RESUMEN
The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG
RESUMEN
T cells are involved in control of COVID-19, but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we assessed the magnitude, function and phenotype of SARS-CoV-2-specific CD4 T cells in 95 hospitalized COVID-19 patients (38 of them being HIV-1 and/or tuberculosis (TB) co-infected) and 38 non-COVID-19 patients, using flow cytometry. We showed that SARS-CoV-2-specific CD4 T cell attributes, rather than magnitude, associates with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity and enhanced HLA-DR expression. Moreover, HIV-1 and TB co-infection skewed the SARS-CoV-2 T cell response. HIV-1 mediated CD4 T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2; and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4 T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mtb-specific CD4 T cells, with possible implications for TB disease progression. There results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.
