Among the various factors responsible for intrinsic resistance to retroviruses are several that inhibit virus entry. Such factors include ERV produced Env glycoproteins that interfere with virus-receptor binding, and mutations in receptor genes that alter their interactions with viruses (Stocking and Kozak, 2008
). For the MLVs, receptor polymorphisms that recognize different Env variants have been identified for the XPR1 receptor that mediates infection of xenotropic and polytropic MLVs (XMVs and PMVs) (Marin et al., 1999
; Yan et al., 2007
), viruses originally described as different host range groups (Hartley et al., 1977
). For the E-MLVs, there are fewer examples of receptor mediated host range variations, in part because, unlike the X/PMVs that can infect all mammals tested, E-MLV infection is limited to rodents. In addition, most known examples of E-MLV entry restrictions are due, at least in part, to glycosylation of cellular proteins. Because glycosylation of the viral Env has also been associated with altered infectivity of multiple viruses including MLVs (Battini et al., 1994
) and other retroviruses (Ogert et al., 2001
), we examined the need for specific N-glycans in Env regions important for entry. Here we show that deletion of specific N-glycan sites from ecotropic MLV env
genes produces viruses with altered host range. This effect is receptor variant specific, virus type specific, and glycan site specific. Deletion of glycans gs1 and gs2 alters the host range of MoMLV and FrMLV but not AKV MLV, and this restriction is observed only in cells expressing two of the four naturally occurring CAT-1 variants.
Virus infectivity was shown to be affected by glycan site deletion but not by the alteration of glycan type or size. This suggests that the role of glycans in host range may be a consequence of their cotranslational placement on the Env protein, a process that may result in conformational changes in the Env glycoprotein that differentially alters recognition by the 4 receptor variants. Glycan additions generally ensure proper folding of proteins, help maintain their stability, and are needed for intracellular routing and further processing. For MLV Env, blockage of glycosylation by tunicamycin halts processing and translocation to the plasma membrane (Schultz and Oroszlan, 1975). That glycan removal affects infectivity due to altered folding rather than a specific role for glycans in entry is consistent with the crystal structure of FrMLV (Fass et al., 1997
) which shows that neither gs1 nor gs2 is positioned near the residues in VRA that form the pocket for virus binding, namely residues S84, D86 and W102 (Davey et al., 1999
Deletion of either of the 2 RBD glycan sites of MoMLV and gs1 of FrMLV produces viruses that identify a functional distinction among the 4 naturally occurring CAT-1 variants. These mutated viruses are much less able to infect dunni and hamster cells, while infectivity on rat XC cells and NIH 3T3 cells is not altered. These infectivity differences are also seen in MA139 ferret cells expressing the dCAT-1 and mCAT-1 receptor variants indicating that receptor polymorphism is the major responsible host cell factor. Virus binding assays further demonstrate there is an association between infectivity and the binding ability of Fr-gs1 compared to the wild type FrMLV in M. dunni cells as well as in the transfected ferret cells. Infectivity can be, however, affected by other, secondary cellular factors as shown by the fact that glycosylation inhibitors can reduce this entry restriction in M. dunni cells.
The three viruses used here have ecotropic host range, but show significant sequence variation in their RDBs and also differ in their patterns of glycosylation (). FrMLV and MoMLV are laboratory isolates that, unlike AKV, are oncogenic in mice. Both viruses were isolated after serial passage of tumors in mice and neither of these viruses have counterparts that are found naturally in laboratory mice or wild mouse species. In contrast, AKV MLVs are naturally occurring MLVs carried as endogenous retroviruses by many inbred laboratory strains and are also found in Asian species of Mus
(Kozak and O’Neill, 1987
). Sequence comparisons of their Env genes indicate closer relationship between FrMLV and MoMLV (88% amino acid identity in RBD) than between either of these viruses and AKV (78% identify). These similarities are reflected in our results; removal of RBD glycan sites from FrMLV and MoMLV similarly compromises infectivity in cells expressing dCAT-1, whereas AKV mutants show no changes in infectivity. Examination of other MLV isolates indicates there is naturally occurring polymorphism at 4 glycan sites (gs3,5,7,8) among ecotropic viruses suggesting that these sites are not critical for producing infectious virus as shown here for gs8.
Mutational changes in the retroviral RBD can lead to the generation of viruses that can use alternative receptors, multiple receptors or even multiple determinants on the same receptor protein (Stocking and Kozak, 2008
). The coevolution of Env and receptor has resulted in their sequence diversity and functional plasticity, and previous studies have identified sequence variations in E-MLVs responsible for variations in receptor usage (Yan et al., 2008
; Masuda et al, 1996
). Such variants show altered infectivity into different mouse or hamster cells, and differential ability to induce multinucleated syncytia. The present study indicates that RBD glycans also serve to control virus host range variation, a control that likely depends on protein folding rather than a direct role in receptor interaction.