We defined 6 variants of X/PMV gammaretroviruses with different species tropisms on rodent cells, and identified critical residues on the XPR1 receptor that mediate their entry. We identified two tropism variants among the PMVs, broad host range MLVs that can infect mouse cells as well as cells of many other species such as human and mink. FrMCF, unlike the other PMVs tested here, is very poorly infectious on rat cells. There are also 2 variants among the XMVs, viruses originally identified by their failure to infect cells of the laboratory mouse; AKR6 and the human derived XMRV, have XMV infectivity patterns on mouse cells, but resemble PMVs in their inability to infect hamster cells after the removal of the glycosylation block to gammaretrovirus infection. The fifth and sixth variants are represented by CasE#1 and Cz524, wild mouse isolates that differ from each other and from XMVs and PMVs in their pattern of infectivity on rodent cells.
Examination of the infectivity of these viruses on hamster cells expressing mutated XPR1 receptors establishes that different critical residues mediate entry of these viruses. As determined previously, K500 in ECL3 and T582 in ECL4 independently mediate entry of XMVs [6
]. These determinants are not, however, functionally equivalent, as T582 but not K500 can function as a receptor for CasE#1, whereas K500 but not T582 provides an efficient receptor for AKR6.
Residues at the C-terminal end of ECL3 are critical for entry of PMVs. PMV receptor function is reciprocally altered in Xpr1p and Xpr1n by substitution of the 4 most C-terminal of the residues that distinguish these receptors. Mutations at one of these sites, position 505 in an apparently unused glycosylation site, do not alter PMV susceptibility. Mutations at the other 3 sites, positions 500 and 507 in ECL3, and position 508 at the boundary of the transmembrane domain, alter PMV infectivity, but substitutions at these sites do not produce equivalent receptors for HIX and FrMCF PMVs. These observations, together with the ability of all PMVs but FrMCF to infect rat cells suggest that different PMVs have different receptor requirements.
Mutations in the PMV critical sites in ECL3 also reduce infectivity by the AKR6 XMV. This, together with the PMV-like failure of this virus to infect deglycosylated hamster cells suggests that AKR6 relies on some critical sites that form the PMV receptor determinant.
Cz524 is a novel wild mouse isolate that is only able to efficiently infect mouse cells carrying one of the 4 Xpr1
. Cz524 resembles XMVs in its ability to infect Xpr1n
modified by E500K or the insertion of T582, but examination of the larger set of mutants indicates that neither of these substitutions is sufficient to produce a Cz524 receptor. The fact that this virus infects cells susceptible to both PMVs and XMVs is not surprising as the Cz524 RBD sequence combines features of XMVs and PMVs. The overall sequence closely resembles that of XMVs, but its VRA shows a 3 amino acid deletion where PMVs have a 4 amino acid deletion. This suggests that this VRA indel is important for receptor interactions. The Cz524 sequence and its unusual tropism also suggest that several regions of the envelope may contact the receptor [18
] and that the cell receptor interface is constructed from both ECLs.
Receptor-mediated resistance and interspecies transmission
The characterization of entry-based virus resistance factors has obvious importance for a broader understanding of how viruses spread and adapt to new hosts, and how natural populations adapt to retrovirus infections. Infectious XMVs and endogenous X/PMVs have been identified in Eurasian mice, and these mice have evolved two protective mechanisms that restrict infection at the level of entry. Receptors can be blocked by Env glycoprotein produced by endogenous retroviruses (ERVs), and ERVs with intact env
genes have been linked to the resistance genes Fv4
]. More commonly, resistance to retrovirus entry is due to polymorphic mutations in the cell surface receptor. The present study indicates that 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/PMVs. Additional functional variants of XPR1 and determinants for X/PMV entry may be identified by expanding this analysis to non-rodent species exhibiting different virus susceptibility profiles [[14
]; CAK, unpublished observations], as recently shown by a recent analysis of human/mouse XPR1 chimeras [15
Receptor-mediated virus restriction can result in the outgrowth of virus variants able to circumvent such blocks by adapting to receptor variation, by using alternative receptors or, as in the case of XMVs, using alternative receptor determinants on the same protein. The panel of variant viruses used in the present study were all the products of such adaptations and included naturally occurring mouse-derived isolates, the human-adapted XMRV, and HIX and FrMCF, variants adapted to cultured cell lines or laboratory-bred animals. These viruses differ from one another at multiple sites within env. Mutagenesis studies focusing on these RBD differences and other env regions implicated in receptor binding and/or fusion should provide further information on the critical residues involved in entry and the factors that limit or extend receptor usage.
Defining genetic factors that underlie resistance to mouse gammaretroviruses is important because retroviruses are capable of trans-species transmission, and retroviruses that cluster with mouse gammaretroviruses are widespread among vertebrates. Martin and colleagues [32
] found MLV-related ERVs in approximately one-fourth of the vertebrate taxa and identified recent zoonotic transmissions from mammals to birds and from eutherians to metatherians. Infectious viruses resulting from transspecies transmissions have been isolated from koalas and gibbon apes [33
]. One of the viruses used in the present study, XMRV, is an infectious MLV-related virus from human prostate cancer patients [16
], and it should be noted that similar viruses have also been reported in cell lines derived from other human tumors [36
]. It would not be surprising to find more examples of interspecies transmissions involving MLVs, since mice have a worldwide geographic distribution and all mammalian species tested have functional XPR1 receptors [[14
]; CAK, unpublished observation]. Thus, the examination of the co-evolution of the XPR1 receptor and the X/PMVs should contribute to an understanding of the natural history of infectious pathogenic gammaretroviruses in their murine hosts and provide a foundation for the study of epizoonotic infections.