A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection

Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope.     

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus.

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.     

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.     

The amphotropic and ecotropic murine leukemia virus envelope TM subunits are equivalent mediators of direct membrane fusion: implications for the role of the ecotropic envelope and receptor in syncytium formation and viral entry.

The murine leukemia virus (MuLV) envelope protein was examined to determine which sequences are responsible for the differences in direct membrane fusion observed with the ecotropic and amphotropic MuLV subtypes. These determinants were studied by utilizing amphotropic-ecotropic chimeric envelope proteins that have switched their host range but retain their original fusion domain (TM subunit). Fusion was tested both in rodent cells and in 293 cells bearing the human homolog of the ecotropic MuLV receptor. The results demonstrate that the amphotropic TM is able to mediate cell-to-cell fusion to an extent equivalent to that mediated by the ecotropic TM, indicating that their fusion domains are equivalent. The "murinized" human homolog of the ecotropic receptor supports syncytium formation as well as the native murine receptor. These findings suggest that interactions between the ecotropic envelope protein and conserved sequences in the ecotropic receptor are the principal determinants of syncytium formation. The relationship of the fusion phenotype to pH-dependent infection and the route of viral entry was examined by studying virions bearing the chimeric envelope proteins. Such virions appear to enter cells via a pathway that is directed by the host range-determining region of their envelope rather than by sequences that confer pH dependence. Therefore, the pH dependence of infection may not reflect the initial steps in viral entry. Thus, it appears that both the syncytium phenotype and the route of viral entry are properties of the viral receptor, the amino-terminal half of the ecotropic envelope protein, or the interaction between the two.     

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.     

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.     

Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.     

Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.     

Introduction of human genomic sequences renders CHO-K1 cells susceptible to infection by amphotropic retroviruses.

To learn more about the nature of the block to infection by amphotropic retroviruses exhibited by Chinese hamster cells (CHO-K1), CHO-K1 cells were made susceptible to amphotropic retrovirus infection by introducing genomic DNA from infectable human cells. A clone, designated CHO18, was obtained and shown to be infected as efficiently as NIH 3T3 fibroblasts. Susceptibility of CHO18 cells to infection was specific to retroviruses and vectors bearing an amphotropic envelope. By comparison to CHO-K1 cells, CHO18 cells may provide a useful model for analysis of the molecular events involved in the retrovirus-receptor interaction.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses.

An epitope common to all classes of murine leukemia viruses (MuLVs) was detected by reactivity of MuLVs with a rat monoclonal antibody (MAb) termed 83A25. The antibody is of the immunoglobulin G2a isotype and was derived after fusion of NS-1 myeloma cells with spleen cells from a Fischer rat immunized with a Friend polytropic MuLV. The antibody reacted with nearly all members of the ecotropic, polytropic, xenotropic, and amphotropic classes of MuLVs. Unreactive viruses were limited to the Friend ecotropic MuLV, Rauscher MuLV, and certain recombinant derivatives of Friend ecotropic MuLV. The presence of an epitope common to nearly all MuLVs facilitated a direct quantitative focal immunofluorescence assay for MuLVs, including the amphotropic MuLVs for which no direct assay has been previously available. Previously described MAbs which react with all classes of MuLVs have been limited to those which react with virion core or transmembrane proteins. In contrast, protein immunoblot and immunoprecipitation analyses established that the epitope reactive with MAb 83A25 resides in the envelope glycoproteins of the viruses. Structural comparisons of reactive and nonreactive Friend polytropic viruses localized the epitope near the carboxyl terminus of the glycoprotein. The epitope served as a target for neutralization of all classes of MuLV with MAb 83A25. The efficiency of neutralization varied with different MuLV isolates but did not correlate with MuLV interference groups.     

Role of Variable Regions A and B in Receptor Binding Domain of Amphotropic Murine Leukemia Virus Envelope Protein

For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.     

Retrovirus molecular conjugates. A novel, high transduction efficiency, potentially safety-improved, gene transfer system

Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans.     

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

A Unique Heparin-Binding Domain in the Envelope Protein of the Neuropathogenic PVC-211 Murine Leukemia Virus May Contribute to Its Brain Capillary Endothelial Cell Tropism

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.     

Properties of the naturally occurring soluble surface glycoprotein of ecotropic murine leukemia virus: binding specificity and possible conformational change after binding to receptor

Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4(r). Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37 degrees C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutant env gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of the Fv-4(r) SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37 degrees C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.     

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.     

Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses.

Viral interference studies have demonstrated the existence of four distinct murine leukemia virus (MuLV) receptors on NIH 3T3 mouse cells. The four viral interference groups are ecotropic MuLV; mink cell focus inducing virus (MCF); amphotropic MuLV; and 10A1, a recombinant derivative of amphotropic MuLV that uses a unique receptor but also retains affinity for the amphotropic MuLV receptor. We report here that 10A1 infects rat and hamster cells, unlike its amphotropic parent. We isolated an infectious molecular clone of 10A1 and present here the sequences of the env genes and enhancer regions of amphotropic MuLV and 10A1. The deduced amino acid sequences of amphotropic MuLV and 10A1 gp70su are remarkably similar to those of MCF and xenotropic MuLV (for which mouse cells lack receptors), with 64% amino acids identical in the four groups. We generated a consensus from these comparisons. Further, the differences are largely localized to a few discrete regions: (i) amphotropic MuLV has two short insertions relative to MCF, at residues 87 to 92 and 163 to 169, and (ii) amphotropic MuLV and MCF are totally different in a hypervariable region, which is greater than 30% proline, at residues approximately 253 to 304. 10A1 closely resembles amphotropic MuLV in its N terminus but contains an MCF-type hypervariable region. These results suggest the possibility that receptor specificity is localized in these short variable regions and further that the unique receptor specificity of 10A1 is due to the novel combination of amphotropic MuLV and MCF sequences rather than to the presence of any novel sequences. The Env proteins of ecotropic MuLV are far more distantly related to those of the other four groups than the latter are to each other. We also found that the enhancer regions of amphotropic MuLV and 10A1 are nearly identical, although 10A1 is far more leukemogenic than amphotropic MuLV.