Mus cervicolor murine leukemia virus isolate M813 belongs to a unique receptor interference group.
ABSTRACT: Murine leukemia virus (MuLV) M813 was originally isolated from the Southeast Asian rodent Mus cervicolor. As with the ecotropic MuLVs derived from Mus musculus, its host range is limited to rodent cells. Earlier studies have mapped its receptor to chromosome 2, but it has not been established whether M813 shares a common receptor with any other MuLVs. In this study, we have performed interference assays with M813 and viruses from four interference groups of MuLV. The infection efficiency of M813 was not compromised in cells expressing any one of the other MuLVs, demonstrating that M813 must use a distinct receptor for cell entry. The entire M813 env coding region was molecularly cloned. Sequence analysis revealed high similarity with other MuLVs but with a unique receptor-binding domain. Substitution of M813 env sequences in Moloney MuLV resulted in a replication-competent virus with a host range and interference profile similar to those of the biological clone M813. M813 thus defines a novel receptor interference group of type C MuLVs.
Project description: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.
Project description:Polytropic murine leukemia viruses (MuLVs) are generated by recombination of ecotropic MuLVs with env genes of a family of endogenous proviruses in mice, resulting in viruses with an expanded host range and greater virulence. Inbred mouse strains contain numerous endogenous proviruses that are potential donors of the env gene sequences of polytropic MuLVs; however, the precise identification of those proviruses that participate in recombination has been elusive. Three different structural groups of proviruses in NFS/N mice have been described and different ecotropic MuLVs preferentially recombine with different groups of proviruses. In contrast to other ecotropic MuLVs such as Friend MuLV or Akv that recombine predominantly with a single group of proviruses, Moloney MuLV (M-MuLV) recombines with at least two distinct groups. In this study, we determined that only three endogenous proviruses, two of one group and one of another group, are major participants in recombination with M-MuLV. Furthermore, the distinction between the polytropic MuLVs generated by M-MuLV and other ecotropic MuLVs is the result of recombination with a single endogenous provirus. This provirus exhibits a frameshift mutation in the 3' region of the surface glycoprotein-encoding sequences that is excluded in recombinants with M-MuLV. The sites of recombination between the env genes of M-MuLV and endogenous proviruses were confined to a short region exhibiting maximum homology between the ecotropic and polytropic env sequences and maximum stability of predicted RNA secondary structure. These observations suggest a possible mechanism for the specificity of recombination observed for different ecotropic MuLVs.
Project description: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.
Project description: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.
Project description: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.
Project description:Murine leukemia viruses (MuLVs) induce leukemias and lymphomas in mice. We have used fluorescence-activated cell sorter analysis to determine the hematopoietic phenotypes of tumor cells induced by a number of MuLVs. Tumor cells induced by ecotropic Moloney, amphotropic 4070A, and 10A1 MuLVs and by two chimeric MuLVs, Mo(4070A) and Mo(10A1), were examined with antibodies to 13 lineage-specific cell surface markers found on myeloid cell, T-cell, and B-cell lineages. The chimeric Mo(4070A) and Mo(10A1) MuLVs, consisting of Moloney MuLV with the carboxy half of the Pol region and nearly all of the Env region of 4070A and 10A1, respectively, were constructed to examine the possible influence of these sequences on Moloney MuLV-induced tumor cell phenotypes. In some instances, these phenotypic analyses were supplemented by Southern blot analysis for lymphoid cell-specific genomic DNA rearrangements at the immunoglobulin heavy-chain, the T-cell receptor gamma, and the T-cell receptor beta loci. The results of our analysis showed that Moloney MuLV, 4070A, Mo(4070A), and Mo(10A1) induced mostly T-cell tumors. Moloney MuLV and Mo(4070A) induced a wide variety of T-cell phenotypes, ranging from immature to mature phenotypes, while 4070A induced mostly prothymocyte and double-negative (CD4- CD8-) T-cell tumors. The tumor phenotypes obtained with 10A1 and Mo(10A1) were each less variable than those obtained with the other MuLVs tested. 10A1 uniformly induced a tumor consisting of lineage marker-negative cells that lack lymphoid cell-specific DNA rearrangements and histologically appear to be early undifferentiated erythroid cell-like precursors. The Mo(10A1) chimera consistently induced an intermediate T-cell tumor. The chimeric constructions demonstrated that while 4070A 3' pol and env sequences apparently did not influence the observed tumor cell phenotypes, the 10A1 half of pol and env had a strong effect on the phenotypes induced by Mo(10A1) that resulted in a phenotypic consistency not seen with other viruses. This result implicates 10A1 env in an active role in the tumorigenic process.
Project description:Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from Mus cervicolor, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (slc5a3) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. slc5a3 encodes sodium myo-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.
Project description:PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.
Project description:A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.
Project description:Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.