Early events in retrovirus XMRV infection of the wild-derived mouse Mus pahari

A novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. Standard Mus musculus laboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whether Mus pahari could serve as a model for XMRV, primary Mus pahari fibroblasts and mice were infected with cell-free XMRV. Infection of cells in vitro resulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV into Mus pahari mice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP(+) CD4(+) T cells and CD19(+) B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3 in vivo. These data demonstrate infection of Mus pahari by XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infection in vivo. These data support the use of Mus pahari as a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.     

Evolution of Functional and Sequence Variants of the Mammalian XPR1 Receptor for Mouse Xenotropic Gammaretroviruses and the Human-Derived Retrovirus XMRV

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.The XPR1 receptor mediates entry for the mouse leukemia viruses (MLVs) with xenotropic and polytropic host ranges (X-MLVs and P-MLVs, respectively). X-MLVs and P-MLVs can be isolated from laboratory mice and are capable of infecting cells of nonrodent species; these viruses are distinguished by the ability of P-MLVs, but not X-MLVs, to infect cells of the laboratory mouse and by the cytopathic and leukemogenic properties of P-MLVs, also termed MCF MLVs (mink cell focus-inducing MLVs) (11, 16, 24). XPR1 is also the receptor for several wild mouse isolates with an atypical host range (6, 48, 49) and for the recently described virus XMRV (xenotropic murine leukemia virus-related virus) (8), isolated from human patients with prostate cancer or chronic fatigue syndrome (27, 37, 43). Studies on the XPR1 receptor have identified residues critical for virus entry and described functionally distinct variants of XPR1 in human and rodent species that differ in their abilities to mediate entry of various virus isolates (18, 29, 31, 48, 49).In Mus, four receptor variants of Xpr1 are found in different taxonomic groups. Xpr1ⁿ was originally described in strains of the laboratory mouse (1, 41, 51), which are largely derived from Mus domesticus (50). Xpr1^c was identified in the Asian species Mus castaneus (29, 31); Xpr1^p is in the Asian species Mus pahari (48); and Xpr1^sxv was found in several Eurasian species (18, 31). These variants are distinguished by their differential susceptibilities to prototype X-MLVs and P-MLVs as well as to two wild mouse isolates, CasE#1 and Cz524 (49); only Xpr1^sxv encodes a receptor that is fully permissive for all isolates. The host range differences of these various virus isolates are due to sequence polymorphisms in both receptor and viral envelope genes.The various mouse X/P-MLV isolates and the humanized XMRV define six different tropism patterns based on infectivity on rodent cells carrying Xpr1 variants (49). These tropisms distinguish the two wild mouse isolates, CasE#1 and Cz524, and identify two P-MLV host range subgroups and two X-MLV/XMRV subgroups. Specific XPR1 residues responsible for entry of these viruses have been identified by analysis of rodent Xpr1 variants and mutants. These receptor determinants lie in two of the four predicted extracellular loops (ECLs) of Xpr1, ECL3 and ECL4 (31, 44, 48, 49). Two critical amino acids have been defined for X-MLV entry: K500 in ECL3 and T582 in ECL4 (31). These two receptor determinants independently produce X-MLV receptors but are not functionally equivalent, as the Δ582Τ insertion into Xpr1ⁿ generates a receptor for CasE#1, but the E500K substitution does not (48). Sensitivity to the six tropism subgroups is further modulated by specific substitutions at ECL3 residues 500, 507, and 508 (49). The sequence variations that distinguish the rodent XPR1 receptors can result in subtle differences in the efficiency of virus infection or complete resistance to specific X/P-MLVs.The characterization of host genes that effect and/or block entry has obvious importance for a broader understanding of how viruses spread in natural populations and are transmitted to new hosts and how those populations adapt to retrovirus infections. The four house mouse species carry endogenous retroviruses (ERVs) for X-MLVs and P-MLVs (XMVs and PMVs, respectively) (3, 20, 42), and three of these species harbor infectious X-MLVs (4, 19, 48, 49). Restrictive variants of the XPR1 receptor have evolved in these virus-infected mice, along with the virus envelope (env) variants that define the tropism subgroups. We thus sought to examine the evolution of Xpr1 in rodent species, and we extended this functional and sequence analysis to nonrodent species for two reasons. First, identification of XMRV in several human patient cohorts (27, 37, 43), the recent detection of P-MLV-related sequences in patients and blood donors (26), and the multiple instances of transspecies transmission of mouse gammaretroviruses (33) support an effort to describe factors that mediate or modulate virus entry in these species. Second, analysis of nonrodent species with novel patterns of virus restriction may uncover different or additional entry determinants. In the present study, we characterized 49 mice of different species or from different geographic locations and 24 other mammalian species for sequence and functional variants of the Xpr1 receptor. We identified a novel 5th functional Xpr1 variation in Mus, showed that restrictive XPR1 receptors in the three MLV-infected house mouse lineages have different deletions in ECL4, demonstrated that XPR1 is under positive selection, identified novel virus restriction phenotypes in nonrodent species, and demonstrated that XMRV relies on unique entry determinants.     

The Avian XPR1 Gammaretrovirus Receptor Is under Positive Selection and Is Disabled in Bird Species in Contact with Virus-Infected Wild Mice

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.     

Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion.

Murine ecotropic leukemia viruses use a common receptor for entry into host cells; however, the site of virus fusion appears to differ with the host cell. Entry in mouse NIH 3T3 fibroblasts is by endocytosis, whereas entry in rat XC sarcoma cells is by surface fusion. We report here the identification of a step common to both entry pathways, as well as of a step unique to the endocytic pathway. Recent demonstration of the clustering of the virus receptor on rat cells suggested a possible interaction of the receptor with the cellular cytoskeleton (M. H. Woodard, W. A. Dunn, R. O. Laine, M. Malandro, R. McMahon, O. Simell, E. R. Block, and M. S. Kilberg, Am. J. Physiol. 266:E817-E824, 1994). We tested the hypothesis that such an interaction might influence receptor function. We found that entry into NIH 3T3 and XC cells was greatly diminished by the disruption of the actin network before but not shortly after virus internalization, suggesting the actin network plays a critical role in an early step common to both entry pathways. Disruption of microtubules before and shortly after virus internalization markedly reduced entry in NIH 3T3 cells, while entry into XC cells remained efficient. These data suggest that intact microtubules are required in a postpenetration step unique to efficient virus entry via endocytosis. The physiological function of the receptor was not affected by disruption of either the actin network or the microtubules, as the uptake of cationic amino acids in NIH 3T3 and XC cells was comparable to that in control cells even when the cytoskeleton remained disrupted for as long as 3 h.     

Rescue of endogenous 30S retroviral sequences from mouse cells by baboon type C virus.

Mus musculus SC-1 cells were infected with M7 baboon type C virus. The progeny of this infection included viral pseudotypes that contained M7 helper virus and endogenous 30S retrovirus-associated sequences derived from SC-1 cells (RAS). The RAS sequences are unrelated by nucleic acid hybridization criteria to previously described types of murine retroviruses and do not code for known murine viral structural proteins. The RAS genome is present in multiple copies in the DNA of laboratory (M. musculus) and Asian (M. caroli and M. cervicolor) mice, is expressed in the RNA of uninfected mouse cells, and can be efficiently rescued by type C, but not type B, viruses. RAS is closely related to 30S virus-associated RNA in NIH/3T3 and BALB/c JLSV-9 cells and may be analogous to the defective 30S RNA sequences found in rats.     

Polymorphisms of the Cell Surface Receptor Control Mouse Susceptibilities to Xenotropic and Polytropic Leukemia Viruses

The differential susceptibilities of mouse strains to xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs, respectively) are poorly understood but may involve multiple mechanisms. Recent evidence has demonstrated that these viruses use a common cell surface receptor (the X-receptor) for infection of human cells. We describe the properties of X-receptor cDNAs with distinct sequences cloned from five laboratory and wild strains of mice and from hamsters and minks. Expression of these cDNAs in resistant cells conferred susceptibilities to the same viruses that naturally infect the animals from which the cDNAs were derived. Thus, a laboratory mouse (NIH Swiss) X-receptor conferred susceptibility to P-MLVs but not to X-MLVs, whereas those from humans, minks, and several wild mice (Mus dunni, SC-1 cells, and Mus spretus) mediated infections by both X-MLVs and P-MLVs. In contrast, X-receptors from the resistant mouse strain Mus castaneus and from hamsters were inactive as viral receptors. These results suggest that X-receptor polymorphisms are a primary cause of resistances of mice to members of the X-MLV/P-MLV family of retroviruses and are responsible for the xenotropism of X-MLVs in laboratory mice. By site-directed mutagenesis, we substituted sequences between the X-receptors of M. dunni and NIH Swiss mice. The NIH Swiss protein contains two key differences (K500E in presumptive extracellular loop 3 [ECL 3] and a T582 deletion in ECL 4) that are both required to block X-MLV infections. Accordingly, a single inverse mutation in the NIH Swiss protein conferred X-MLV susceptibility. Furthermore, expression of an X-MLV envelope glycoprotein in Chinese hamster ovary cells interfered efficiently with X-MLV and P-MLV infections mediated by X-receptors that contained K500 and/or T582 but had no effect on P-MLV infections mediated by X-receptors that lacked these amino acids. In contrast, moderate expression of a P-MLV (MCF247) envelope glycoprotein did not cause substantial interference, suggesting that X-MLV and P-MLV glycoproteins interfere nonreciprocally with X-receptor-mediated infections. We conclude that P-MLVs have become adapted to utilize X-receptors that lack K500 and T582. A penalty for this adaptation is a reduced ability to interfere with superinfection. Because failure of interference is a hallmark of several exceptionally pathogenic retroviruses, we propose that it contributes to P-MLV-induced diseases.     

Rmcf2, a xenotropic provirus in the Asian mouse species Mus castaneus, blocks infection by polytropic mouse gammaretroviruses

Cells from the Asian wild mouse species Mus castaneus are resistant to infection by the polytropic host range group of mouse gammaretroviruses. Two factors are responsible for this resistance: a defective XPR1 cell surface receptor for polytropic murine leukemia viruses (P-MLVs), and a resistance factor detectable only in interspecies hybrids between M. castaneus and mice with an XPR1 variant that permits infection by xenotropic MLVs (X-MLVs) as well as P-MLVs. This second novel virus resistance phenotype has been associated with expression of viral Env glycoprotein; Northern blotting with specific hybridization probes identified a spliced X-MLV env message unique to virus-resistant mice. These observations suggest that resistance is due to expression of one or more endogenous X-MLV envelope genes that interfere with infection by exogenous P-MLVs. M. castaneus contains multiple X-MLV proviruses, but serial backcrosses reduced this proviral content and permitted identification of a single proviral env sequence inherited with resistance. The resistance phenotype and the provirus were mapped to the same site on distal chromosome 18. The provirus was shown to be a full-length provirus highly homologous to previously described X-MLVs. Use of viral pseudotypes confirmed that this resistance gene, termed Rmcf2, prevents entry of P-MLVs. Rmcf2 resembles the virus resistance genes Fv4 and Rmcf in that it produces Env glycoprotein but fails to produce infectious virus; the proviruses associated with all three resistance genes have fatal defects. This type of provirus Env-mediated resistance represents an important defense mechanism in wild mouse populations exposed to endemic infections.     

Caveola-dependent endocytic entry of amphotropic murine leukemia virus

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t(1/2) approximately 5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.     

Common inbred strains of the laboratory mouse that are susceptible to infection by mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV

Laboratory mouse strains carry endogenous copies of the xenotropic mouse leukemia viruses (X-MLVs), named for their inability to infect cells of the laboratory mouse. This resistance to exogenous infection is due to a nonpermissive variant of the XPR1 gammaretrovirus receptor, a resistance that also limits in vivo expression of germ line X-MLV proviruses capable of producing infectious virus. Because laboratory mice vary widely in their proviral contents and in their virus expression patterns, we screened inbred strains for sequence and functional variants of the XPR1 receptor. We also typed inbred strains and wild mouse species for an endogenous provirus, Bxv1, that is capable of producing infectious X-MLV and that also contributes to the generation of pathogenic recombinant MLVs. We identified the active Bxv1 provirus in many common inbred strains and in some Japanese Mus molossinus mice but in none of the other wild mouse species that carry X-MLVs. Our screening for Xpr1 variants identified the permissive Xpr1(sxv) allele in 7 strains of laboratory mice, including a Bxv1-positive strain, F/St, which is characterized by lifelong X-MLV viremia. Cells from three strains carrying Xpr1(sxv), namely, SWR, SJL, and SIM.R, were shown to be infectable by X-MLV and XMRV; these strains carry different alleles at Fv1 and vary in their sensitivities to specific X/P-MLV isolates and XMRV. Several strains with Xpr1(sxv) lack the active Bxv1 provirus or other endogenous X-MLVs and may provide a useful model system to evaluate the in vivo spread of these gammaretroviruses and their disease potential in their natural host.     

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.     

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.     

Nucleotide Sequence Relationship Between Intracisternal Type A Particles of Mus musculus and an Endogenous Retrovirus (M432) of Mus cervicolor

Intracisternal type A particles are retrovirus-like structures found in embryonic cells and many tumors of Mus musculus but having no clear relationship with other retroviruses of this mouse species. We have observed a partial nucleotide sequence homology between the high-molecular-weight (32S and 35S) RNA components of intracisternal A-particles from a neuroblastoma cell line and the 70S RNA fraction from M432, a type of retrovirus endogenous to the Asian mouse Mus cervicolor. M432 complementary DNA (cDNA) was hybridized to the extent of 30% by the A-particle RNAs. The hybrids showed a lower thermal stability (DeltaT(m), 7 degrees C) than those formed with homologous RNA. The reaction was commensurate with that found between M432 cDNA and divergent sequences in the M. musculus genome. The capacity to hybridize M432 cDNA was closely correlated with the concentration of A-particle sequences in the cytoplasmic RNA of several M. musculus cell types. The major RNA fraction of M432 virus showed a reciprocal partial reaction with the A-particle cDNA's; the virus, which was grown in NIH/3T3 (M. musculus) cells, also contained a small proportion of apparently authentic A-particle nucleotide sequences. A subset of A-particle sequences seemed to be almost totally lacking in the main M432 RNA. The A-particle cDNA's hybridized extensively with divergent sequences in M. cervicolor cellular DNA, indicating that this mouse species may contain not only the partially homologous M432 virogene, but also a more complete genetic equivalent of the intracisternal A-particle.     

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.     

Human plasma membrane-derived vesicles inhibit the phagocytosis of apoptotic cells - Possible role in SLE

Plasma membrane-derived vesicles (PMVs) also known as microparticles, are small membrane-bound vesicles released from the cell membrane via blebbing and shedding. PMVs have been linked with various physiological functions as well as pathological conditions such as inflammation, autoimmune disease and cardiovascular disease. PMVs are characterised by the expression of phosphatidylserine (PS) on the plasma membrane. PS, also expressed on apoptotic cells (ACs) enables macrophages to phagocytose ACs. As it is widely known that PMV production is increased during apoptosis, we were able to show that PMVs could compete dose dependently with ACs for the PS receptor on macrophages, so reducing phagocytosis of ACs. In a clinical setting this may result in secondary necrosis and further pathological conditions. In SLE in which there are raised PMV levels, there is an anti-phospholipid-mediated increase in PMV release, which can be abrogated by depletion of IgG. Our work provides an insight into how PMVs may play a role in the aetiology of autoimmune disease, in particular SLE.     

Sodium-dependent myo-inositol transporter 1 is a cellular receptor for Mus cervicolor M813 murine leukemia virus

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.     

Membrane fusion and cell entry of XMRV are pH-independent and modulated by the envelope glycoprotein's cytoplasmic tail

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.     

The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.

Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.     

Molecular cloning of Mus dunni endogenous virus: an unusual retrovirus in a new murine viral interference group with a wide host range.

Mus dunni endogenous virus (MDEV) is activated from cells of the Asian wild mouse M. dunni (also known as Mus terricolor) in response to treatment with either 5-iodo-2'-deoxyuridine or hydrocortisone. MDEV represents a new murine retrovirus interference group and thus appears to use a different receptor for entry into cells than do other murine retroviruses. Here we show that MDEV is also not in the gibbon ape leukemia virus or RD114 virus interference groups. A retroviral vector with an MDEV pseudotype was capable of efficiently infecting a wide variety of cells from different species, indicating that the MDEV receptor is widely expressed. We isolated a molecular clone of this virus which exhibited no hybridization to any cloned retrovirus examined, suggesting that MDEV has an unusual genome. One copy of a possible retrovirus element that weakly hybridized with MDEV was present in the genomes of laboratory strains of mice, while no such elements were present in other species examined. A virus activated by 5-iodo-2'-deoxyuridine from cells of a BALB/c mouse, however, was not related to MDEV by either hybridization or interference analyses.