High-Mannose But Not Complex-Type Glycosylation of Tetherin Is Required for Restriction of HIV-1 Release

Abstract

Tetherin is an interferon-inducible antiviral protein that inhibits the release of a broad spectrum of enveloped viruses by retaining virions at the surface of infected cells. While the role of specific tetherin domains in antiviral activity is clearly established, the role of glycosylation in tetherin function is not clear. In this study, we carried out a detailed investigation of this question by using tetherin variants in which one or both sites of N-linked glycosylation were mutated (N65A, N92A, and N65,92A), and chemical inhibitors that prevent glycosylation at specific stages of oligosaccharide were added or modified. The single N-linked glycosylation mutants, N65A and N92A, efficiently inhibited the release of Vpu-defective human immunodeficiency virus type 1 (HIV-1). In contrast, the non-glycosylated double mutant, N65,92A, lost its ability to block HIV-1 release. The inability of the N65,92A mutant to inhibit HIV-1 release is associated with a lack of cell-surface expression. A role for glycosylation in cell-surface tetherin expression is supported by tunicamycin treatment, which inhibits the first step of N-linked glycosylation and impairs both cell-surface expression and antiviral activity. Inhibition of complex-type glycosylation with kifunensine, an inhibitor of the oligosaccharide processing enzyme mannosidase 1, had no effect on either the cell-surface expression or antiviral activity of tetherin. These results demonstrate that high-mannose modification of a single asparagine residue is necessary and sufficient, while complex-type glycosylation is dispensable, for cell-surface tetherin expression and antiviral activity.

N-Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells

“…All these KO cell lines showed increased resistance to PTC3 W14 treatment compared with control cells (Figs 1F-1H and S1D). Furthermore, we also utilized kifunensine, as a potent and selective inhibitor of class I α-mannosidases, to pharmacologically block the synthesis of N-glycans without interfering with cell viability [24,25]. Consistently, pretreatment with kifunensine was also shown to significantly inhibit intoxication by PTC3 W14 (Fig 1I and 1J).…”

Regulation of Viral Restriction by Post-Translational Modifications

“…Thus, depletion of both sites and/or inhibition of N-glycosylation greatly impairs BST2 restriction activity against HIV-1 [172,178] and XMRV [175]. This defect of activity is correlated with a decrease in BST2 expression [172,178] and relocalization of BST2 in subcellular compartments [175]. Together, this suggests that glycosylation of at least one N-glycosylation site of BST2 is required for its efficient transport to the PM where it can trap viruses (Figure 3A).…”

“…Thus, depletion of both sites and/or inhibition of N-glycosylation greatly impairs BST2 restriction activity against HIV-1 [172,178] and XMRV [175]. This defect of activity is correlated with a decrease in BST2 expression [172,178] and relocalization of BST2 in subcellular compartments [175].…”

“…Hence, the relevance of incomplete N-glycosylated BST2 expression is important to be determined. Indeed, a complementary approach with chemical inhibitors of the transfer of high-mannose oligosaccharides revealed that complextype glycosylation is dispensable for both BST2 cell surface expression and antiviral activity toward HIV-1 [178].…”

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX

“…Many previous reports have shown that the release of a broad spectrum of enveloped viruses is significantly blocked in the presence of tetherin also known as bone marrow stromal antigen 2 (Bst-2) [37][38][39]. As such, "physical tethering" by tetherin prevents effective secretion of viral particles and restricts cell-cell transmission of viruses.…”