Although no cellular function has been identified for mammalian XPR1, retrovirus receptors control the spread of elements responsible for genetic variation and disease and thus have important roles in the evolution of host species subject to infection (30
). In this study, we describe XPR1 sequence and functional variation in the natural hosts of these gammaretroviruses and also in other mammalian species that could serve as virus reservoirs or targets for trans-species transmission. This analysis has allowed us to address two questions: (i) how receptor polymorphism helps natural populations adapt to exposure to infectious disease-inducing retroviruses and (ii) whether the potential for transspecies transmission in different mammalian species is restricted or facilitated by functional XPR1 polymorphisms. In the course of these studies, we characterized a novel restrictive Xpr1
allele in Mus
and identified a correlation between exposure to MLV infection and appearance of restrictive XPR1s caused by deletion. We used mutagenesis and phylogenetics to evaluate the functional contributions made by constrained, variable, and deleted residues in XPR1.
There are numerous examples of trans-species transmissions of retroviruses. The most well-known example is, of course, the derivation of HIV-1 from simian lentiviral precursors, but there are other examples, and retroviruses that cluster with mouse gammaretroviruses are found in multiple vertebrates. Martin and colleagues (33
) found MLV-related ERVs in approximately one-fourth of vertebrate taxa and identified recent zoonotic transmissions from mammals to birds and from eutherians to metatherians. It would not be surprising to find more examples of interspecies transmissions involving MLVs, since MLV-infected house mouse species have a worldwide geographic distribution (32
), and all mammalian species tested are permissive to infection by some or all X/P-MLVs.
The XMRV virus found in some human patients may have been acquired directly from mice or after transmission from mice to another species in contact with humans. We therefore examined the reported worldwide incidence of prostate cancer (7
) relative to the geographic distribution of the various Xpr1
alleles of house mice. The highest rates of this disease are found in the United States, and the lowest rates are found in Asian countries like Japan, India, and China. In Europe, rates are highest in Austria, France, and Scandinavia and lowest in Eastern Europe. This distribution roughly corresponds to the distribution of Xpr1
receptor variants in mouse populations, with the most permissive allele, sxv
, found in high-tumor-incidence areas like America and Western Europe, and with the allele most restrictive of X-MLVs, Xpr1m
, found in low-tumor areas like Japan and Eastern Europe. This Eurasian group of mice also carries receptor-blocking genes (47
) that further suggest that these mice might provide a poor reservoir for zoonotic transmission to humans. While the American and Western European mice with Xpr1sxv
carry only poorly expressed endogenous PMVs and few or no endogenous XMVs, wild mice are also known to carry viruses that have not become endogenized (12
), and at least one virus in the X/P-MLV family, CasE#1, was isolated from a mouse trapped in California. Clearly, more studies are needed to examine wild mouse populations for infectious virus.
The isolation of XMRV from human patients raises the question of whether adaptation to this species has altered its tropism. XMRV shows 95% overall identity to X-MLVs and 93% identity in the 5′ env receptor binding domain. XMRV, as shown here, can utilize the receptor determinants for X-MLVs defined by residues K500 and T582 to enter mouse cells, although K500 is a more effective receptor for XMRV. Unlike the mouse X-MLVs that are able to infect all mammals, however, XMRV is restricted by hamster and gerbil cells and these XPR1 genes share two substitutions, T583A and L/K585Q. Residues A583 and Q585 are not found in other mammals, except for a few rodents that have not been tested for XMRV susceptibility. Other evidence implicates these two codons in virus entry: both residues are deleted in Xpr1c, 583 is under positive selection, and the T583K substitution found in M. molossinus was associated with P-MLV restriction. This XMRV tropism difference demonstrates that the XMRV-receptor interaction differs from that of the mouse viruses and raises the possibility that this may represent an XMRV adaptation acquired through contact with humans or with an as-yet-undiscovered species before transmission to humans.
There are six XPR1 codons important for virus entry identified by mutagenesis of Mus
alleles (500, 507, 508, 579, 582, and 583) (31
). Expanding this receptor analysis to other mammals should be fruitful; analysis of the gerbil XPR1 has implicated A583 and Q585 in XMRV entry. That further studies on the human and other XPR1 genes could provide additional information on the receptor virus interface is suggested by several observations: the extensive sequence variation in permissive mammalian XPR1s in the regions involved in entry; the fact that various mammalian cells, like bat and dog cells, show resistance patterns not found in mice; the fact that studies on human/hamster Xpr1
chimeras implicate as yet unidentified ECL4 residues in P-MLV entry (44
); and the fact that X/P-MLV interference patterns differ in cells with different Xpr1
; C. A. Kozak, unpublished data).
Phylogenetic analysis of Xpr1
indicates that it is under diversifying selection in rodents, suggesting that this gene has had a defensive role in rodent evolution. There is ample evidence that some wild mouse populations have been exposed to viruses that use the XPR1 receptor: Some species carry MLV ERVs acquired from past infections (20
), some of which can produce infectious virus (4
), and some wild mice also carry infectious MLVs that have not become endogenized (12
). For such populations, survival is enhanced by host factors that restrict virus, and the XPR1 receptor is clearly one of those factors. Among the five Mus
XPR1 variants, the one with the broadest susceptibility phenotype, Xpr1sxv
, is widely distributed among the Eurasian Mus
species, and the sxv
ECL4 sequence is also found in multiple species that predate Mus
. The species with this ECL4 sequence either lack X/P-MLV ERVs or carry only PMVs, ERVs not known to produce infectious virus (Fig. ) (17
The four restrictive Mus
XPR1 polymorphisms appeared at two distinct points in Mus
evolution. First, Xpr1p
appeared about 7.5 million years ago (MYA), shortly after the divergence of Mus
from other Murinae
), but this allele is confined to two species of Southeast Asian mice. The presence of this restrictive receptor in mice that do not carry XMV or PMV ERVs (20
) suggests that either the fixation of this variant is unrelated to receptor function, or these mice were exposed to an as-yet-undescribed retrovirus infection. The other three restrictive XPR1s arose later in Mus
evolution, in the house mouse complex, which appeared about 0.5 MYA (13
). House mice are distinguishable from other Mus
species by two notable features. First, these mice are behaviorally different from other Mus
species in their dependence on humans; these mice live in our houses, barns, warehouses, and ships and travel wherever we go. Second, these house mice have all been exposed to MLV infection and carry numerous endogenous copies of X/P-MLVs, some of which have remained active. Acquisition of these germ line viral sequences is roughly coincident with the appearance of the restrictive house mouse variants Xpr1m
, both of which, like the ERV sequences in these species, show an apparent species-wide distribution. Thus, the mutations in these two variants restricting Xpr1
receptor function likely provided a survival advantage in the face of endemic infection by potentially mutagenic and pathogenic gammaretroviruses and may have contributed to the “arms race” between virus and host by providing the selective pressure that produced viral variants with altered receptor specificities.
We had expected Xpr1n
to be widespread in wild M. domesticus
for several reasons. First, Xpr1n
was initially identified in laboratory mice, and although M. musculus
, M. castaneus
, and M. domesticus
all contributed to the fancy mouse colonies used to generate the common inbred strains, M. domesticus
is, by far, the most significant contributor to the laboratory mouse genome (50
). Second, the complete absence of XMV env
genes in M. domesticus
is consistent with Xpr1n
restriction of X-MLV infection. The failure to identify Xpr1n
in any M. domesticus
mouse trapped in geographically disparate locations in Western Europe and the Americas indicates, however, that this allele is not responsible for the absence of XMVs in these mice, an absence that also marks all other species with Xpr1sxv
. The failure to identify Xpr1n
in the wild may be a consequence of limited sampling, but the fact that Xpr1n
does not show the apparent species-wide distribution of Xpr1m
suggests that this allele evolved only recently in Mus
, perhaps in the fancy mouse colonies that provided progenitors of the common laboratory strains (50
). Testing of additional Eurasian wild mouse populations and laboratory breeding stocks may identify the origin of this restrictive allele.
The presence of different deletion mutations in the 13-residue ECL4 of the three restrictive alleles found in house mice is unusual in the natural history of this gene. No ECL4 deletion mutations were found in XPR1 genes of nonrodent mammals, and none appeared during the 7 million years of Mus evolution prior to the house mouse radiation. These independent deletion mutations are thus unlikely to have resulted from inherent structural features in this region of the gene, because such deletions would not then be restricted to house mice. The appearance of these variants, each of which blocks two or more viruses in the XPR1 family, coincides with the acquisition of germ line MLV env genes, suggesting that, in the face of infection, replacement mutations may not be an effective way to disable or alter receptor function. After all, permissive receptors have sustained multiple ECL4 substitutions. That all three of the restrictive alleles carry deletion mutations suggests either that these six residues are critical for entry or that the size of the ECL4 loop may affect receptor function.
Finally, these studies do not provide any special insight into the origins or evolutionary relationships of the two major host range variants of MLVs that use XPR1, X-MLV and P-MLV. The fact that P-MLVs are found in M. domesticus
that were thought to carry Xpr1n
prompted the reasonable suggestion that these viruses may have evolved in response to XPR1 mutations that enabled European mice to evade X-MLV infections (31
). Our present studies, however, indicate that the acquisition of P-MLV ERVs predates Xpr1n
. The observed distribution of ERVs and XPR1 variants also does not explain why P-MLVs integrated selectively into European mice carrying Xpr1sxv
but are not found in other Eurasian species carrying the same receptor variant.