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

Abstract

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.

Fig. 1

N-linked glycosylation in Env of 3 ecotropic gammaretroviruses A) Location of N-glycan sites in the Env genes of MoMLV, FrMLV57 and AKV MLV. Six mutants were generated that lack gs1, gs2 or gs8 as indicated. The domain structure of Env is indicated at the top. RBD: receptor binding domain; VRA, VRC and VRB: variable regions A, C and B; PRD: proline-rich domain; LS: leader sequence. B) Western immunoblot analysis of protein lysates from virus infected cells. NIH 3T3 cells were infected with the indicated viruses. Panels at the left and top right were reacted with goat anti-gp70; the panel in the lower right was reacted with MoMLV specific MAb538.

Figure 2

Infectivity of wild type ecotropic MLVs and mutated viruses that lack specific sites for N-glycans A) Amino acid sequence comparisons of rodent variants of the third extracellular loop of the CAT-1 receptor. mCAT-1 is found in laboratory mouse strains and dCAT-1 is found in the Asian wild mouse species M. terricolor (M. dunni). choCAT-1 and xcCAT-1 are the Chinese hamster and rat XC cell variants. Sites for N-glycosylation are shaded. Glycosylation sites were removed from dCAT-1 to produce dCAT-1-g as described previously (Yan et al., 2008). References or GenBank accession Nos. are provided for each sequence. B) Viruses were tested on 5 cell lines carrying 4 naturally occurring variants of the CAT-1 receptor. Virus titers were determined by the XC overlay test in which the indicated cells were infected with virus dilutions, irradiated and overlaid with XC cells to identify virus infected cells (Rowe et al., 1970; Yan et al., 2008). Virus titers are given as the log₁₀ number of XC PFU in 0.2 ml. Chinese hamster E36 cells were resistant to infection by all viruses (not shown). Results for each set of viruses are from one representative experiment; each set was tested at least 3 times. SD of the data are also shown. Arrow: in 8 trials, XC plaques were produced once for the two viruses, with log₁₀ virus titers of 0.2 for Mo-gs1 and 0.3 for Mo-gs2. X: not done.

Figure 3

Western immunoblot analysis of lysates from MoMLV-infected cells. A) NIH 3T3 (N) or hamster Lec8 (L) cells infected with MoMLV or FrMLV and probed with goat anti-gp70. B) Cell lysates from NIH 3T3 cells treated with the indicated inhibitors. After the reaction with MAb538 (on the bottom), the filter was stripped and probed with anti-gp70. Lanes separated by a space were from the same exposure of a single filter.

Figure 4

Virus titers of ecotropic MLVs on mouse cells and on mouse and ferret transfectants expressing specific CAT-1 receptors. Virus titers were measured as the number of XC PFU in 0.2 ml using the XC overlay test. Results are from a representative experiment. SD of the data are also shown. Arrow: no XC plaques detected in 5 trials. X, not done.

Figure 5

Virus binding assay performed on NIH3T3 (A, B), MDTF (C, D) FerrM (E) and FerrD (F) cells. Dead cells were excluded by gating 7-AAD negative cells. Viruses used in these experiments included Mo-MuLV (black line, panel A and C) and its Mo-gs1 glycosylation mutant (broken line, panel A and C) and Friend MuLV (black line, panel B, D, E and F) and its Fr-gs1 glycosylation mutant (broken line, panel B, D, E and F). Cells treated similarly in absence of virus suspension served as negative control (grey histogram in all panels).