNV turkey virus which can only be amplified by the line D-17-Rev-A, which in addition only produces infectious virions in turkey and dog cells, the infection efficiency in other avian species being very low.

 More specifically, the subject of the present invention is a viral vector for integration and expression of at least one heterologous gene in avian cells, constituted from a proviral genome originating from avian AEV retroviruses and related avian retroviruses in particular of RAV or RSV type and comprising between two avian LTR sequences the said heterologous gene (s) which is (are) under the control of heterologous internal promoter (s), characterized in that deletions or mutations in the 3 'LTR have been carried out so as to inactivate the viral promoters present in the LTRs, said 3' LTR coming from the retroviruses RAV-0, RAV-1, RAV-2.

 The structural modifications of the viral genome made by the vectors according to the invention totally inactivate the viral promoters present in the LTRs of the proviral forms integrated into the target cells of the transfer.

 By heterologous internal promoter is meant a promoter which is not found naturally in the genome of an avian retrovirus and which has been placed between the two LTR sequences.

 In one embodiment according to the invention, the promoter or the enhancer sequence, as the case may be, of the 3 ′ LTR of the viral genome are deleted in whole or in part. The virus originating from this genome introduces into the genome of the infected cells a provirus whose LTR 5 ′ is copied onto LTR 3 ′ according to the conventional process of synthesis of proviral DNA from virionic RNA.

More specifically, a deletion is carried out in the U3 region of the 3 'LTR. In particular, it is possible to carry out point mutations or deletions in the CAAT and TATA boxes or to eliminate said boxes.

 Since the U3 region of the 3 'LTR serves as a matrix for the synthesis of the corresponding region of the 5' LTR in reverse transcription, any mutation in U3 in the 3 'LTR appears in both LTRs after a replication cycle viral. This is why the mutation which destroys the promoter of the LTR 3 'gives rise to a provirus which is inactive in the infected cells. It has been observed, however, that transcription from an internal promoter can still take place.

 If we use a 3 'LTR from a virus whose

LTRs are naturally very little active, so it suffices to carry out specific deletions or mutations to inactivate the viral promoters present in LTRs. As the LTR 3 *, which naturally has very little activity, that of the RAV-0 virus can be mentioned.

The deletions in the 3 'LTR influence not only the inactivation of the vectors but also the efficiency of the transfer in the cells, which is why, according to another characteristic of the present invention, the vectors carry in 5' the LTR of the R5V virus. (Rous sarcoma virus). This LTR has a very high transcriptional activity, notably higher than that of the RAV-1 and RAV-2 viruses. These constructs improve the transcription of the viral genome during the transfection of heiper cells and therefore increase the number of viral particles produced.

 In the vectors according to the invention, the heterologous genes

(selection genes or useful gene) are suitably under the control of internal heterologous promoters. However, in the case of the selection gene, this can be directly animated by the 5 'LTR and not associated with its own internal promoter, the useful heterologous gene being under the control of heterologous internal promoters.

In the genomes of the INS vectors according to the invention, it is possible to integrate transcriptional units in which the heterologous gene is associated with its own promoter in its original genome, as a heterologous internal promoter. In infected cells, only these internal promoters will then be functional.

 The correct functioning of these vectors has been checked and the results are presented below.

 On the one hand, the expression of the inserted genes is initiated at the level of their respective internal promoters. On the other hand, in infected cells the vector LTRs become inactive. Finally, the vectors cannot come out of the infected cells, even after infection of the latter with a competent helper virus.

 The vectors according to the invention therefore provide great safety in use since, in the infected cells on the one hand, the inactive LTRs cannot alter the functioning of the genetic sequences adjacent to the insertion site of the provirus, and on the other hand the provirus can no longer generate a transmissible virus.

 In a particular embodiment, the vectors were constructed from genome elements derived from the RAV-1, RAV-2 and / or AEV retroviruses with regard to the part between the two LTRs.

 Mention may in particular be made of vector constructs constituted essentially from the genome of the RAV-2 retrovirus in which the CAAT and TATA boxes have been deleted in the 3 ′ LTR of said genome. In one embodiment, the vector comprises as LTR 3 ′:

- the U ₃ sequence of the RAV-2 LTR in which the boxes

CAAT and TATA have been deleted, and

- the R and U ₅ sequences of the RAV-1 LTR.

Suitably, the vectors according to the invention comprise two heterologous genes, namely a selection gene and a useful gene.

 When the vector is constructed from the genome of the avian erythroblastosis retrovirus (AEV), the heterologous genes are advantageously placed in positions of the oncogenes v-erbA and v-erbB.

By selection gene is meant a resistance marker gene, in particular resistance to a drug analogous to an antibiotic. Mention may in particular be made of the hygro ^R gene for resistance to hygromycin G418 and the neo ^R gene for resistance to neomycin. Of course, the marker genes which have been cited are only by way of purely illustrative examples. It is possible to use any other resistance gene as a marker gene.

 By useful gene is meant a gene coding for a protein of interest other than a selection gene.

 In an appropriate embodiment, the heterologous genes are placed under the control of heterologous internal promoters, in particular the simier promoter SV40 or human metallothionein MTIIa.

 In an embodiment of the vectors according to the invention, the selection gene is placed under the control of the simian promoter SV40 and the useful heterologous gene is placed under the control of the human metallothionein promoter lIa (MTIIa).

 In another embodiment, the selection gene is directly animated by the 5 ′ LTR and not associated with a specific promoter.

 The direction of transcription of a heterologous gene inserted into the vector must be opposite to that of the LTR in the case where said heterologous gene contains a poiyadenylation control sequence. Otherwise, the heterologous gene may be inserted in the direction of transcription of the LTR, the transcript of said gene then using the polyA signal supplied by the 3 'LTR. In the case where the inserted heterologous gene contains introns, this gene must necessarily be inserted in the reverse transcription direction, in order to avoid any interference between the splicing control signals of this gene and those of the viral genome.

 In one embodiment, the selection gene is inserted in the direction of retroviral transcription and the useful gene is inserted in the opposite direction to that of retroviral transcription.

The gene encoding histone H5 has been used as an example of a useful heterologous gene and it has been shown that the expression of H5 in normal or transformed quail or chicken fibroblasts has not significantly affected the properties of transformed cell growths, but inhibited the proliferation of normal fibroblasts. According to another example, the heterologous gene is the E. Coli gene coding for beta-gaiactosidase.

 In particular, vectors carrying a 3 'LTR derived from the RAV-0 virus, naturally devoid of enhancer sequences, have been constructed in which the CAAT box is deleted:

 These vectors carry the following sequences from 5 'to 3':

- a 5 'LTR of the RSV virus,

 - a leader sequence of the RAV-1 virus,

- a resistance gene, such as Neo ^R ,

a heterologous gene of interest, such as the E. Coli gene coding for a beta-galactosidase provided with a nuclear targeting signal, under the control of the promoter of the 5V40 virus,

 - the 3 'non-coding sequence and the 3' LTR of the RAV-0 virus deleted from the CAAT box.

 These vectors have been tested and are functional and offer maximum safety for inactivation since the 3 'RAV-0 LTR has been deleted in the CAAT promoter sequence.

 Given that avian retroviruses are RNA viruses, virus terminology may sometimes refer to the proviral genome in the form of DNA, as in some cases the foreign gene may be in the form of RNA, particularly when is inserted into the genome of a virion. The techniques allowing to pass from a DNA form to the RNA form and vice versa, are known and are transcription and reverse transcription. The recombinant virus can be used as such in the form of RNA virions or of a proviral genome in the form of DNA comprising the insertion of a foreign gene also in the form of DNA and corresponding to the reverse transcription of the virus's RNA, may be integrated into a DNA vector such as a plasmid, a cosmid, or a phage for example.

For reasons of convenience, the constructions were firstly carried out on plasmids comprising a composite DNA sequence corresponding to the reverse transcript of a retroviral RNA vector of a virus as described above. The present invention also relates to a process for the preparation of a vector according to the invention, characterized in that deletions or mutations are carried out in the avian LTR 3 ′ so as to inactivate the viral promoters present in said LTRs and in that one inserts said heterologous gene (s) under the control of a heterologous internal promoter or of the promoter of the LTR 5 ′.

 In one embodiment of the method, a transcriptional cassette for a heterologous gene is inserted into the vector pDA1 at the Xbal site.

 For example, the transcriptional cassette for the heterologous gene consists of the vector pBOB as represented in FIG. 6 in which said heterologous gene is inserted at the restriction sites of the poiylinker comprised between the transcription initiation sequence in the form of the MTIIa promoter and the transcription termination and polyadenylation sequence derived from the histone H5 gene, said cassette being framed by two Xbal sites allowing its isolation and then its insertion at the unique Xbal site of the vector pDA1.

 The present invention further relates to a method of modifying cells characterized in that a transfection or infection is carried out, as the case may be, of said cells with vectors according to the invention.

The present invention therefore relates to cells transfected or infected with the vectors according to the invention, whether they are in the form of a plasmid or of a virus respectively. It is obvious that the primary transfection will generally be carried out with plasmid type vectors, that is to say that the avian retrovirus will be in the form of the DNA of its genome but in the presence of the DNA of a virus. assistant of the transfected cells will in this case transform and release the recombinant viruses which will be able to transfect other cells. This type of process also makes it possible to build up stocks of vector viruses which can subsequently be used in place of the plasmid vectors. Retroviruses such as AEV, RAV-1, RAV-2 are defective for their replication. To replicate, these viruses require the presence in the same host cell of a functional helper virus as is known to those skilled in the art.

 The present invention also relates to a method for modifying cells, characterized in that co-infection or co-transfection is carried out, as the case may be, with an assistant virus which comprises the genes ensuring the replication of said vector.

 The cells modified according to the invention can be diverse but are preferably avian cells, of hematopoietic, germinal or fibrobiastic type.

 The preferred cells for implementing the vectors of the invention are the chicken or quail embryo fibroblasts. However, the vectors according to the invention make it possible to modify cells of turkey, duck and other birds.

 Finally, the invention relates to the process for the preparation of a specific protein in which cells modified by a vector according to the invention are cultivated in which a foreign gene codes for said protein and in that the protein from the cellular culture.

 Other advantages and characteristics of the present invention will appear in the light of the detailed description of an embodiment given purely by way of illustration and as a model for the integration and expression of a heterologous gene, using the gene. histone H5.

The following description is made with reference to FIGS. 1 to 8. Figure 1 Diagram illustrating the transfer of the deletion from 3 'LTR to 5' LTR. (1) A retroviral vector deleted in the 3 'LTR promoter and carrying a foreign gene (X) with its own promoter (P), is transfected into helper cells. (2) After integration, the proviral DNA is transcribed from the 5'LTR promoter. and the viral transcripts containing the deletion in the U3 region are packaged in viral particles

infectious. (3) In cells infected with these helper-free viral particles, the viral RNA is transcribed by reverse transcription into a double-stranded DNA molecule showing a deleted U3 region in the two LTRs. Thus, in these cells, viral transcription is abolished and only the

transcription from the internal promoter can take place. The triangle induces deletion; hatched box: region U3, dotted box region U5; clear box: region R

Figure 2 (A). Structure of normal LTRs (top) and deletions (bottom). The localization of the transcriptional regulation signals is indicated. The hatched and pomtilla areas in the U3 region represent the regions of the enhancer and of the promoter respectively. The deleted U3 region (originating from the proviral DNA RAV-2) has been linked, by the ClaI linkers, to the R and U5 regions of the RAV-1 LTR. The dotted triangle indicates the extent of the deletion. (B) Structure of retroviral vectors. In pTXN3 ', derived from AEV, the neomycin resistance gene (neo) is transcribed from the viral LTR, whereas in pDAl, it is transcribed from the internal SV40 promoter (SV) pDAl, essentially derived from RAV -2. contains a U3 region deleted in the 3'LTR pDAHY and pDAH5 were derived from pDAl by inserting the genes nph (resistance to hygromycin) and K5 respectively. The arrows indicate the transcriptional direction of the different genes. In pDAHY. the region coding for hph, linked to polyadenylation site of the thymidine kinase (Tk) gene, was placed under the control of the SV40 promoter. In pDAH5, the human metallothioneine promoter MT-II _A (MT) was used to transcribe the H5 gene containing its own signal.

polyadenylation (Pol H5). Hatched, clear and domed boxes: LTR from AEV respectively. RAV-2 and RAV-1; SD and SA:

splice donor and acceptor respectively; ΔU3: deleted U3 region; Xo: Xhol; X: Xbal; C: Clal. The scale (in pairs of kilobases: kb) is indicated below.

Figure 3 Southern blottmg analysis of QT6 cells infected with DAH5. 15 μg of DNA, isolated from infected cells (bands 5 and 7) and not infected (bands 4 and 6), were digested with Xbal (bands 4 and 6) or Xhol (bands 6 and 7). After electrophoresis and transfer to nitrocellulose, the transfer range ("blot") was hybridized with a specific H5 probe. The amount of restriction fragment corresponding to 10, 1 and 0.1 copies of H5 gene per genome (bands 1, 2 and 3 respectively) were processed in parallel. Lambda DNA digested with EσoRI and HindIII was used as a size marker. A schematic representation of the DAH5 restriction map is shown at the bottom of the figure

Figure 4 S1 nuclease protection assay of cytoplasmic RNA transcribed in QT6 cells. 20 µg of cytoplasmic RNA. isolated from uninfected cells (lane 1) and cells infected with DAH5 (lanes 2 and 5), were hybridized with the H5 probe In lanes 3 and 4, the cells were treated with 50 mM and 100 mM respectively ZnClo, before isolation of RNA. In lane 5, infected cells were maintained for 50 generations without selection by G418 before extraction. A 1025 bp fragment isolated from pMTH5 and representing the H5 probe (band 6) protected 315 nucleotides after S1 digestion The figures in the left margin

represent the sizes, in nucleotides, of the fragments used as molecular weight markers Figure 5: Detection by Western blotting of H5 in infected QT6 cells. The nuclear proteins, isolated from QT6 cells infected with DAH5 (bands 1, 2 and 3) or from uninfected QT6 cells (bands 4, 5 and 6), were subjected to electrophoresis on polyacrylamide-SDS gel transferred to nitrocellulose. H5 was detected after incubation with anti-H5 antibodies and protein A ^-125 I. The cells were treated with

100 mM ZnCl ₂ (bands 2 and 5). Lanes 7, 8 and 9 represent the equivalent of 0.5 μg, 1 μg and 2 μg of purified H5, and bands 10, 1 1 and

12 show the amount of H5 present in 5.10, 10.10 and 20.10 ⁶ chicken erythrocytes.

 Figure 6: represents pBOB. This plasmid was constructed from the H5 transcription cassette containing the MTIIA promoter, the H5 coding sequence and the polyA sequence. The H5 coding sequence was eliminated by EcoRI + SmaI digestion and replaced by a synthetic oligonucleotide shown in FIG. 6. one can consider replacing the promoter

MTIIA by another promoter.

 Figure 7: Structure of OVA retrovirus vectors. The black triangles indicate the positions of the internal transcription promoters, while the white triangles indicate the position of the U3 deletions. The presence or absence of an enhancer element, a CAAT box and a TA TA box in each downstream LTR is indicated on the right.

Figure 8: represents the construction of OVA type vectors.

 EXAMPLE 1.

 The transfer and expression of histone H5 were carried out in normal avian fibroblasts and transformed by a retroviral SIN eur vect. The vector, called pDAH5, was obtained by mutation in the 3 'LTR by eliminating the CAAT and TATA boxes. The H5 coding sequence inserted into pDAH5 was controlled by the human metallothionein MT-IIA promoter.

Infection of QT6 quail cells with the DAH5 virus led to the integration of a copy of a foreign H5 gene by genome and to the formation of correctly initiated mRNA and H5 protein transcripts. However, the expression of the exogenous H5 sequence was constitutive and not inducible by Zn ⁺⁺ ions. The level of H5 in QT6 cells was equivalent to that of a mature chicken erythrocyte. Expression of H5 in DAH5 infected QT6 cells or AEV-ES4 transformed chicken embryo fibroblasts had only slight effects on growth rate and did not inhibit cell replication. Conversely, the expression of H5 in normal quail and chicken fibroblasts was spectacular: the cells acquired the appearance of senescent fibroblasts, they grew very slowly and the nuclei appeared compact, they were often devoid of cytoplasm.

 Analysis of the post-translational modification of H5 in QT6 cells has shown that phosphorylation may be involved in the differential effects of the protein in normal and transformed cells.

I - MATERIAL AND METHODS

1. Construction of a retroviral vector for the transfer and expression of the coding sequence for H5

 The principle of the SIN vectors is illustrated in FIG. 1. The construction of the vector pTXN3 'has been published recently (3 and WO 89/03877). The genome of pDAl is derived from the genome of the virus associated with Rous-2 sarcoma (RAV-2) after modification of the 3 'LTR.

 A mutated avian LTR was constructed by deletion of the U3 region isolated from the avian leukosis virus RAV-2 LTR between nucleotides -149 and -15 (opposite the transcriptional cap site: nucleotide

+ 1). That is, the sequence downstream of the Sphi site in U3 has been deleted. This deletion eliminated the CAAT and TATA boxes but left intact the enhancer region located upstream of position -149 (6,16, 22). The deleted RAV-2 U3 region was then linked to the R and U5 regions isolated from the RAV-1 LTR. That is, the detected sequence of U3 has been replaced by the Taql-Sstl fragment derived from the LTR of RAV-1. This fragment essentially contains the R and U5 sequences and 14 residual U3 nucleotides. The iinker Clal was inserted at the junction of the fragments derived from RAV-1 and RAV-2. The hybrid LTR therefore has a deletion of 134 nucleotides which has eliminated the CAAT and TATA boxes. The deleted LTR thus reconstituted was used to replace the normal 3 'LTR in the vector pDAl. The vector pDAl was constructed from RAV-2 and it carries a transcription cassette containing the neo gene linked to SV40 early promoter inserted in the same direction of transcription of viral transcription (Figure 3). More specifically, the internal Xhol fragmen of the RAV-2 genome was eliminated and replaced by a cassette containing, in order, gag sequences thus carrying Xbal, the enhancer and early promoter of the simian virus SV40 and the sequence coding for the gene. bacterial neomycin phosphotransferase (neo) (25).

 As a control virus, the pDAHY genome derived from pDAl was constructed by inserting a cassette containing the sequence coding for hygromycin B phosphotransferase (hph) between the early promoter of 5V40 and the poiyadenylation signal of H5V Tk. This transcription cassette was inserted in the opposite direction with respect to the neo gene. More specifically, the vector pDAHY was derived from pDAl by inserting at the Xbal site a cassette containing the sequence coding for bacterial hygromycin B phosphotransferase (hph) (11). This gene was put under the control of the enhancer and the early promoter of SV40. The polyadenylation signal of the herpes virus (HSV) thymidine kinase (Tk) gene was added to the 3 'end.

 As an additional control, the pTXN3 'virus, a vector which expresses the neo gene of the viral LTR, was used in parallel.

 The genome of the vector pDAH5 was constructed from pDA1 by inserting a transcription cassette containing the sequence coding for H5 linked to the promoter of the gene for human metallothionein MT-IIA. In this construct, the H5 sequence provides its own polyadenylation signal and it has been inserted in an anti-sense direction relative to retroviral transcription. More specifically, the pDAH5 genome was constructed from pDAl by inserting at Xbal a cassette containing the sequence coding for H5 with its own poiyadenylation signal linked to the MT-IIA promoter (13). The H5 transcription unit as the hph gene transcription cassette was inserted in the opposite direction to the LTR-controlled transcription.

All of these retroviral genomes were transfected into the helper G cell line which released only retroviral vectors devoid of helper free viruses (24). The helper free DAH5 titer was 100 rfu / ml (resistance forming unit / ml) as determined by its ability to induce resistance to G418 in recipient QT6 quail cells. 2. Cells. Chicken embryo fibroblasts (CEF) were prepared and cultured as previously described (9). The

Quail embryo fibroblasts (QEF) were prepared and cultured under the same conditions. Chicken cells

AEV-ES4, QT6 quail fibroblasts (17) and quail G helper cells (24) were cultured in CEF culture medium. AEV-ES4 cells were derived from CEF infected with the AEV-ES4 virus (9); these cells were kept in culture for several months before being used. The selection for the colonies of neomycm-resistant cells was made in the presence of G418 (Gibco) at a concentration of 200 μg / ml.

3. Transfeotions and infections. The helper G cells were transfected by the polybrene-dimethyl sulfoxide method (14). Viral suspensions were collected for 12 hour periods from producer cells

sub-confluent (3 ml per 100 mm dish), then centrifuged for 15 min at 2500 g and filtered on binder filters

weakly proteins (Milliporei. The cells, seeded at the rate of 5 10 ⁵ cells per 60 mm dish, were infected with 1 ml of viral solution for twelve hours, puls

selected in G418. Resistant colonies were assessed 10 to 15 days after infection. 4. Induction by zinc of the prosecutor of MT-II _Δ in the infected cells. The cells were incubated for 24 h in the CEF culture medium supplemented with 50 μM or 100 μM of ZnCl ₂ (Merck; ZnCl ₂ was prepared in the form of a 10 mM reserve solution in an isotonic saline buffer and stored at + 4 ºC

5. Kinetics of growth of infected cells. The cells were seeded at a rate of 5 10 ⁵ cells per dish of 60 mm and the medium was then changed every day. At each delay, one or three boxes per cell type were

trypsinized and cells were counted. 6. Analysis of ADH and ARM. High molecular weight DNA was extracted as previously described (10). After digestion of 15 μg with appropriate restriction enzymes, the fragments were separated by electrophoresis on 0.8% agarose gels

The DNA was then transferred to nitrocellulose filters and hybridized with a labeled probe primed at random. The filters were finally washed under extremely rigorous conditions and exposed to a Kodak-X-Omat film. The probe used was an EcoRI-Xmnl fragment derived from pMTH5. The total cellular RNA was purified after lysis of the cells in a buffer containing 1 mM Dithiotrèitol and 0.2% Nonidet P40 essentially as previously described (10). Of

S1 mapping experiments were performed in accordance with Favaloro et al. (8). A BamHI-Pstl restriction fragment containing the sequence coding for H5 was isolated from pMTH5 and labeled at the 5 ′ end with [γ ^-32 P] ATP and the polynucleotide kinase. The probe was coprecipitated with 10 μg of total RNA and resuspended in the hybridization buffer S1 containing 40 mM of Pipes [piperazme NN 'bis (2-ethane sulfonic acid, pH 6.4), 400 mM of NaCl , 1 mM EDTA] Hybridization was carried out for 6-9 h at 56ºC. then the hybrids were digested with 300 U of nuclease S1 per ml of digestion buffer (30 mM sodium acetate pH 4.5, 250 mM NaCl, 2 mM ZnSO ₄ ) for 30 min at 31 ° C. S1 nuclease resistant DNA was finally passed through a 6 polyacrylamide gel and exposed to film

 Kodak-X-Omat. The identity of the protected fragments was determined by comigration with markers of appropriate molecular weight.

7. Electrophoretic transfer of proteins onto nitrocellulose paper. Total histones were extracted with

H ₂ SO ₄ of the purified nuclei (19). The collected extracts have been dialyzed and lyophilized. The histones were analyzed by electrophoresis on polyacrylamide-SDS gel (sodium dodecyl sulfate) (15). The transfer of the polyacrylamide gel onto nitrocellulose paper was carried out for 1 hour at 24 V and the filters were finally incubated with anti-H5 antibodies and protein A labeled with ¹²⁵ I, 1-10 × 10 ⁵ cpm (Amersham Corp .) The production of antibodies in rabbits immunized with H5 has been reported previously (18) The anti-H5 antibodies were purified immunospecifically by affinity chromatography with H5 conjugated to Sepharose 4B. Their immunoreactivity was

controlled by solid phase radioimmunoassay (19).

8. Immunofluorescence. The cells were cultured on Lab Tek slides (Miles Laboratories), then fixed in absolute ethanol followed by acetone at room temperature. After washing with PBS-PMSF [10 mM sodium phosphate buffer, pH 7.4, 0.15 M NaCl, 0.1 mM phenyl methyl sulphonyl fluoride], the cells were incubated for 30 min at 4ºC with anti- H5 affinity purified. Fluorescently labeled anti-rabbit antibodies were then added and the stained cells were examined under an Olympus microscope equipped with an epi-illumination. Photomicrographs were taken with an Olympus camera and Kodak color film. Constant exposure and development times were used for printing.

9. Analysis of phosphorylation of the H5 protein. The cells were incubated for 4 h in a minimal essential phosphate-free medium, then they were labeled for 4 h in the same medium with 2 mCi of H ₃ PO ₄ labeled with 32 p (Amersham Corp. Lysine-rich histones have and extracted with 5% perchlorcihe acid and subjected to electrophoresis as described above The phosphorylation of H5 was detected after transfer of the gel onto nitrocellulose paper and autoradiography of the transfer range ("blot"). same transfer range ("blot") was used for the detection of the H5 protein with an anti-H5 antibody and a protein A-peroxidase conjugate horseradish (Amersham) H5 was finally visualized by incubation in 10 mM Tris, pH 7.4, 0.025% H ₂ O ₂ , 0.04%

4-aminoantipyrine (Sigma) and 0.2% phenol.

II - RESULTS

1. Analysis of ADH and viral RNA in QT6 cells infected with DAH5. QT6 cells, a line of

chemically transformed quail fibroblasts (17) have been

infected with a reserve of DAH5 virus devoid of helper-free pus selected by G418 and amplified in cultures

polyclonal. These cells did not release viruses and

supernatants from cultures superinfected with a normal helper virus were unable to transmit resistance to G413.

Since no virus could be found, we conclude that in cells infected with DAH5, the integrated provirus is inactive from the point of view of transcription. To determine the presence, organization and number of copies of the introduced DNA vector, high molecular weight DNA from infected or uninfected QT6 cells was prepared. 15 μg of DNA were digested with restriction enzymes and were examined by the Southern blottmg method with a probe containing the region coding for H5 (fig. 3). The restriction enzyme Xhol cuts twice inside the vector and the presence of a fragment of 4.6 kilobases (kb) is therefore characteristic of the genes.

intact H5 and neo. The expected fragment of 4.6 kb has been detected

in infected cells (lane 7), but it was not present in the DNA of uninfected cells (lane 6). To test more precisely the presence of an intact MTII-H5 fragment, the DNA of infected and uninfected GT6 cells was cut by Xbal.

As expected, a 2.1 kb fragment was detected in the infected cells (lane 5) and was not detected in the uninfected cells (lane 4) These data show that the inserted proviruses were not subjected to major rearrangements and presented the expected structure. The high molecular weight fragments observed in infected and uninfected cells

correspond to the endogenous H5 gene. From the intensities of the bands at 2.1 and 9.5 kb in the infected cells, we have

estimates that an exogenous H5 gene had integrated by genome

To confirm this estimate, the intensity of the 2.1 kb band was compared to that given by a known quantity of H5 sequences. 15 μg of avian genomic DNA represents 6 10 ⁶ avian genomes. Bands 1, 2 and 3 contain

respectively 6 10 ⁵ , 6.10 ⁶ and 6.10 ⁷ copies of plasmid pMTH5 digested with EcoRI. If we compare the intensity of hybridization of the 0.3 kb fragment of band 2 to the 2.1 kb fragment of band 5, we confirm that a copy of the DAH5 provirus has been inserted by genome

To determine the levels of specific transcripts of the H5 gene in QT6 cells infected with DAH5 and not infected, a quantitative analysis with nuclease S1 was used (FIG. 4) The DNA probe was a BamHI-Pstl fragment of 1026 bp isolated of the plasmid pMTH5. This fragment contains the promoter of MT-II _A and 216 nucleotides of the sequence coding for H5. Transcripts initiated to the MT-II _A promoter must be protected over a length of 316 bp (13) As expected, a protected fragment of nearly 315 nucleotides was detected with the RNA of QT6 cells infected with DAH5 (bands 2 to 5) A protected fragment of the same size was detected in QT6 cells infected with DAH5 maintained in culture for 50 generations without selection by G418 (lane 5) No protected fragment was detected with the RNA of uninfected QT6 cells (strip 1). The human MT-II _A promoter can be induced by heavy metals such as Zn ⁺⁺ (23). To test regulated expression of the H5 gene from the internal promoter, infected QT6s were exposed for 24 h to 50 μM (lane 3) or 100 μM of ZnCl ₂ (lane 4) before isolation of the RNA. The amount of H5 mRNA did not significantly increase compared to the RNA of uninfected cells (lane 2). We can

conclude that transcription of the sequence coding for H5 was efficiently and correctly initiated in QT6 cells and that the addition of Zn ⁺⁺ did not increase the degree of transcription. In addition, the sequence coding for H5 once transferred into QT6 cells was expressed stably during at least 50 generations of cultures released from selection by G418.

2. Expression of the H5 protein in QT6 fibroblasts infected with the DAH5 virus. The synthesis of histone H5 was measured in QT6 cells infected with DAH5 and not infected. Total histones were extracted from purified nuclei isolated from the respective cells. Proteins were analyzed by polyacrylamide gel electrophoresis, transferred to

nitrocellulose and the presence of H5 was detected after

incubation of the transfer range ("blot") with an anti-H5 antibody and the protein A- ¹²⁵ I (FIG. 5). This antibody gave no signal when tested against the total histones of uninfected QT6 cells treated with 0 (lane 6), 50 μM (lane

5) or 100 μM of ZnCl ₂ (band 4). A clearly visible signal with the same mobility of purified H5 (bands 7, 8, 9) or H5

total histones of chicken erythrocytes (bands 10, 11, 12), was detected with the total histones of 5.10 ⁶ QT6 cells infected with DAH5 treated with 100 μM of ZnCl ₂ (band 1). 50 μM of ZnCl ₂ (band 2) or no ZnCl ₂ (band 3). In an attempt to quantify the production of H5 in infected QT6 cells, known quantities of purified H5, 0.5 μg (lane 7). 1 μg (band 8) and 2 μg (band 9), or of total histones extracted from 5 10 ⁶ (band 10), 10.10 ⁶ (band 11) or 20.10 ⁶ (band 12)

erythrocytes from adult chickens have been subjected to

éiectrophorese on the same gel. From these results, it can be concluded that the amount of H5 present in 5 10 ⁶ QT6 cells infected with DAH5 is equivalent to the amount of H5 produced by 5.10 ⁶ erythrocytes from adult chickens and that ZnCl ₂ did not increase the degree of H5 production. The

nuclear localization of H5 in QT6 cells infected with DAH5 has been confirmed by immunofluoreecence.

3. Properties of QT6 cells and fibroblasts transformed by AEV-ES4 expressing the H5 protein. Cultures of QT6 infected with DAH5 have developed large dense foci containing cells phenotypically not different from non-infected QT6 cells or cells infected with the control virus TXN3 Likewise, chicken fibroblasts transformed by the avian retrovirus AEV-ES4 , were infected with DAH5 These cells developed foci which cannot be distinguished from those of cells infected with TXN3 'and they could not be amplified (not shown). To further investigate whether the H5 protein could have some effect on transformed cells, the growth kinetics of uninfected QT6 cells were compared to that of QT6 infected with DAH5 or TXN3 '. The three cell types were seeded individually at the same density in boxes of

60 mm and grown under the same conditions. Then, at different times, the cells were trypsinized and counted.

It can be seen that QT6 cells infected with DAH5 showed slower growth than QT6 cells not infected or infected with TXN3. The generation times could be estimated at

20 h for control QT6 cells and 25 h for QT6 cells producing the H5 protein It can therefore be concluded that the expression of the K5 protein seems to have little effect on the growth of QT5 cells and the fibroblasts transformed by

AEV-E34

4. Expression and effects of the H5 protein in normal fibroblasts Chicken embryo fibroblasts (CEF) and quail embryo fibroblasts (QEF) were infected either by the DAH5 virus or by the control viruses, TXN3 'or DAHY. Ten days after selection in a medium containing G418, large dense clones of fibroblasts were observed in the cultures of quail and chicken cells infected with each control virus. In all infected cultures

by DAH5 only a few small aggregates of dispersed cells were observed. These cells were growing very

slowly and they could not be effectively transplanted, therefore they could not be amplified. In addition, most of these cells appeared diseased and had a flattened star-shaped morphology identical to that of senescent fibroblasts. The same observations were made in six independent experiments using fibroblasts.

secondary chicken or quail. Since the cells infected with the DAHY control virus appeared to be as healthy as normal fibroblasts, the effect observed with the DAH5 virus is most probably due to the expression of the sequence coding for H5. As these cells developed very poorly, molecular analysis could not be performed and consequently the expression of the H5 protein was analyzed by immunofluorescence;

 Giemsa Wright staining of the same field was performed to distinguish the nuclei and the cytoplasm.

 Normal chicken fibroblasts infected with DAH5 exhibited heterogeneous fluorescence when treated with anti-H5. All cells had

fluorescent nuclei and the intensity of fluorescence correlates well with the structure of chromatin. The

highly condensed nuclei exhibited the strongest fluorescence, while nuclei with less condensed chromatin exhibited weak fluorescence.

 Certain highly condensed nuclei appeared to be expelled from the cells Conversely, the non-infected normal fibroblasts did not show significant fluorescence and they

contained nuclei with a typical dispersed chromatism 5. H5 is phosphorylated in QT6 cells. It appears from the results obtained that the expression of the H5 protein has only a slight effect on the growth of transformed cells. As a next step, we looked for possible post-translational modifications of this protein leading to its inactivation in these cells. Since phosphorylation could be involved in the inactivation of H5 in the stages

early in normal erythropoiesis (27), we examined

the extent of H5 phosphorylation in QT6 cells. QT6 cells infected with DAH5 and not infected were incubated in the presence of H ₃ PO ₄ labeled with ³² p. The lysine-rich histones were then extracted, subjected to electrophoresis on a polyacrylamide-SDS gel, transferred to nitrocellulose and

the importance of phosphorylation was directly analyzed by autoradiography of the transfer range ("blot"). A

phosphorylated protein. absent in control QT6 cells

(lane 1) was detected in QT6 cells infected with DAH5 (lane 2) To identify this protein, the same range of

transfer ("blot") was treated with an antι-H5 antibody, a protein A-peroxidase conjugate and 4-amino-antipyrine as substrate. The H5 protein was detected in QT6 cells infected with DAH5 (lane 4) and in total histones

extracted from chicken erythrocytes (lane 5), while no zone was observed in the control QT6 cells (lane 3). The phosphorylated protein detected in band 2 has roughly the same mobility as the H5 protein identified in bands 4 and 5

We can therefore conclude that the H5 protein is phosphorylated in QT6 cells infected with DAH5.

6. Discussion

This example demonstrates the efficient transfer and expression of histone H5 in normal fibroblasts and

The SIN vector described in this study was

built from avian leukosis virus RAV-2 La deletion of the promoter sequences in the 3 'LTR leads to the integration of an inactive provirus in the infected cells.

Vector transferred copy

of foreign H5 sequence in the genome of each cell

infected It was found that this transfer is very stable since the inserted H5 sequence was still present in cells cultured for 50 generations without selective pressure. In cells infected with the INS vector of

DAH5, H5 sequences were transcribed from the internal MT-II _A promoter. Since no vector virus could be recovered from infected cells even after superinfection with a helper virus, no transcription was

initiated from the 5 'LTR. The SIN vector described here

 is completely reliable and efficient.

No effects were observed

significant of Zn ⁺⁺ ions on the transcription efficiency of H5 in infected cells. Even without stimulation, the integrated H5 transcription unit was active. The constitutive activity of the MT-II _A promoter and its lack of response to Zn ⁺⁺ ions in the DAH5 vector could be inherent in this promoter in an avian cell, or could be due to the stimulatory effects created by the SV40 promoter proximal or LTR

The amount of H5 produced per QT6 cell infected with DAH5 is similar to that found in mature avian erythrocytes Therefore, the effects of H5 in infected cells can be compared to native physiological conditions

Expression of H5 in transformed fibroblasts, i.e. quail QT6 fibroblasts or chicken embryo fibroblasts transformed with AEV-E54. did not inhibit their growth in vitro. DAH5-infected QT6s show little or no increase in their generation time, from 20 to 25 hours, but their ability to grow as an established line has not been diminished. This result shows explicitly that the constant production of H5 has little effect on the phenotype of transformed fibroblasts. Normal quail or chicken fibroblasts expressing the exogenous H5 protein had very limited growth potential and could not be transplanted or developed. In addition, these cells resemble senescent cells with a

flattened morphology, vacuolated cytoplasm and condensed nuclei. The condensation of chromatin in these cells was directly related to the expression of H5 since the more condensed nuclei exhibited more intense fluorescence when treated with the anti-H5 antibody In addition, some nuclei with highly condensed chromatin was expelled from the cells. These data therefore show that H5 exhibits very different effects in normal and transformed fibroblasts.

The biological role of H5 has been regularly linked to the idea that it is a general repressor. This view is confirmed by the fact that. when the nuclei have been reactivated by the fusion of chicken erythrocytes with HeLa cells, the early observed events include decondensation of the chromium. initiation of RNA synthesis and disappearance of H5 (2) A recent study carried out by micro-injection of H5 into proliferating L6 rat myoblasts, a cell line which preserves the potential to differentiate into myotubules. showed that the migration and localization of H5 in the nuclei were linked to the decrease in cell size, vacuolation of the cytoplasm, packing of the nuclei and inhibition of transcription and replication (4).

The fact that H5 is highly phosphorylated in infected transformed GT6 cells recalls the process observed in normal erythropoiesis. The presence of H5 has been detected at a reduced level in erythroblasts in a state of early division. In these cells, the newly synthesized Droteme is transported into the nucleus where it is modified by a process involving continuous unidirectional phosphorylation during which nine phosphorylated groups can be introduced. In the terminal stages of erythrocyte maturation, the phosphorylated protein is dephosphorylated and this latter event is in good correlation with the cessation of DNA and RNA synthesis and the condensation of the chromium (5, 26). The extent of H5 phosphorylation in normal DAH5 infected fibroblasts could not be analyzed since these cells could not be amplified.

The exact mechanism of interaction of H5 with DNA in the chromium is not known. However, one can envision a mechanism that allows the cell to process large amounts of H5 without deteriorating cell metabolism. The

phosphorylation of H5 produces a protein which has less affinity for DNA, by neutralization of charges. This is why, the modified amino acids must be located in areas which are decisive for this interaction, that is to say the C-term region (residues 100-182) which is the region which contributes most to the condensation of the chromium by partial neutralization of the DNA phosphates. The unmodified H5 showing more affinity for higher order structures of

chromium and DNA, would bind tightly to DNA, especially to regions rich in A-T, by condensing and / or changing its conformation (20) and, in this way, blocking the binding of the machinery of transcription and replication.

It will be interesting to study the metabolic pathways which are directly responsible for the phosphorylation of H5 in transformed cells and their relationship with the expression of oncogenes Since the fibroblasts QT6 and AEV-ES4 are transformed by different initial events, it is likely that the two processes activate common H5-kinases

Analysis of H5 changes in various cells infected with DAH5 may be helpful in defining the role of this protein in cellular replication and differentiation and in the knowledge of oncogenic transformation mechanisms.

 EXAMPLE 2

Vectors carrying sequences of the RAV-0 virus and a 5 'LTR of RSV

 Two vectors carrying a 3 'LTR derived from the RAV-0 virus, naturally devoid of enhancer sequences, were constructed.

 The vector OVA-ZZ carries the following sequences from 5 'to 3':

- A RSV virus LTR,

 - A leading sequence of the RAV-1 Virus,

 - The NeoR resistance gene,

 - The E. Coli gene coding for a beta-galactosidase provided with a nuclear addressing signal, under the control of the promoter of the SV40 virus (SVnlslacZ gene, supplied by JF Nicolas of the Pasteur Institute in Paris, of. Bonnerot et al. 1987, Proc. Natl. Acad. Sci. USA 84. 6795-6799),

 - The 3 'non-coding sequence and the LTR of the RAV-0 virus.

 The vector OVA-Zdel is identical to the previous one except in its 3 ′ part where the LTR 3 ′ of RAV-0 deleted by 50 nucleotides comprising its CAAT box.

 The OVA-Zdel virus can be produced at a titer of 1000 particles / ml conferring beta-galactosidase activity on infected cells. On the other hand, the NeoR gene is not expressed in the infected cells, which confirms the self-inactivating character (SIN) of this vector.

The vectors OVA-ZZ and OVA-Zdel were transfected into the Ispere helper line. The supernatants of pools of helper cells resistant to G418 were titrated on QT6 cells on the one hand by selection with G418 (title NeoR) and on the other hand by in situ revelation of the beta-galactosidase activity (title lacZ). The report of the two titles reveals that only OVA-Zdel is capable of significant self-inactivation: OVA-ZZ gives 100 G418rfu / ml and 100 lacZfu / ml. The activity of resistance to G418 of cells infected with this vector undoubtedly results from the transcription initiated at the level of the 5 'LTR, which therefore remains fairly active. We therefore built OVA-Zdel, a vector similar to OVA-ZZ but whose 3 'LTR, still from RAV-0, has been deleted from its CAAT box. Titration was carried out using 20 clones of transfected helper cells. One of the clones produces 1000 lacZfu / ml and none (<20 / ml) G418ru. This result demonstrates the efficacy and safety of the OVA-Zdel vector.

 Subsequent experiments have made it possible to better understand the factors that control the efficiency of gene transfer and self-activation, by comparing OVA-ZZ and OVA-Zdel with a construct carrying a 3 'RSV LTR (OVA-D):

 - The transient expression of the nislacZ gene in transfected QT6 cells is reduced by 5 to 10 times by the deletion of the RAV-0 LTR.

 - The level of messenger RNA initiated at 5 'LTR in helper cells resistant to G418 is not affected by the 3' sequences.

 - The packaging of the RNAs carrying the RAV-0 sequences is reduced by a factor of 2 or 3.

 These results explain why the lacZ titer obtained with OVA-Zdel remains 100 times lower than that obtained with OVA-D.

 Vectors derived from avian leukosis were constructed with an internal transcription promoter and various non-coding sequences in

3 '. Deletions are introduced into the downstream U3 LTR element (3 ') to obtain self-inactivation of the LTR-mediated transcription after a replication cycle. However, the 3 ′ non-coding sequences seem to determine not only the self-inactivation ability of the vectors, but also the efficiency of gene transfer. Further analysis reveals the influence of these sequences both on the expression of the internal gene and on the packaging of the RNA. Elements built with a 5 'LTR of RSV make it possible to carry out an efficient gene transfer while strongly inactivating the transcription promoted by LTR in 5 ′.

 The ability of self-inactivating vectors to produce a high titer viral preparation and to fully inactivate the LTR promoter is primarily based on the mutation that is created. However, the consequences of mutating non-coding 3 'viral sequences on RNA transcription, processing, translation, packaging and reverse transcription are very unpredictable (R34, R43, R47).

 The construction of several vectors derived from avian leukosis with various non-coding 3 'sequences is described here. The influence of these 3 'sequences on the expression of the virus is analyzed further, and reveals its various consequences on the expression and replication of the viruses. MATERIALS AND METHODS

Construction of OVA plasmids (Figures 7, 8)

 DA1 is constructed by assembling in pBR322 the following fragments: an Xhol fragment from pRAV-2 (R45) carrying a fully active RAV-2 LTR and a leader sequence, an Xhol-Xbal from pTXN3 'carrying part of the GAG sequence avian erythroblastosis virus (R29), an AccI-BglII fragment of pSV2Neo (R50) in direct orientation, and a deleted 3 'LTR. The deleted LTR is created by assembling the 3 'part of the RAV-2 genome (from Xhol to a ClaI linker inserted in the SphI site which is located 149 nucleotides upstream from the capsule site). This deletion removes the TATA and CAAT boxes.

OVA is constructed by assembling the polylinker of pBluescript (Stratagene SanDiego), the following fragments: the Ndel-EcoRI fragment of pUT507 (R52) (carrying 0.5 kb of RSV sequences strain Schmidt-Ruppin D, covering part of the v gene -src and the upstream part of U3), the EcoRI-Xhol of pRAV-1 (R35) (from U3 to GAG) an Xhol-Xbal of pTXN5 '(R29). (NeoR gene) and the Sall-BamHI fragment of pMuMLVSVnlslacZ (R24) (the promoter SV40) followed by a mutated version of the lacZ gene of E. coli coding for a protein targeted at the nucleus of the cell). Given the insertion

small fragments of pBluescript polylinker, this structure is flanked by two Spel restriction sites.

 To build OVA-D, we transfer the

Ndel-Xhol fragment of OVA between the Saml sites and

Xhol from pBluescript (to provide a complete 3 'non-coding sequence in RSV type), then insert

the OVA Spel fragment in the upstream Spel site in the right orientation. OVA-ZZ is constructed by transferring the HindIII-BamHI fragment from pGJ18 (R19) carrying two copies of RAV (O) LTR into pBluescript (giving BluZZ), then inserting the OVA Spel fragment into the HindIII site of BluZZ in orientation correct, after partial filling of the projecting ends with Klenow polymerase. OVAZdel is obtained by deleting BluZZ by digestion with Ndel and religation at low DNA concentration (giving BluZdel) and by inserting the OVA Spel fragment at the HindIII site.

Cellular culture

 All reagents are purchased from Gibco. Isold helper cells (R44) are cultivated in a Williams medium supplemented with 5% fetal calf serum, 2% chicken serum, NaCl

(60 mM), penicillin (1000 u / ml), streptomycin

(0.05%), phleomycin (50 μg / ml) and hygromycin

(50 μg / ml). QT6 (R36) and chicken embryonic fibroblasts prepared from from 10-day-old N / A SPAFAS embryos (CEF, R45) in HamF10 medium supplemented with 5%

calf serum, 1% chicken serum, 2% phosphate tryptose broth, penicillin and streptomycin. We change the environment every three days.

Transfection, virus production and titration

 The purified plasmid DNA is transfected by production of CsCl bands (R39) in Isold cells either by precipitation with calcium phosphate (R40) or with polybrene (R41). Cells are selected (250 μg / ml) with G418 (Gibco) 24 hours

after transfection, for at least 10 days. We

harvest viral supernatant from cells

subconfluent 16 hours after changing the medium, and centrifuged (2000 g, 5 minutes) to remove cellular debris. For the titration, we add

 50 μl to 500 μl of helper cell supernatant

QT6 or CEF cells (5 x 10 ⁵ cells per 25 cm ² ). Cells infected with G418 are selected after 24 hours, or stained to determine the activity of nlslacZ after 48 hours (R53).

RNA analysis

 The RNA is extracted from helper cells or from a centrifuged supernatant (45 minutes, 35,000 rpm in a Backman SW41 rotor) by proteinase K digestion and phenol-CHCl3 extraction as

it says (R39). The slotted and Northern blot products are hybridized with an in vitro synthesized RNA sample labeled with 32 P using standard techniques. The SI card is established as described (R39). RESULTS

 A significant deletion in the promoter elements of the LTR in 3 'reduces the titer of the virus

 FIG. 7 shows the structure of DA1, a vector carrying the NeoR gene promoted by the SV40 promoter and inserted in the direct orientation (with respect to the transcription of the virus). The U3 sequence of the LTR is deleted in 3 ′ from the nucleotide -149 to -15 (to the capsule site; see materials and methods) This deletion comprises the CAAT box and the TATA box but neither the enhancer sequence nor the poiyadenylation signal. Several other vectors are derived from DA1 by insertion of an additional gene between the 5 'LTR and the SV40 promoter, in the reverse orientation.

 These constructs are transfected into helper cells, which are then selected with G418, and the recombinant viruses are titrated from the cell supernatant. Viral titers are low, ranging from 3U G418 rfu / ml to 200 G418 rfu / ml when testing clonal cell populations. Resistant infected cells are superinfected with a helper virus competent for replication to save non-inactivated viruses.

As no recombinant virus is saved in a group of approximately 100 infected cell clones, it is concluded that the deletion effectively inactivates the LTR promoter in 5 ′ after a cycle of viral replication.

S1 RNA analysis fails to detect LTR and SV40 promoted RNA in cells G418 resistant transfected helper. (A control experiment has been successfully carried out with

pTXN3 ', previously described vector (R29)). Thus, it is likely that the stability and / or processing at the 3 'end of the RNA is affected by the LTR mutation at 3'. The addition of a SV40 polyadenylation signal downstream of the LTR in 3 'fails to restore the viral titer (data not shown). Therefore, an approach similar to that which has been successfully used to generate self-inactivating viruses derived from rat spleen necrosis virus results in poor success with viruses derived from avian leukosis.

Modification from non-coding sequences in 3 'only

 To allow direct comparison

of various non-coding 3 'sequences and studying precisely their influence on the efficiency of gene transfer, we develop a series of vectors which differ only in their 3' end. We

achieves this by constructing OVA, a plasmid which can provide, in the form of a single restriction fragment, a cassette carrying (from 5 ′ to 3 ′) a fully active LTR of RSV type, a leader sequence RAV (1), the coding sequence NeoR , and the nlslacZ gene controlled by the

SV40 transcription promoter (as the SV40-nlslacZ unit is placed in the forward orientation, it is conceivable that translation reinitiation can occur at low frequency on the transcripts promoted by LTR in 5 ', and reflect a weak part of the translation of nlslacZ).

The OVA cassette is inserted upstream of various LTRs which provide the signal necessary for 3 'end processing of LTR-promoted transcripts

in 5 'and SV40.

 As a positive control, use the fully active RSV LTR as the 3 'LTR and obtain

OVA-D. OVA-ZZ has unmutated RAV (O) LTR as a 3 'sequence. This LTR is naturally

present in an endogenous provirus which can be cultivated at a relatively high titer (R32) and has been shown to possess promoter, terminator activity, but not to enhance (R14, R19). We therefore expect

construction product OVA-ZZ a

very limited self-inactivation but transfer

effective gene. To increase the ability to self-inactivate, OVA-Zdel is also constructed, endelecting 50 nucleotides comprising the CAAT box.

from RAV (O).

 Viral titer is influenced by 3 'non-coding sequences

 Virus titrations based on in situ revelation of beta-galactosidase activity or of G418 selection are carried out from helper cells transfected in a stable manner (Table I).

The titer expressed in the form of lac + fu / ml (titer lacZ) results from the combined efficiencies of the transfer of

gene and internal gene expression. The comparison with the titer expressed in the form G418rfu / ml (NeoR titer) gives an estimate of the self-inactivation. OVA-D seems to give a high titer in both

assays, confirming that avian leukosis viruses such as murine leukemia viruses can harbor

the internal promoter. To our surprise, the efficiency gene transfer is dramatically reduced with OVA-ZZ (100 lac + fu / ml) and the vector shows no signs of self-inactivation (100G418rfu / ml).

The deletion of the CAAT box in 3 'in OV-Zdel

results in an improvement in the efficiency of gene transfer and an increase in the ratio between

the title lacZ and NeoR. To improve both the efficiency of gene transfer and self-inactivation, 20 clones of helper cells transfected with OVA-Zdel are isolated and the expression of both nlslacZ and NeoR is titrated therein. 10 of these clones do not give any virus (<20 lac + or G418rfu / ml). The results, obtained with the four clones which give the highest lacZ titer, are reported in Table II. The ratio between the two viral titers (ie the efficacy of self-inactivation) varies widely from one clone to another and can exceed 60. Consequently, OVA-Zdel can be considered as a vector of

effective self-inactivating retrovirus.

 Self-inactivation is further confirmed in a virus rescue experiment that fails to give a recombinant virus after superinfection

by RAV (1) (<20 lac + fu / ml).

3 'sequences influence expression

transient of the internal gene

 To acquire additional information on the factors which determine the production of virus in helper cells, Isold or QT6 cells are transfected with the OVÀ plasmids.

and the transient expression of the SV40-nlslacZ gene is assayed 48 hours later by in situ staining

(Table III). Under these conditions, OVA-D and OVA- ZZ give similar results. The deletion

of U3 in OVA-Zdel alters the transient expression

from SV40-nlslacZ. These results support the idea

that the modest viral titer obtained with OVA-Zdel

is linked to a defect in the expression of SV-nlslacZ and that the deleted part of the element RAV (O) U3

carries an important signal for the expression of the viral gene. On the other hand, the low viral titer obtained

with OVA-ZZ does not correlate with the transient expression of nlslacZ, which indicates that a

post-transcriptional stage of viral production

is also upset when using the 3 'RAV (O) sequence. Transcripts promoted by

 SV40 are less abundant than transcripts promoted by 5 'LTR in stably transfected cells but the constant level of both

 RNA in these cells is not clearly dependent on the 3 'non-coding sequences.

 Although this observation is an argument against a possible engagement of the 3 'sequences in the viral transcription, it can be biased by the fact that only the cells producing enough viral RNA to resist G418 selection are considered in the latter case.

Clonal variation in gene expression

SV40-nlslacZ in infected cells

 Clones of infected cells resistant to G418 are stained (clones G418R) to determine the activity of β-galactosidase. Although this analysis is carried out on a limited number of clones, it

leads to several interesting observations: - Some G418R clones do not contain

no lac + cell cells. This could be due to a mutation occurring in the nlslacZ gene. However, since the G418R / lac helper cells can produce a recombinant lac + virus (data not

shown) and as a low level of NeoR gene expression is sufficient to provide resistance to

G418 (see above and R29), it is more likely that G418R / lac cells actually express

nlslacZ, but below the threshold level necessary for positive staining in the in situ assay.

A position effect may explain the reason why the phenotype is almost constant in a single G418R clone. It is therefore likely that lacZ staining in situ will lead to an underestimation of the effective efficiency of gene transfer.

 - The relative proportion of the G418R / lac- and G418R / lac + clones depends on the vector which is used to infect the cells. As expected from transient dosages, this ratio is similar for OVA-D and OVA-ZZ and is higher than that obtained with OVA-Zdel.

 RNA analysis reveals that the 3 'non-coding sequences influence the efficiency of RNA enveloping.

 A low viral titer may result either from reduced production of viral particles carrying vector DNA by helper cells, or

a low level of viral gene expression in infected cells. To identify the limiting step in the present situation, we analyzes the retrovirus RNA in helper cells stably transfected with OVA-D, OVA-ZZ and OVA-Zdel (clone A) and in the viral particles which they produce. The slotted blot analysis shows that the viral titer correlates with the amount of retrovirus RNA enveloped in the virus particles but not with the constant level of vector RNA present in the cells. Quantitative analysis reveals that the OVA-ZZ and OVA-Zdel transcripts initiated at the 5 'LTR are underrepresented in the viral particles. Envelopment of RNA therefore seems to be a limiting step in the production of viruses for OVA-ZZ and OVA-Zdel.

 It is shown here that the 3 'non-coding sequences influence the ability to inactivate retrovirus vectors but also the efficiency of retroviral mediated gene transfer in cells - avians. As the U3 deletions that have been tested affect the viral titer, we are led to assume that the LTR sequences play a role in this phenomenon.

 However OVA-Zdel with a 5 'RSV LTR allows us to produce 100 lacZfu / ml while fully inactivating the LTR promoter. Table I: Virus titration

 The helper cells are transfected and subjected to selection by G418. The cell supernatant is then titrated on QT6 and CEF cells for the expression of NeoR as of lacZ. The viral titer is averaged from at least 3 different transfection experiments.

Table II: Clonal variation in the viral titer obtained with OVA-Zdel

Twenty clones of helper cells stably transfected with OVA-Zdel are titrated for expression of the NeoR gene as lacZ. A, B, C and D are the four clones which give the highest lacZ titer. Table III: Transient expression of SVnlslacZ in OVA vectors.

 Transient expression: QT6 cells are transfected by precipitation with calcium phosphate or with polybrene (see materials and methods). The cells are fixed after 48 hours and are stained to determine the activity of β-galactosidase. When more than 500 cells are positive for the transitive expression of nlslacZ on a single dish, the total number of lac + cells is extrapolated from an exhaustive count carried out on a limited area. The last track indicates the average reports relating to OVA-D.

BIBLIOGRAPHY

1. Affolter, M., J. Cote, J. Renaud and A. Ruiz-Carillo. 1987. Regulation of histone and B ^A -globin gene expression during differentiation of chicken erythroid cells. mol. Cel. Biol. 7, 3663-3672.

 2. Appels, R., L. Bolund and R.N. Ringertz. 1974. Biochemical analysis of reactivated chick erythrocyte nuclei isolated from chick HeLa heterokaryons. J. Mol. Biol. 85: 182-190.

 3. Benchaibi, M., F. Mallet, P. Thoraval, P. Savatier, J.H. Xiao, G. Verdier, J. Samarut and J.V. Nigon. 1989. Avian retroviral vectors derived from avian defective leukemia virus: role of the translational context of the inserted gene on efficiency of the vectors. Viroiogy 169: 15-26.

4. Bergman, M.G., E. Wawra and M. Winge. 1988. Chicken histone H5 inhibits transcription and replication when introduced into proliferating cells by microinjection. 3. Cell Sci. 91: 201-209.

 5. Briand, G., D. Kmiecik, P. Sautiere, D. Wouters, O. Borie-Loy, G. Biserte, A. Mazen and M. Champagne. 1980. Chicken erythrocyte histone H5. FEBS Lett. 112: 147-

6. Cullen, B.R., K. Raymond and G. Ju. 1985. Functional analysis of the transcription control region located within the avian retroviral long terminal repeat. Mol. Cell. Biol. 5: 438-447.

 7. Emerman, M. and H.M. Temin. 1984. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39: 459-467.

8. Favaloro, J., R. Treisman and R. Kamen. 1980. Transcription maps of polyoma virus specific RNA: analysis by two dimensional nuclease S I gel mapping. enzymol methods. 65: 718-749.

9. Gandrillon, O., P. Jurdic, M. Benchaibi, J.H. Xiao, J. Ghysdael and J.

Samarut. 1987. Expression of the v-erb A oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo. Cell 49: 687-697. 10. Garcia, M., R. Wellinger, A. Vessaz and H. Diggelman. 1986. A new site of integration for mouse mammary tumor virus proviral DNA common to Balb / c (C3H) mammary and Kidney adenocarcinomas. 1: 127-134.

1 1. Gritz, L., and J. Davies. 1983. Plasmid-encoded hygromycin B resistance: the sequence of hydromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25: 179-188.

 12. Hwang, L. S. and E. Gilboa. 1984. Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection. J. Virol. 50: 417-424.

13. Karin, M. and R.I. Richards. 1982. Human metallothionein genes- primary structure of the metaliothionein-II gene and a related processed gene. Nature (London) 299: 797-802.

 14. Kawai, S. and M. Nishizawa. 1984. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol. Cell. Biol. 4: 1 172-1 174.

15. Laemli, V.K. 1970. cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 256: 680-685.

 16. Laimins, L.A., P. Tsichlis and G. Khoury. 1984. Multiple enhancer domains in the 3 'terminus of the prague strain of Rous sarcoma virus. Nucleic Acids Res. 12: 6427-6442.

 17. Moscovici, D., M. G. Moscovici, H. Jimenez, M.M.C. Lai, M. J. Hayman and P.K. Vogt. 1977. Continuous tissue culture cell lines derived from chemically induced tumors of japanese quail. Cell 11: 95-103.

 18. Mura, C.V. and B.D. Stollar. 1981. Fluorescence-activated sorting of isolated nuclei. Exp. Cell. Res. 135: 31-37.

 19. Mura, C.V. and B.D. Stollar. 1981. Serological detection of homologies of H10 with H5 and H1 histones. J. Biol. Chem. 256: 9767-9769.

20. Mura, CV and BD Stollar. 1984. Interaction of H1 and H5 histones with polynucleotides of B- and Z- conformation Biochem. 23: 6141-6152. 21. Mura. CV, G. Setterfield and JM Neelin. 1982. Immunofluorescent localization of lysine-rich histones in isolated nuclei from aduit and embryonic chicken erythrocytes. Can. J. Biochem. 60: 215-223.

 22. Norton, P. and J.M. Coffin. 1987. Characterization of Rous Sarcoma virus sequences essential for viral gene expression. J. Virol. 61: 1,171 -1,179.

23. Richards, R.I., A. Heguy and M. Karin. 1984. Structural and functional analysis of the human metaiiothionein-IA gene: differentiai induction by metal ions and glucocorticoids. Cell 37: 263-272.

 24. Savatier, P., C. Bagnis, P. Thoraval, D. Poncet, M. Belakebi, F. Mallet, C. Legras, F.L. Cosset, J.L. Thomas, Y. Chebloune, C. Faure, G. Verdier,

J. Samarut and V. Nigon. 1989. Generation of a helper cell Une for packaging avian leukosis virus-based vectors. J. Virol. 63: 513-522.

25. Southern, P.J. and P. Berg. 1982. Transformation of mammalian cells to antibiotic rresistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Broom. 1: 327-341.

 26. Sung, M. T. and E. F. Freedlender. 1978. Sites of in vivo phosphorylation of histone H5. Biochem. 12: 1864-1869.

 27. Sung, M. T., J. Harford, J. Bundman and G. Vidaiakas. 1977. Metabolism of histones in avian erythroid cells. Biochem. 16: 279-285.

28. Varmus, H. and R. Swanstrom. 1982. Replication of retrovirueses. In (R.

 Weiss, N. Teich, H. Varmus and J. Coffin, Eds.), RNA Tumor Viruses

Molecuiar bioiogy of Tumor Viruses "pp. 369-512. Cold Spring Harbor

Laboratory, Cold Spring Harbor, N.Y.

29. Wu, RS, HT Panusz, CL. Hatch and WM Bonner. 1986. histones and their modifications. CRC Crit. Rev. Biochem. 20: 201-210.

BIBLIOGRAPHICAL REFERENCES FOR EXAMPLE 2

-R3: Gaileo. D. S., G. E Gray, G. C. Owens. J. Majors and J. A. Sanes. 1990. Neurons and glia arise from a common progenitor in chicken optic tectum: Demonstration with two retroviruses and cell-type specific antibodies. Proc. Natl. Acad. Sci. USA 87: 458-462.

 - R4: Darlix, J-L. 1986. Circularization of retroviral genomic RNA and the control of RNA translation, packaging and reverse transcription. Biochemistry 68: 941-949.

 - R7: Darlix, J-L. 1986. Control of Rous sarcoma virus translation and packaging by the 5 'and 3' untranslated sequences. J. Mol. Biol. 189: 421-434.

- R 10: Flanagan, J.R., A. M. Krieg, E. E Max and A. S. Kahn. 1989. Negative control region at the 5 'end of munne leukemia virus long terminal repeats. Mol. Cell. Biol. 9: 739-746.

 - R 11: Murphy, J. E .. and S. P. Goff. 1989. Construction and analysis of deletion mutations in the U5 region of Moloney munne leukemia virus: Effects on RNA packaging and reverse transcription.

 - R 13: Ludw, P. A., J. M. Bishop, H. E. Varmus and M. R. Capecchi. 1983. Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA. Cell 33: 705-716.

 - R14: Herman, S. A., and J. M. Coffin. 1986. Differential transcription from the long terminal repeats of integrated avian leukosis virus DNA. J. Virol. 60: 497-505.

 - R16: Emerman, M. and H. M. Terrin. 1986. Quantitaive analysis of gene suppression in integrated retrovirus vectors. Mol. Cell. Biol. 6: 792-800.

 R17: Swain, A. and J. M. Coffin. 1989. Polyandenylation at correct sites in genome RNA is not required for retrovirus replication of genome encapsidation. J. Virol. 63: 3301-3306.

 R18: Norton, P. A. and J. M. Coffin. 1987. Caracterization of Rous sarcoma virus sequences essential for viral gene expression. J. Virol. 61: 1171-1179.

 R19: Cullen. B. R., A. M. Skalka and G. Ju. 1983. Endogenous avian retroviruses contain deficient promoter and leader sequences. Proc. Natl. Acad. Sci. USA 80: 2946-2950.

 - R23: Dougherty, J. P. and H. M. Temin. 1987. A promotertess retroviral vector indicates that there are sequences in U3 required for 3 'RNA processing. Proc. Natl. Acad. Sci. USA 84: 1,197-1201.

 - R24: Bonnerot. C. D. Rocancourt, P. Briant, G. Grimber and J.F. Nicolas. 1987. A β- galactosidase hybrid protein targeted to nuciei as a marker for developmental studies. Proc. Natl. Acad. Sci. USA 84: 6795-6799.

 - R29: Benchaibi, M., F. Mallet, P. Thoraval, P. Savatier, J. H. Xiao, G. Verdier. J. Samarut and V. M. Nigon 1989. Avian retroviral vectors derived from avian detective leukemia virus: Role of the translational context of the inserted gene on efficiency of the vectors. Virology 169: 15-26.

- R30: Cone. R. C, Weber-Benarous A., Baorta, D and RC Mulligan. 1987. Regulated expression of a complete human β-globin gene encoded by a transmissible retrovirus vectors. Mol. Cell. Biol. 7: 887-897 - R31: Emerman, M. and HM Temin. (1984). Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39: 459-467.

 - R32: Sorge, J. A .. W. Ricci and S. H. Hughes. 1983. Cis acting RNA packaging locus in the 1 15- nucleotide direct repeat of Rous sarcoma virus. J. Virol. 48: 667-675.

 -R33: RNA tumor viruses. 1985 R. Weiss, N. Teich, H. Varmus and J. Coffin eds. Cold Spring Harbor Laboratory. Cold Spring Harbor. N. Y.

- R34: Yu, S. F., T. Von Rüden, P. Kantoff, C. Garber, M. Seiberg. U. Rüther, W. F. Anderson, E. F. Wagner and E. Gilboa. 1986. Setf-inactivating retroviral vectors designed fortransfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. USA 83: 3194-3198.

- R35: Payne, G. S., S. A. Courtneidge, L B. Crittenden, A M. Fadly, J. M. Bishop and H. M. varmus. Analysis of avian leukosis DNA and RNA in bursal tυmors: Viral gene expression is not required for the maintenance of the tumor state. Cell 23. 311 1981

 - R36: Moscovici. C., M. C. Moscovici, H. Jimenez, M. M. C. Lai, M. J. Hayman and P. K. Vogt. 1976. Continuous tissue culture cell lines derived from chemically induced tumors of japanes quail. Cell 11: 95-103.

 - R39: Maniatis, T., E. F. Fritsch and J. M. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold spring harbor laboratory, Cold Spring Harbor. N. Y.

 - R40: Dusty Miller, A., D. R. Trauber and C. Buttimore; 1986. Factors invotved in production of helper virus-free retrovirus vectors. Som. Cell. Mol. Broom. 12: 175-183.

 -R41: Chaney, W. G.. D. R. Howard. J. W. Pollard, S. Sallustio and P. Stanley. 1986. High-frequency transfection of CHO cells using polybrene. Som. Cell. Mol. Broom. 12: 237-244.

- R 43: Yee. J. K., J. C. Moores, O. J. Jolly, J. A. Wolff and J. G. Respess. 1987. Gene expression from transcriptionally disabled retroviral vectors. Proc. Natl. Acad. Sci. USA 84: 5197-5201.

 - R44: Cosset, F-L., C. Legras. Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J.

Samarut. V. M. Nigon and G. Verdier. 1990. A new avian leukosis virus -based packaging cell une that uses two separate transcomplementing helper genomes. J. Virol. 64: 1070-1078.

- R45: Bizub D., R. A. Katz and A. M. SSkalka 1984 Nucleotide sequence of non coding regions in Rous associated virus -2: comparison delineate conserved regions important in replication and oncogenesis.

 -R46: Hughes. S. H., in Viral vectors. 1988. Y. Gluzman and S. H. Hughes eds. Cold Spring Harbor Laboratory. Cold Spring Harbor. N. Y.

 -R47: Iwakaza K. and H. M. Temin. 1990. The U3 region is not necessary for 3 'end formation of spleen necrosis virus RNA. J. Virol. 64: 6329-6334.

 - R48: Denome R. M. and C. N. Colle. 1988. Pattem of polyadenylation site selection in gene constructs containing multiple polyadenylation signal. Mol. Cell. Biol. 8: 4849-4839.

- R49: Katz RABR Cullen. R. Malavarca and AM Skalka. 1986. Role of the avian retrovirus mRNA leader in expression: Evidence for novel translational control. Mol. Cell. Biol. 6: 372-379. - R50: Southern PJ and P. Berg. 1982. Transformation of mammaiian cenns to antibiotic resistance with a bacterial gene under control of the SV40 earty promoter region.

 J. Mol. Appl. Broom. 1: 327-341.

 - R51: Sealy, L., M. L. Privalsky. G. Moscovici, C: Moscovici and J. M. Bishop. 1983. Site- specfic mutagenesis of avian erythroblastosis virus: erbB is required for oncogenicity. Viroiogy 130: 155-178.

 -R52: Gorman CM., G.T. Merlino, M.C. Willingham, I. Pastan and B.H. Howard. 1982. The Bous sarcoma virus long terminal repeat is a strong promoter when introduced in a variety of eukariotic cells by DNA -mediated transfection. Proc. Natl. Acad. Sci. USA 79: 6777-6781.

 -R53: Sanes J.R., J.L.R. Rubenstein and J.F. Nicolas. 1986. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5: 3133-3142.

Claims

1. Self-inactivating viral vector for integration and expression of at least one heterologous gene in avian cells, consisting of the proviral genome originating from avian retroviruses, essentially

AEV and / or related retroviruses in particular of the RAV type, and comprising between two avian LTR sequences the said heterologous gene (s) which is (are) under the control of a heterologous internal promoter , characterized in that deletions or mutations in the 3 'LTR have been carried out so as to inactivate the viral promoters present in the LTR, the

3 'LTR from the RAV-0, RAV-1, RAV-2 retroviruses.

2. Vector according to claim 1, characterized in that the promoter or the enhancer sequence of the LTR 3 'have undergone a complete or partial deletion.

3. Vector according to claim 2 or 3, characterized in that the LTR sequence has undergone deletions in the U3 region.

4. Vector according to one of the preceding claims, characterized in that the CAAT and TATA boxes have been deleted.

5. Vector according to one of the preceding claims, characterized in that it is constructed essentially from the genome of the RAV-2 retrovirus whose LTR 3 ′ comprises the sequence U, from LTR of

RAV-2 in which the CAAT and TATA boxes have been deleted and the R and U ₅ sequences of the RAV-1 LTR.

6. Vector according to one of claims 1 to 3, characterized in that the LTR 3 'is derived from a RAV-0 virus whose LTRs are naturally very little active and has undergone point deletions or mutations.

7. Vector according to claim 6, characterized in that the 3 'LTR of RAV-0 has undergone a deletion in the CAAT promoter sequence.

8. Vector according to one of claims 1 to 7, characterized in that the 5 'LTR consists of ia 5' LTR of the RSV virus.

9. Vector according to one of the preceding claims, characterized in that it comprises two heterologous genes, namely a useful gene placed under the control of a heterologous internal promoter and a selection gene placed under the control of a promoter internal heterologous or 5 'LTR promoter.

10. Vector according to the preceding claim, characterized in that said heterologous genes replace the oncogenes v-erbA and v-erbB.

1 1. Integration and expression vector of a foreign gene characterized in that it is of the plasmid type and comprises a DNA reverse reverse transcribed DNA sequence of a viral vector RNA according to one of claims 1 to 10.

12. Method for preparing a vector according to one of the preceding claims, characterized in that one or more deletion (s) or mutation (s) in the avian LTR 3 'so as to inactivate the viral promoters present into said LTRs, and inserting said heterologous genes under the control of a heterologous internal promoter or the 5 'LTR promoter.

13. A method of modifying cells, characterized in that an infection or a transfection of said cells is carried out with retroviral vectors according to one of claims 1 to 10.

14. The method of claim 13, characterized in that one carries out a co-infection or co-transfection with an assistant virus which comprises the genes ensuring ia replication of said retroviral vector.

15. Cell modified by the implementation of a method according to one of claims 13 and 14.

16. Cell according to claim 15, characterized in that it is avian cells.

17. Cell according to claim 16, characterized in that it is chicken or quail embryonic fibroblasts.

18. A method of preparing at least one protein determined by cell culture according to one of claims 15 to 17, the gene being a foreign gene coding for said protein, the protein being recovered after culture.