Genomes of murine leukemia viruses isolated from wild mice.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.     

Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts

Background

The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice.

Results

The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains.

Conclusion

The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.     

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.     

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

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.     

Diverse wild mouse origins of xenotropic, mink cell focus-forming, and two types of ecotropic proviral genes

We analyzed wild mouse DNAs for the number and type of proviral genes related to the env sequences of various murine leukemia viruses (MuLVs). Only Mus species closely related to laboratory mice carried these retroviral sequences, and the different subclasses of viral env genes tended to be restricted to specific taxonomic groups. Only Mus musculus molossinus carried proviral genes which cross-reacted with the inbred mouse ecotropic MuLV env gene. The ecotropic viral env sequence associated with the Fv-4 resistance gene was found in the Asian mice M. musculus molossinus and Mus musculus castaneus and in California mice from Lake Casitas (LC). Both M. musculus castaneus and LC mice carried many additional Fv-4 env-related proviruses, two of which are common to both mouse populations, which suggests that these mice share a recent common ancestry. Xenotropic and mink cell focus-forming (MCF) virus env sequences were more widely dispersed in wild mice than the ecotropic viral env genes, which suggests that nonecotropic MuLVs were integrated into the Mus germ line at an earlier date. Xenotropic MuLVs represented the major component of MuLV env-reactive genes in Asian and eastern European mice classified as M. musculus molossinus, M. musculus castaneus, and Mus musculus musculus, whereas Mus musculus domesticus from western Europe, the Mediterranean, and North America contained almost exclusively MCF virus env copies. M. musculus musculus mice from central Europe trapped near the M. musculus domesticus/M. musculus musculus hybrid zone carried multiple copies of both types of env genes. LC mice also carried both xenotropic and MCF viral env genes, which is consistent with the above conclusion that they represent natural hybrids of M. musculus domesticus and M. musculus castaneus.     

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.     

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.     

Evidence against expression of an endogenous murine leukemia virus causing cellular resistance to lysis by activated macrophages

In previous studies we observed that resistance of murine SV40-transformed fibroblast cell lines to cytolysis by activated macrophages was frequently associated with cellular expression of the gp70 of an endogenous ecotropic murine leukemia virus (MuLV). The work described here was initiated to test directly for a causative relationship between MuLV expression and resistance to lysis by macrophages. Northern blot analysis revealed that macrophage-resistant cells contain full length retroviral RNA. A panel of mAb which distinguish among host-range classes of MuLV detected only a non-recombinant ecotropic gp70 in these cells. The ecotropic MuLV from two independently derived macrophage resistant cells were isolated by limiting dilution cloning on Mus dunii fibroblasts. These viruses were then used to infect macrophage-sensitive cell lines and the resultant MuLV-positive cells tested for sensitivity to macrophage cytolysis. The MuLV-infected lines remained highly sensitive to macrophage lysis despite their high levels of cell surface gp70 and release of infectious MuLV. Thus, although we cannot rule out the possibility that MuLV or a product thereof is necessary for development of macrophage resistance in transformed cells, expression of MuLV per se is not sufficient to create the resistant phenotype.     

Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.     

Specific hybridization probes demonstrate fewer xenotropic than mink cell focus-forming murine leukemia virus env-related sequences in DNAs from inbred laboratory mice.

We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.     

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments.     

Amphotropic proviral envelope sequences are absent from the Mus germ line.

We derived an amphotropic murine leukemia virus (MuLV) type-specific probe for use in Southern blot hybridizations with cloned and genomic DNAs. A 133-base-pair RsaI-RsaI fragment from the 5' env region of the amphotropic viral isolate 4070A was subcloned into M13mp18 and radiolabeled in vitro. The probe detected the proviral DNAs in mink cells infected with seven different amphotropic MuLV isolates. The probe did not cross hybridize with the DNAs of molecular clones of ecotropic, mink cell focus-forming, or xenotropic MuLVs; nor did it anneal to the proviral DNAs of four xenotropic or six mink cell focus-forming viral isolates grown in mink cells. DNAs of 12 inbred laboratory mouse strains and more than 15 different wild mouse species and subspecies were examined for the presence of endogenous amphotropic env-related fragments. Amphotropic env-related sequences were found only in the DNAs of wild mice trapped in southern California in an area previously shown to harbor mice producing infectious amphotropic virus. Restriction enzyme analyses of DNAs from these mice showed that amphotropic sequences were not present as germ line copies but were the result of congenital or horizontal infection or both in this population. The DNAs of 11 various mammalian and avian species, including both natural predators of mice and squabs from the farms with virus-positive mice, lacked amphotropic envelope-related sequences.     

Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus.

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.     

Wild mouse ecotropic murine leukemia virus infection of inbred mice: dual-tropic virus expression precedes the onset of paralysis and lymphoma.

NFS/N mice inoculated at birth with an ecotropic murine leukemia virus (Cas-Br-MuLV) obtained from wild mice developed hind limb paralysis beginning at 7 weeks of age and nonthymic lymphomas beginning at more than 20 weeks of age. Studies of 1- to 7-week-old Cas-Br-M MuLV-infected mice showed the following: (i) a marked increase in nonecotropic MuLV-related antigens on spleen cells but not thymocytes beginning at 2 weeks; (ii) the appearance of dual-tropic mink cell focus-forming (MCF) MuLV-related gp70 in spleen but not thymus or brain cells at 4 weeks; and (iii) the isolation of infectious MCF MuLV from spleen cells of 7-week-old mice. A role for MCF MuLV in Cas-Br-M MuLV-induced nonthymic lymphomas is indicated by these studies, and a role for recombinant MuLV in neurological disease is considered.     

Neurotropic Cas-BR-E murine leukemia virus harbors several determinants of leukemogenicity mapping in different regions of the genome.

The infectious virus derived from the molecularly cloned genome of the neurotropic ecotropic murine Cas-BR-E retrovirus was previously shown to have retained the ability to induce hind-limb paralysis and leukemia when inoculated into susceptible mice (P. Jolicoeur, N. Nicolaiew, L. DesGroseillers, and E. Rassart, J. Virol. 45:1159-1163, 1983). To map the viral sequences encoding the leukemogenic determinant(s) of this virus, we used chimeric viral genomes constructed in vitro between cloned viral DNAs from the leukemogenic Cas-BR-E murine leukemia virus (MuLV) and from the related nonleukemogenic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered from NIH 3T3 cells microinjected with these DNAs, were inoculated into newborn NIH Swiss, SIM.S, and SWR/J mice to test their leukemogenic potential. We found that each chimeric MuLV, harboring either the long terminal repeat, the gag-pol, or the pol-env region of the Cas-BR-E MuLV genome, was leukemogenic, indicating that this virus harbors several determinants of leukemogenicity mapping in different regions of its genome. This result suggests that the amphotropic 4070-A MuLV has multiple regions along its genome which prevent the expression of its leukemogenic phenotype, and it also shows that substitution of only one of these regions for Cas-BR-E MuLV sequences is sufficient to make it leukemogenic.     

Mammary tumorigenesis in feral Mus cervicolor popaeus.

A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors.     

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.     

Sequences determining the pH dependence of viral entry are distinct from the host range-determining region of the murine ecotropic and amphotropic retrovirus envelope proteins.

The entry of ecotropic and amphotropic murine leukemia retroviruses (MuLV) into cells was investigated by using viral vector particles carrying chimeric amphotropic-ecotropic envelope glycoproteins on their surface. Chimeras were made by joining, at or near the polyproline hinge, the N-terminal portion of the amphotropic (4070A) gp70 onto the C-terminal portion of the ecotropic (Moloney) gp70 and p15E (constructs AE2 and AE4) or vice versa (AE12). Transduction efficiency of the constructs was tested on target cells that either have only ecotropic receptors (CHO-2 and CHO-11 cells), only amphotropic receptors (mink lung fibroblasts and Cos 1 cells), or both types of receptors (NIH 3T3 cells). The assay made use of the fact that the mechanism for viral entry of ecotropic viruses is pH dependent while that of amphotropic viruses is pH independent. Treatment of target cells with NH4Cl, which prevents the reduction of pH within endosomes, reduced the titers of viral particles bearing the C-terminal moiety from the ecotropic envelope but did not reduce the titers of particles which had a C-terminal moiety from the amphotropic envelope. In addition, in contrast to other low-pH-dependent enveloped viruses, brief acid treatment did not allow surface-bound viruses to bypass the NH4Cl block. The results indicate that the pH dependence of viral entry is a property of the sequences C terminal to the polyproline hinge.     

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.