Stabilization of the Metastable Prefusion Conformation of SARS-CoV-2 Spike Glycoprotein through N-linked Glycosylation on S2 Subunit (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic, represents a serious threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using α-glycosidase inhibitors, castanospermine and NB-DNJ, we confirmed that disrupting the N-linked glycosylation process blocked the maturation of the S protein, leading to impairment of S protein-mediated membrane fusion. Single substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation, resulting in reduced S protein-mediated cell-cell fusion and viral entry. Of note, N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T/hACE2-KO cells, and removal of the furin-cleavage site partially compensated instability of N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the mutants N669Q and N1080Q exhibited increased fusogenic activity and induced syncytia formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are of great importance in maintaining the prefusion state of the S protein. The study revealed the critical roles of N-glycans in S protein maturation and stability, which have implications for the design of vaccines and anti-viral strategies.

Coagulation factors and COVID-19 severity: Mendelian randomization analyses and supporting evidence (preprint)

BackgroundThe evolving pandemic of COVID-19 is arousing alarm to public health. According to epidemiological and observational studies, coagulopathy was frequently seen in severe COVID-19 patients, yet the causality from specific coagulation factors to COVID-19 severity and the underlying mechanism remain elusive. MethodsFirst, we leveraged Mendelian randomization (MR) analyses to assess causal relationship between 12 coagulation factors and severe COVID-19 illness based on two genome-wide association study (GWAS) results of COVID-19 severity. Second, we curated clinical evidence supporting causal associations between COVID-19 severity and particular coagulation factors which showed significant results in MR analyses. Third, we validated our results in an independent cohort from UK Biobank (UKBB) using polygenic risk score (PRS) analysis and logistic regression model. For all MR analyses, GWAS summary-level data were used to ascertain genetic effects on exposures against disease risk. ResultsWe revealed that genetic predisposition to the antigen levels of von Willebrand factor (VWF) and the activity levels of its cleaving protease ADAMTS13 were causally associated with COVID-19 severity, wherein elevated VWF antigen level (P = 0.005, odds ratio (OR) = 1.35, 95% confidence interval (CI): 1.09-1.68 in the Severe COVID-19 GWAS Group cohort; P = 0.039, OR = 1.21, 95% CI: 1.01-1.46 in the COVID-19 Host Genetics Initiative cohort) and lowered ADAMTS13 activity (P = 0.025, OR = 0.69, 95% CI: 0.50-0.96 in the Severe COVID-19 GWAS Group cohort) lead to increased risk of severe COVID-19 illness. No significant causal association of tPA, PAI-1, D-dimer, FVII, PT, FVIII, FXI, aPTT, FX or ETP with COVID-19 severity was observed. In addition, as an independent factor, VWF PRS explains a 31% higher risk of severe COVID-19 illness in the UKBB cohort (P = 0.047, OR per SD increase = 1.31, 95% CI: 1.00-1.71). In combination with age, sex, BMI and several pre-existing disease statues, our model can predict severity risks with an AUC of 0.70. ConclusionTogether with the supporting evidence of recent retrospective cohort studies and independent validation based on UKBB data, our results suggest that the associations between coagulation factors VWF/ADAMTS13 and COVID-19 severity are essentially causal, which illuminates one of possible mechanisms underlying COVID-19 severity. This study also highlights the importance of dynamically monitoring the plasma levels of VWF/ADAMTS13 after SARS-CoV-2 infection, and facilitates the development of treatment strategy for controlling COVID-19 severity and associated thrombotic complication.