Generation of novel syncytium-inducing and host range variants of ecotropic moloney murine leukemia virus in Mus spicilegus

Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SUenv. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni.     

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.     

Mink epithelial cell killing by pathogenic murine leukemia viruses involves endoplasmic reticulum stress

We previously demonstrated that mink cells undergo apoptosis after MCF13 murine leukemia virus (MLV) infection. In this study, we observed that virus-infected mink epithelial cells had significantly larger amounts of steady-state levels of MCF13 MLV envelope precursor protein (gPr80(env)) than did Mus dunni fibroblasts, which are resistant to virus-induced cytopathicity. Infection of mink cells with the noncytopathic NZB-9 MLV did not result in the accumulation of gPr80(env). MCF13 MLV infection of mink cells produced low cell surface expression of envelope glycoprotein and less efficient spread of infectious virus. Western blot analysis of mink epithelial cells infected with MCF13 MLV showed an increase in GRP78/BiP, which was not observed for either mink cells infected with NZB-9 MLV or M. dunni fibroblasts infected with MCF13 MLV. MCF13 MLV infection of mink cells also resulted in a significant upregulation of CHOP/GADD153. These results indicate that the accumulation of MCF13 MLV gPr80(env) triggers endoplasmic reticulum stress, which may mediate apoptosis in mink epithelial cells.     

Characterization of recombinant nonecotropic murine leukemia viruses from the wild mouse species Mus spretus

The wild mouse species most closely related to the common laboratory strains contain proviral env genes of the xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs). To determine if the polytropic proviruses of Mus spretus contain functional genes, we inoculated neonates with Moloney MLV (MoMLV) or amphotropic MLV (A-MLV) and screened for viral recombinants with altered host ranges. Thymus and spleen cells from MoMLV-inoculated mice were plated on Mus dunni cells and mink cells, since these cells do not support the replication of MoMLV, and cells from A-MLV-inoculated mice were plated on ferret cells. All MoMLV-inoculated mice produced ecotropic viruses that resembled their MoMLV progenitor, although some isolates, unlike MoMLV, grew to high titers in M. dunni cells. All of the MoMLV-inoculated mice also produced nonecotropic virus that was infectious for mink cells. Sequencing of three MoMLV- and two A-MLV-derived nonecotropic recombinants confirmed that these viruses contained substantial substitutions that included the regions of env encoding the surface (SU) protein and the 5' end of the transmembrane (TM) protein. The 5' recombination breakpoint for one of the A-MLV recombinants was identified in RNase H. The M. spretus-derived env substitutions were nearly identical to the corresponding regions in prototypical laboratory mouse polytropic proviruses, but the wild mouse infectious viruses had a more restricted host range. The M. spretus proviruses contributing to these recombinants were also sequenced. The seven sequenced proviruses were 99% identical to one another and to the recombinants; only two of the seven had obvious fatal defects. We conclude that the M. spretus proviruses are likely to be recent germ line acquisitions and that they contain functional genes that can contribute to the production of replication-competent virus.     

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag–pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

A Mus dunni cell line that lacks sequences closely related to endogenous murine leukemia viruses and can be infected by ectropic, amphotropic, xenotropic, and mink cell focus-forming viruses

A Mus dunni cell line has been developed that is permissive for all four classes of murine leukemia viruses (MuLV): ecotropic, amphotropic, xenotropic, and mink cell focus-forming viruses. The M. dunni cells contain fewer MuLV-related sequences than do feral or domestic mouse, rat, or mink cells. Infection of the line by ecotropic MuLV induces a distinct cytopathic effect, and the cells can be readily transfected by MuLV DNA. The M. dunni line has been used to isolate an endogenous MuLV from the SC-1 feral mouse cell line.     

Murine retroviruses use at least six different receptors for entry into Mus dunni cells.

Murine retroviruses have been divided into six interference groups that use different receptors for cell entry: the ecotropic, xenotropic, polytropic, amphotropic, 10A1, and Mus dunni endogenous virus groups. Some interference is observed between xenotropic and polytropic viruses and between amphotropic and 10A1 viruses, indicating some overlap in receptor specificity between these groups, but otherwise these interference groups appear completely independent. In contrast, one study found interference among many of these groups when Mus dunni wild mouse cells were examined with an immunofluorescence assay to detect infection by the challenge virus. Here we have used a more direct assay for cell entry by using pseudotyped retroviral vectors to measure interference in M. dunni cells, and we find no evidence for extensive interference between members of different murine retrovirus groups. Indeed, our results in M. dunni cells are consistent with interference results observed in other cell types and indicate that the anomalous interference results previously observed in M. dunni cells with the immunofluorescence assay were most likely due to factors other than those that affect receptor-mediated virus entry. In summary, our results show that murine retroviruses use at least six different receptors for entry into M. dunni cells.     

Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes.

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag-pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

Mus spicilegus endogenous retrovirus HEMV uses murine sodium-dependent myo-inositol transporter 1 as a receptor

We sought to determine the relationship between two recent additions to the murine leukemia virus (MLV) ecotropic subgroup: Mus cervicolor isolate M813 and Mus spicilegus endogenous retrovirus HEMV. Though divergent in sequence, the two viruses share an Env protein with similarly curtailed VRA and VRB regions, and infection by both is restricted to mouse cells. HEMV and M813 displayed reciprocal receptor interference, suggesting that they share a receptor. Expression of the M813 receptor murine sodium-dependent myo-inositol transporter 1 (mSMIT1) allowed previously nonpermissive cells to be infected by HEMV, indicating that mSMIT1 also serves as a receptor for HEMV. Our findings add HEMV as a second member to the MLV subgroup that uses mSMIT1 to gain entry into cells.     

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.     

Identification of the myelin protein plasmolipin as the cell entry receptor for Mus caroli endogenous retrovirus

The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.     

Contribution of virus-receptor interaction to distinct viral proliferation of neuropathogenic and nonneuropathogenic murine leukemia viruses in rat glial cells

The efficiency of receptor-mediated entry of pseudotyped virus carrying the surface protein (SU) of clone A8, a neuropathogenic variant of Friend murine leukemia virus (FrMLV), to rat glial cell line F10 was 1 order of magnitude greater than that of pseudotyped virus carrying SU of nonneuropathogenic FrMLV clone 57. Introduction of the gene coding for ecotropic MLV receptor on F10 cells (F10-ecoR) into SIRC cells, which are naturally resistant to FrMLV infection, also revealed the difference in receptor recognition between the A8 and the 57 viruses. Our results show that the difference in receptor utilization between A8-SU and 57-SU only partially explains the 3-order-of-magnitude difference in proliferation between A8 and 57 viruses in F10 cells.     

Analysis of the unique hamster cell tropism of ecotropic murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV). Previous studies from our laboratory demonstrated that unlike the parental F-MuLV, PVC-211 MuLV can infect rat brain capillary endothelial cells efficiently and that it has acquired genetic changes responsible for its expanded cellular tropism. To determine if PVC-211 MuLV also has expanded its host range, we tested its infectivity on Chinese hamster ovary-derived CHO-K1 cells, which are generally resistant to ecotropic MuLV. The results indicated that PVC-211 MuLV, but not F-MuLV, was highly infectious for CHO-K1 cells. Studies using glycosylation inhibitors and glycosylation mutants of CHO-K1 cells, as well as interference studies, suggested that PVC-211 MuLV has acquired the ability to interact with the ecotropic MuLV receptor on CHO-K1 cells that has undergone glycosylation-dependent modification. Using chimeric viruses between PVC-211 MuLV and F-MuLV, we were able to localize the viral genetic element crucial for CHO-K1 cell tropism within the env gene of PVC-211 MuLV and show that glycine at position 116 and lysine at position 129 of the envelope glycoprotein SU were important. These viral determinants also appear to confer tropism for other hamster cells resistant to ordinary ecotropic MuLVs. Further studies on the interaction between PVC-211 MuLV and the receptor on hamster cells may provide novel insights into the molecular mechanisms for receptor recognition and binding by viral envelope glycoproteins.     

Complementation of a binding-defective retrovirus by a host cell receptor mutant

The entry of ecotropic murine leukemia virus (MLV) into cells requires the interaction of the envelope protein (Env) with its receptor, mouse cationic amino acid transporter 1 (mATRC1). An aspartic acid-to-lysine change at position 84 (D84K) of ecotropic Moloney MLV Env abolishes virus binding and infection. We recently identified lysine 234 (rK234) in mATRC1 as a residue that influences virus binding and infection. Here we show that D84K virus infection increased 3,000-fold on cells expressing receptor with an rK234A change and 100,000-fold on cells expressing an rK234D change. The stronger complementation of D84K virus infection by rK234D than by the rK234A receptor suggests that although the major reason for loss of infection of D84K and D84R virus is due to steric hindrance and charge repulsion, the loss of an interaction of D84 with receptor appears to contribute as well. Taken together, these results indicate that D84 is very close to rK234 of mATRC1 in the bound complex and there is likely an interaction between them. The definitive localization of the receptor binding site on SU should facilitate the design of chimeric envelope proteins that target infection to new receptors by replacing the receptor binding site with an exogenous ligand sequence.     

A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.