Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity

Abstract

Functional and epidemiological data suggest that N -linked glycans on the SARS-CoV-2 Spike protein may contribute to viral infectivity. 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. Some of these mutations, particularly N61Q and N801Q, reduced Spike incorporation into Spike-pseudotyped lentivirus and authentic SARS-CoV-2 virus-like particles (VLPs). These mutations also reduced pseudovirus and VLP entry into ACE2-expressing cells by 80 to 90%. In contrast, glycan mutations had a relatively minor effect on cell surface expression of Spike, ACE2 binding, and syncytia formation. A similar dichotomy in function was observed when virus was produced in host cells lacking ER chaperones, calnexin and calreticulin. Here, while both chaperones regulated pseudovirus function, only VLPs produced in calnexin KOs were less infectious. Overall, Spike N -glycans are likely critical for SARS-CoV-2 function and could serve as drug targets for COVID-19.

SARS-CoV-2 evolution influences GBP and IFITM sensitivity

“…S5 C shows that PNGase treating 293T cell lysates abolished the spike band shift, such that the S2 subunit migrated at the same molecular weight +/− GBP. Taken together, these data indicate that GBP expression inhibits furin cleavage and also influences spike N-linked glycosylation ( 29 , 30 ) resulting in reduced particle infectivity.…”

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…NP is essential for the early replication of the virus in the host cell, which plays a major role in packaging viral RNA into a ribonucleoprotein (RNP) complex called nucleocapsid. 325,326 When the virus enters the host cell, NP supports the replication of the viral RNA and releases the virus particles into the host cell. 327,328 However, because NP is highly conserved among all coronaviruses, the specificity of its detection is low.…”

Widespread exposure to SARS-CoV-2 in wildlife communities

“…We mapped the second missense mutation, G 798 D, in the S2 subunit of the S protein (residues 686 to 1,273) and within the fusion peptide domain (residues 788 to 806) near the N 801 glycosylation site. When glycosylated, the N 801 site significantly enhances viral entry, with very low mutation rates observed in adjacent residues (e.g., within 2-3 residues from position 801) 31 . Interestingly, D 798 creates a small, charged pocket on this solvent-accessible S2 loop, decreasing the probability of glycosylation at N 801 (0.48) compared to BA.2 [0.61] 32 .…”