Yuhe Yan

Six host range variants of the xenotropic/polytropic gammaretroviruses define determinants for entry in the XPR1 cell surface receptor

BackgroundThe evolutionary interactions between retroviruses and their receptors result in adaptive selection of restriction variants that can allow natural populations to evade retrovirus infection. The mouse xenotropic/polytropic (X/PMV) gammaretroviruses rely on the XPR1 cell surface receptor for entry into host cells, and polymorphic variants of this receptor have been identified in different rodent species.ResultsWe screened a panel of X/PMVs for infectivity on rodent cells carrying 6 different XPR1 receptor variants. The X/PMVs included 5 well-characterized laboratory and wild mouse virus isolates as well as a novel cytopathic XMV-related virus, termed Cz524, isolated from an Eastern European wild mouse-derived strain, and XMRV, a xenotropic-like virus isolated from human prostate cancer. The 7 viruses define 6 distinct tropisms. Cz524 and another wild mouse isolate, CasE#1, have unique species tropisms. Among the PMVs, one Friend isolate is restricted by rat cells. Among the XMVs, two isolates, XMRV and AKR6, differ from other XMVs in their PMV-like restriction in hamster cells. We generated a set of Xpr1 mutants and chimeras, and identified critical amino acids in two extracellular loops (ECLs) that mediate entry of these different viruses, including 3 residues in ECL3 that are involved in PMV entry (E500, T507, and V508) and can also influence infectivity by AKR6 and Cz524.ConclusionWe used a set of natural variants and mutants of Xpr1 to define 6 distinct host range variants among naturally occurring X/PMVs (2 XMV variants, 2 PMVs, 2 different wild mouse variants). We identified critical amino acids in XPR1 that mediate entry of these viruses. These gammaretroviruses and their XPR1 receptor are thus highly functionally polymorphic, a consequence of the evolutionary pressures that favor both host resistance and virus escape mutants. This variation accounts for multiple naturally occurring virus resistance phenotypes and perhaps contributes to the widespread distribution of these viruses in rodent and non-rodent species.

Origin, antiviral function and evidence for positive selection of the gammaretrovirus restriction gene Fv1 in the genus Mus

The Fv1 virus resistance gene is a coopted endogenous retrovirus (ERV) sequence related to the gag gene of the MuERV-L ERV family. Three major Fv1 resistance alleles have been identified in laboratory mice, and they target virus capsid genes to produce characteristic patterns of resistance to mouse leukemia viruses (MLVs). We identified Fv1 in 3 of the 4 Mus subgenera; its absence from Coelomys and 1 of 3 species of Pyromys indicate Fv1 was acquired shortly after the origin of the Mus genus. We sequenced Fv1 genes from 21 mice representative of the major taxonomic groups of Mus. Two lines of evidence indicate that Fv1 has had antiviral function for 7 million years of evolution. First, 2 species of African pygmy mice (subgenus Nannomys) show an Fv1-like MLV resistance, and transduced cells expressing the Nannomys Fv1 gene reproduce this resistance pattern. Second, sequence comparisons suggest that Fv1 has been involved in genetic conflicts throughout Mus evolution. We found evidence for strong positive selection of Fv1 and identified 6 codons that show evidence of positive selection: 3 codons in the C-terminal region including 2 previously shown to contribute to Fv1 restriction in laboratory mice, and 3 codons in a 10-codon segment overlapping the major homology region of Fv1; this segment is known to be involved in capsid multimerization. This analysis suggests that Fv1 has had an antiviral role throughout Mus evolution predating exposure of mice to the MLVs restricted by laboratory mouse Fv1, and suggests a mechanism for Fv1 restriction.

Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.

Wild Mouse Variants of Envelope Genes of Xenotropic/Polytropic Mouse Gammaretroviruses and Their XPR1 Receptors Elucidate Receptor Determinants of Virus Entry

ABSTRACT Mouse xenotropic and polytropic leukemia viruses (XMVs and PMVs) are closely related gammaretroviruses that use the XPR1 receptor for entry. To identify amino acid residues in XPR1 important for virus entry, we tested mouse cells derived from evolutionarily divergent species for susceptibility to prototypical PMVs, XMVs, and the wild mouse isolate CasE#1. CasE#1 has a variant XMV/PMV host range, and sequence analysis of the CasE#1 env gene identifies segments related to PMVs and XMVs. Cells from the Asian mouse species Mus pahari show a unique pattern of susceptibility to these three viruses; these cells are susceptible to XMVs and CasE#1 but are resistant to PMVs, whereas NIH 3T3 cells show the reciprocal pattern, susceptibility to only PMVs. The M. pahari XPR1 gene differs from that of NIH 3T3 in the two extracellular loops (ECLs) previously shown to mediate virus entry (M. Marin, C. S. Tailor, A. Nouri, S. L. Kozak, and D. Kabat, J. Virol. 73:9362-9368, 1999, and N. S. Van Hoeven and A. D. Miller, Retrovirology 2:76, 2005). Using transfected hamster cells expressing chimeric and mutated XPR1s, we demonstrated that the susceptibility differences between NIH 3T3 and M. pahari cells are receptor mediated, that PMV entry requires residues in ECL3, that the CasE#1 entry determinant is in ECL4, and that determinants for XMV entry are in both ECL3 and ECL4. Additional substitutions in ECL3 and ECL4 modulate virus susceptibility and suggest that ECL3 and ECL4 may contribute to the formation of a single virus receptor site. The position of M. pahari at the base of the Mus phylogenetic tree indicates that XPR1-mediated susceptibility to XMVs is the ancestral type in this genus and that the phenotypic variants of mouse XPR1 likely arose in conjunction with exposure to gammaretrovirus infections and coevolutionary adaptations in the viral envelope.

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

ABSTRACT 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.

Rmcf2, a Xenotropic Provirus in the Asian Mouse Species Mus castaneus, Blocks Infection by Polytropic Mouse Gammaretroviruses

ABSTRACT 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.

Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

BackgroundWe previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range.ResultsWe expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry.ConclusionVirus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific.

Novel Postentry Resistance to AKV Ecotropic Mouse Gammaretroviruses in the African Pygmy Mouse, Mus minutoides

ABSTRACT Cells of Mus minutoides, an African pygmy mouse of the subgenus Nannomys, are susceptible to ecotropic Moloney and Friend mouse leukemia viruses (MLVs) but not to AKV-type MLVs. Transfected MA139 ferret cells expressing the mCAT-1 cell surface receptor, with the minCAT-1 substitutions K222Q and V233L, did not restrict AKV MLV. The resistance of M. minutoides cells to AKV MLV was not relieved by inhibitors of glycosylation or by the introduction of NIH 3T3 mCAT-1. Resistance is thus not mediated by receptor sequence variation, expression level, or glycosylation. M. minutoides cells are also infectible with LacZ pseudotypes having AKV Env and Moloney MLV (MoMLV) Gag proteins, further indicating that AKV Env sequence variations do not contribute to the observed block. The pattern of virus resistance in M. minutoides differs from that of the known variants of the Fv1 postentry resistance gene; M. minutoides is equally resistant to N-, B-, and NR-tropic AKV viruses and is equally susceptible to NR- and NB-tropic Friend MLVs. This novel resistance blocks replication before reverse transcription, whereas Fv1 generally restricts replication after reverse transcription; M. minutoides cells produce 2-long-terminal-repeat viral DNA circles and linear viral DNA after infection with MoMLV but not with AKV MLV. Analysis of MoMLV-AKV MLV chimeras determined that the target of resistance is in the virus capsid gene. Mutagenesis demonstrated that restriction is mediated by two amino acid substitutions, H117L and A110R; substitutions at these sites can also be targeted by the resistance genes Fv1 and TRIM5α. M. minutoides cells thus have a novel postentry resistance to AKV MLVs.

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.

Mouse "xenotropic" gammaretroviruses, XMRV and their XPR1 receptor

Background Xenotropic/polytropic mouse leukemia viruses (XPMLVs) and the xenotropic murine leukemia virusrelated virus (XMRV) identified in human patient samples rely on the XPR1 receptor for entry into cells. There are 5 functional variants of Xpr1 in Mus species and laboratory mouse strains that differ in their ability to support entry of XMRV and various isolates of XPMLVs. Additional receptor variants are found in nonrodent vertebrate species.