![]() Overall, Spike N- glycans are likely critical for SARS-CoV-2 function and could serve as drug targets for COVID-19. For example, calnexin and calreticulin alternate binding between Glc 1 Man 9 GlcNAc 2 and Man 9 GlcNAc 2 protein-linked glycans to mediate substrate folding. Here, while both chaperones regulated pseudovirus function, only VLPs produced in calnexin KOs were less infectious. A similar dichotomy in function was observed when virus was produced in host cells lacking ER chaperones, calnexin and calreticulin. In contrast, glycan mutations had a relatively minor effect on cell surface expression of Spike, ACE2 binding, and syncytia formation. These mutations also reduced pseudovirus and VLP entry into ACE2-expressing cells by 80 to 90%. Some of these mutations, particularly N61Q and N801Q, reduced Spike incorporation into Spike-pseudotyped lentivirus and authentic SARS-CoV-2 virus-like particles (VLPs). The essential nature of the pathway is also illustrated by the lethal outcome of an inherited glucosidase I deficiency involving a neonate born with severe generalized hypotonia and dysmorphic features. ![]() We also determined that a soluble form of calnexin (residues 1387) can functionally replace its membrane-bound. Transgenic mice devoid of calreticulin die on embryonic day 18. Calnexin and calreticulin, which are lectin-type molecular chaperones, play important roles in glycoprotein folding. Indeed, the functions of calnexin and calreticulin were largely interchangeable. To investigate this, we created a panel of N-to-Q mutations at N- glycosylation sites proximal to the Spike S1-S2 (N61, N603, N657, and N616) and S2' (N603 and N801) proteolysis sites. The calnexin-calreticulin cycle seems to be essential in vivo. ![]() Functional and epidemiological data suggest that N-linked glycans on the SARS-CoV-2 Spike protein may contribute to viral infectivity. ![]()
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