Research Of The Glycosylation Modification Of BST-2 In Antiviral Ability In Intracellular Vesicles

Humans and other mammals are equipped with endogenous cellular defense proteins as host restriction factors to provide resistance to infection, which must be overcome by viruses to facilitate their optimal replication. BST-2/tetherin is such an interferon-inducible antiviral glycoprotein. BST-2 inhibits the release of various enveloped viruses by tethering nascent virions at the cell surface, with its GPI anchors incorporated into the virion envelope and TM domains embedded in the host cell membrane to exert antiviral effects via a “physical tethering” model that requires its structural domains and specific amino acid sites. BST-2 can be modified by multiple N-linked glycosylation at two conserved asparagine residues in its extracellular domain. The detailed contribution of N-linked glycosylation to this model is controversial. Recent studies have provided the novel finding that BST-2 restricts hepatitis B virus(HBV) at intracellular vesicles including multivesicular bodies(MVBs). However, whether N-linked glycosylation is critical for this newly discovered antiviral function is also unknown.To better understand the functional significance of BST-2 glycosylation, asparagines 65 and 92 of human BST-2 were mutated to alanine yielding mutants N65 A, N92 A and N65/92 A. 293 T cell lines transduced by BST-2 variants were established by transfection of p LVX-puro-BST-2 WT, BST-2 N65 A, BST-2 N92 A. The stably-expressed BST-2 exhibited similarities with that of the endogenous protein, although multiple glycosylation forms were present. In the cellular fractionation test, the highly and lowly glycosylated forms showed an unbalanced distribution in the subcellular regions with different densities. The mutation of glycosylation sites resulted in more abundant lower-glycosylated of BST-2 proteins, which were found in fractions with larger densities compared with the higher-glycosylated form. The Immunofluorescence analysis found that BST-2 N65/92 A and BST-2 N92 A both exhibited as larger puncta which accumulated in CD63-positive compartments. This BST-2 N65/92 A mutant exhibited more antiviral activity against HIV and HBV release in cellular. Both WT BST-2 and glycosylation mutants exhibited considerable co-localization ratios with EEA1 and LAMP1, implying that the mutation of glycosylation sites affected only the localization in the CD63-positive compartment, without affecting the degradation pathway. So N-linked glycosylation affects subcellular distribution of BST-2, and BST-2 lacking glycosylation accumulated at the intracellular CD63-positive vesicles with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and HBV. A balance could be controlled through regulating BST-2 glycosylation by related cellular machinery to maintain an effective inhibition of virion production from intracellular vesicles and the plasma membrane.Through investigating the effect of glycosylation on the subcellular distribution and antivirial ability of BST-2, we find that BST-2 lacking glycosylation accumulated at the intracellular CD63-positive vesicles, revealing the effect of glycosylation on the subcellular distribution of BST-2. Accumulation at the intracellular CD63-positive vesicles lead to the phenotype potently inhibited the release of multivesicular body-targeted virions, illustrating the importance of glycosylation of BST-2 from the mechanism, and insipiring us to study the other functions in CD63-positive vesicles of BST-2.