Date Published: October 23, 2011
Publisher: Hindawi Publishing Corporation
Author(s): Christine A. Kozak.
Gammaretroviruses of several different host range subgroups have been isolated from laboratory mice. The ecotropic viruses infect mouse cells and rely on the host CAT-1 receptor. The xenotropic/polytropic viruses, and the related human-derived XMRV, can infect cells of other mammalian species and use the XPR1 receptor for entry. The coevolution of these viruses and their receptors in infected mouse populations provides a good example of how genetic conflicts can drive diversifying selection. Genetic and epigenetic variations in the virus envelope glycoproteins can result in altered host range and pathogenicity, and changes in the virus binding sites of the receptors are responsible for host restrictions that reduce virus entry or block it altogether. These battleground regions are marked by mutational changes that have produced 2 functionally distinct variants of the CAT-1 receptor and 5 variants of the XPR1 receptor in mice, as well as a diverse set of infectious viruses, and several endogenous retroviruses coopted by the host to interfere with entry.
The various inbred strains of laboratory mice and wild mouse species differ in their susceptibility to mouse gammaretrovirus infection and to virus-induced diseases. Host resistance is due to numerous constitutively expressed antiviral factors that target specific stages of the retroviral life cycle. These host restriction factors can block entry, postentry uncoating and reverse transcription, trafficking, integration, assembly, and release . The first step in the replicative cycle is entry, and this process relies on host-encoded receptors. Host cell factors that can interfere with virus entry include genetic variations of the cell receptor as well as other host factors such as envelope (Env) glycoproteins produced by endogenous retroviruses (ERVs).
The first gammaretrovirus receptor gene to be cloned was the CAT-1 receptor for E-MLVs . This gene (gene symbol Slc7a1) encodes a glycoprotein with 14 putative transmembrane domains, and it functions as a cationic amino acid transporter [19, 20] (Figure 1(a)). Ten additional gammaretrovirus receptors have now been cloned; all of these gammaretrovirus receptors are multi-transmembrane proteins, and the receptors with known functions are all transporters of small solutes (reviewed in [21–26]). The human orthologue of mouse CAT-1 does not function as an E-MLV receptor, and the key sites in the mouse protein critical for virus entry lie in the third extracellular loop along with two consensus recognition sites for N-linked glycosylation [27, 28] (Figures 1(a) and 1(b)). CAT-1 is modified posttranslationally by glycosylation, and N-glycans are added to both of the CAT-1 loop 3 glycosylation sites . All E-MLVs rely on the CAT-1 receptor for entry, although initial binding and the efficiency of entry may be influenced by other factors at the cell surface, such as heparin [30, 31].
There has been no systematic attempt to screen for CAT-1 receptor variation in mice, but 3 sequence variants have been identified in Mus (Figure 1(b)). The prototype receptor, mCAT-1, was cloned from NIH 3T3 cells . Two sequence variants have been identified in the wild mouse species M. dunni and M. minutoides [13, 44]. Limited testing suggests that the M. minutoides CAT-1 functions like the laboratory mouse mCAT-1 receptor, but the receptor of M. dunni, dCAT-1, differs from mCAT-1. M. dunni cells are relatively resistant to infection by Moloney E-MLV (MoMLV), although these cells are fully susceptible to other E-MLV isolates . dCAT-1 differs from mCAT-1 by 4 residues, two of which are in the receptor determining third extracellular loop; one, I214V, is a substitution, and the second is a glycine insertion within the NVKYGE virus binding site  (Figure 1(b)).
Polymorphisms that alter virus-receptor interactions can affect pathogenesis as well as entry. Cytopathic variants are common among the retroviruses that induce disease in their hosts, including HIV-1 as well as avian leukosis viruses and some pathogenic bovine and feline leukemia viruses [50–52]. These viruses can produce large multinucleated syncytia in cultures of susceptible cells. In contrast, mouse gammaretroviruses rarely produce syncytia although there are three exceptional cytopathic E-MLVs. The MoMLV variant, Spl574 and a FrMLV variant, F-S MLV, both induce syncytia and cell death in M. dunni cells [45, 48]. The third cytopathic virus, TR1.3, is a neuropathic FrMLV variant that also induces syncytia in SC-1 cells .
The retroviral Env is glycosylated, as are cellular proteins involved in entry. Many viruses use the glycans on cell surface glycoproteins as attachment factors , but glycosylation of the CAT-1 receptor is not required for virus entry. CAT-1 continues to support virus entry after both loop 3 N-glycan sites have been removed by mutagenesis . However, host cell glycans can modulate entry of some E-MLVs. Thus, resistance of M. dunni cells to MoMLV, resistance of NIH 3T3 cells to Spl574, and resistance of primary rat fibroblasts and hamster cells to E-MLVs are relieved by inhibitors of glycosylation [49, 62–66]. It is not clear whether the responsible glycoprotein is CAT-1 or other host glycoproteins, like the secreted factor associated with resistance to gibbon ape leukemia virus in hamster cells . There is, however, some evidence that the restriction of E-MLV infection in rat cells may be regulated by the glycosylation of rat CAT-1. The CAT-1 of rat XC sarcoma cells lacks one of the glycosylation sites found in the CAT-1 gene of other rat cells (Figure 1(b)), and heterologous cells expressing xcCAT-1 were found to be more susceptible to MoMLV than cells expressing rCAT-1 .
Exposure to E-MLV gammaretroviruses occurred only recently in the evolution of Mus . Although E-MLV ERVs are found in few of the 40 Mus species, wild mice carry three distinctive Env subtypes of E-MLVs (Figure 3). Sequence identity in SUenv among these virus types is 70–77%. The first E-MLV type, the AKV E-MLVs of the laboratory mouse, is found as ERVs in multiple inbred strains . Many of these proviruses are capable of producing infectious virus , and the widely used laboratory virus strains MoMLV, FrMLV, and Rauscher MLV are derived from AKV MLV  (Figure 3). Among the wild mouse species, AKV MLV ERVs are found in the Asian species M. molossinus and in M. musculus of Korea and China but not eastern Europe [76, 80]. A second E-MLV subtype was initially identified in California wild mice [81, 82]. Proviruses with this CasBrE Env type have also been found in the Asian species M. castaneus, and these virus-infected mice were likely introduced to California by passive transport from Asia [76, 80, 83, 84]. A third E-MLV subtype, HoMLV, was isolated from the eastern European species M. spicilegus, but is transmitted only as an exogenous virus .
Two subgroups of nonecotropic MLVs have been isolated from laboratory mice. These viruses were originally described as having distinct host ranges, but they use the same receptor, XPR1. X-MLVs and P-MLVs are both capable of infecting cells of nonrodent species, and although P-MLVs can efficiently infect mouse cells, X-MLVs were initially identified as incapable of infecting their natural hosts [2, 87, 88]. X-MLVs and P-MLVs are closely related viruses, and sequence differences in env and LTR are responsible for their differences in species tropism, for their nonreciprocal interference patterns, and for the pathogenicity of P-MLVs in mice [11, 89–92]. Although it is clear that the RBD VRA region is the major determinant of P-MLV and X-MLV host range , the critical VRA residues involved in XPR1 receptor recognition have not been identified, although 2 residues outside VRA can influence the ability of these viruses to infect cells of other mammalian species (Figure 4) . Viruses in the XP-MLV family are highly variable in the Env segment containing the RBD (Figure 4), and the wild mouse viruses, CasE#1 and Cz524, show atypical host range patterns that distinguish them from prototypical P-MLVs and X-MLVs (Table 1) [16, 89, 94].
The genus Mus includes about 40 species, and all available species have been screened for sequence and functional variants of Xpr1 (Figures 5 and 6). Of the 5 sequence variants found in wild mice, 4 show unique receptor phenotypes based on their ability to support entry of different virus isolates that rely on this receptor (Table 1). The most common receptor variant among wild mouse species was originally termed Sxv (susceptibility to xenotropic virus) . This variant is found in many Asian species as well as western European house mice [17, 99], and mice with Sxv were introduced into the Americas by European immigrants and explorers (Figure 7). Sxv is also carried by several of the common inbred strains of laboratory mice . Sxv is the most permissive of the Xpr1 alleles and supports entry of all XP-MLV host range variants (Table 1). The second most geographically widespread Xpr1 allele, Xpr1m, is found in two house mouse species, M. musculus, which ranges from central Europe to the Pacific, and M. molossinus, found in Japan . This variant is highly restrictive, allowing inefficient entry of X-MLVs, while restricting all other XP-MLVs. A third allele, Xpr1c, is found in the southeast Asian mouse, M. castaneus, and is responsible for resistance to infection by P-MLVs [15, 99]. A fourth wild mouse Xpr1 allele is restricted to the Asian species M. pahari; these mice are susceptible to X-MLVs and to CasE#1  (Table 1).
Initial studies on Xpr1 receptor function focused on sequence differences between the phenotypic variants identified in NIH 3T3 cells (Xpr1n) and M. dunni (Xpr1sxv) . Two critical amino acids were identified for X-MLV entry that lie in different putative extracellular loops (Figure 5). The restrictive Xpr1n carries a substitution, K500E, in its third extracellular loop (ECL3), and a deletion, T582Δ, in the fourth loop (ECL4). Corrective mutations at either of these sites produce functional receptors for X-MLVs without compromising P-MLV receptor function . Subsequent studies on the mouse receptor showed that these 2 critical residues are not equivalently used by the XP-MLVs, as CasE#1 can use Xpr1n-Δ582T but not Xpr1n-E500K . Mutational analysis of other polymorphic sites in the various Mus Xpr1s identified residues at additional sites that modulate virus entry: ECL3 positions 500, 507, and 508 and ECL4 positions 579 and 583 (Figure 5) [17, 94].
Although it is clear that MLV entry is typically mediated by specific cell surface receptors, some MLVs are capable of bypassing the need for their cognate receptors and can infect cells that lack receptors and may also be able to infect cells in which those receptors are downregulated by superinfection [106, 107]. Such alternative entry mechanisms seem to be particularly important for P-MLVs, viruses that are less able to establish effective superinfection immunity against further infection [99, 108] either because they may have lower binding affinity for the XPR1 receptor than the X-MLVs or because Env-bound receptors may recycle rapidly into acidic compartments where the Env-receptor complex is disrupted allowing the freed receptor to recycle back to the plasma membrane. This ineffective or delayed establishment of interference to exogenous infection has been linked to the massive accumulation of viral DNA in P-MLV-infected mice  and to the ability of P-MLVs to induce cytopathic responses in mink lung cells in which superinfection induces an ER stress response and apoptosis [4, 110, 111].
The species distribution of the Mus XPR1 variants indicates that polymorphic, virus-restrictive receptors appeared when mice were exposed to XP-MLVs, especially X-MLVs (Figure 6). For most of the 8 million years of Mus evolution, species carried the permissive Xpr1sxv allele. Mice were subjected to XP-MLV infection about 0.5 MYA, and this exposure is marked by the acquisition of MLV ERVs in the 4 house mouse species [76, 119, 120]. M. domesticus carries P-MLV ERVs, whereas M. castaneus, M. musculus, and M. molossinus carry predominantly X-MLVs . The common laboratory mouse strains, which are mosaics of these wild mouse species, generally carry multiple copies of X-MLVs and P-MLVs [121, 122]. The acquisition of these germline ERVs, and specifically X-MLVs, roughly coincides with the appearance of restrictive Xpr1 alleles: Xpr1c in M. castaneus, Xpr1m in M. molossinus and M. musculus, and Xpr1n in laboratory mice (Figure 6). Each of these restrictive receptors carries a unique deletion in the XPR1 ECL4 (Figure 5) . M. domesticus, the species carrying only inactive P-MLV ERVs, maintains the full-length, permissive Xpr1sxv receptor common to ancestral species of Mus. The fact that restrictive receptors have evolved in X-MLV infected mice suggests that this host pathogen interface has been an important evolutionary battleground. This also suggests that X-MLV infection is deleterious for mice, although the consequences of X-MLV infection have not yet been described because mouse gammaretroviruses have been studied largely in X-MLV-resistant laboratory mice. The discovery of mouse species and strains with XPR1 variants that efficiently support X-MLV entry now provides the basis for studies on pathogenesis of these viruses, and such studies have now been initiated .
Virus entry can be inhibited by receptor mutants but can also be blocked by members of a second set of genes found in E-MLV- or X-MLV-infected wild mice. This family of resistance genes governs production of MLV Env glycoproteins that are thought to restrict virus through receptor interference. These genes include Fv4, which blocks E-MLVs , and the genes Rmcf and Rmcf2 which restrict XP-MLVs and, in the case of Rmcf, inhibit P-MLV-induced disease [118, 124–126]. There is also evidence suggesting that additional Rmcf-like XPR1 receptor blocking genes are present in M. castaneus . Specific ERVs have been mapped to 3 of these resistance genes, all of which are defective for virus production but have intact env genes. Fv4 is a truncated provirus, Rmcf has a major deletion spanning gag-pol [124, 128], and Rmcf2 has a stop codon that prematurely terminates integrase . It has been proposed that the products of Fv4, Rcmf, and Rcmf2 reduce or downregulate activity of their cognate receptors, and Fv4 also has a defect in the fusion peptide of TMenv, so incorporation of this Env into virions in virus-infected cells results in their reduced infectivity .
The XP-MLVs are capable of infecting cells of other species, including humans (Figure 9). Cells of nearly all mammals are permissive to infection by X-MLVs, whereas a subset of these species is also susceptible to P-MLVs. This suggests that X-MLVs have less stringent receptor requirements than P-MLVs [17, 87, 88]. Some mammalian species show distinctive patterns of virus susceptibility not found in mice, for example, the restriction of P-MLVs and both wild mouse XP-MLVs by dog and buffalo cells (Figure 9) . Analysis of mammalian XPR1 genes reveals significant sequence variability especially in the receptor determining ECL4, although this 13 residue segment contains 3 nonvariant residues, S578, T580, and G589. These conserved residues do not contribute to the receptor attachment site . Further analysis of these functionally distinctive XPR1 genes may provide insight into the factors that facilitate transspecies transmission.
Mice are important vectors of diseases that infect humans and their livestock , and MLV-infected house mouse species have a worldwide geographic distribution . The horizontal transfer of infectious MLVs between individuals has been documented in wild mouse populations and in laboratory mice [82, 137], and MLV-related viral sequences, proteins, and antibodies have been reported in human blood donors and patients with prostate cancer and chronic fatigue syndrome [138–140]. An infectious virus first identified in prostate cancer patients, termed XMRV (xenotropic murine leukemia virus-related virus), shows close sequence homology with XP-MLVs , uses the XPR1 receptor , and has xenotropic host range . Although XMRV origin by transspecies transmission is consistent with the evidence of MLV transmission between mice and evidence of transmission of mouse C-type viruses to other species [142–144], several recent studies on XMRV have implicated laboratory contamination [145–148]. Additional studies aiming to resolve the origins issue are focused on patient samples and the characterization of mice for XMRV-related sequences .
Retrovirus entry is dependent on the presence and accessibility of specific cell surface receptors. Mutational changes in these receptors and in the receptor attachment sites in the virus Env can alter the very first step in the virus life cycle and can thus have profound consequences for virus replication. Inhibition of virus entry has been a particularly effective antiviral tactic in mice infected with MLVs as well as with other gammaretroviruses . Entry is also the target of host restrictions in other species subject to retrovirus infection as shown by the discovery of interfering ERV Envs in multiple species [132–134] and by the discovery of inhibitory mutations in other receptors, such as the HIV-1 CCR5 coreceptor .