Relevance of the viral Spike protein/cellular Estrogen Receptor-α interaction for endothelial-based coagulopathy induced by SARS-CoV-2 (preprint)

Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor-α (ERα). Given the implications of ERα in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ERα, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo , without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER binding mode. In cultured cells, S DNA transfection increased ER cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.