SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

Abstract

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1–S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell–cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

“…In addition, ACE2 and DPP4 receptor transfection allows for more efficient entry in poorly permissive cells lines, such as HEK-293T cells. The method is highly adaptable to other viral envelope glycoproteins and has been used extensively 48,49,50,51,52,53,55,56,57,58,59 , often to complement other assays like biochemical analyses or native virus infections.…”

Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells

“…They also suggest that several regions within S2 could act in a concerted fashion to mediate the membrane fusion process. Further studies have been conducted taking a more functional approach and considering other criteria that define fusion peptides, such as proximity to a cleavage site and sequence conservation (Madu et al, 2009a(Madu et al, , 2009b. The starting point for these studies was the identification of a second cleavage site in the SARS-CoV S S2 domain called S2′ (at R 797 residue) (Belouzard et al, 2009).…”

“…More recently this domain has been subjected to extensive characterization by electron spin resonance (ESR) spectroscopy (along with CD spectroscopy) to show membrane ordering and the critical involvement of the LLF motif . Another mutagenesis study based on cell-cell fusion and pseudovirion infectivity assays has also found that a pair of highly conserved cysteines (C822 and C833) and the flanking residues D830 and L831 located C-terminally to the 798-815 region played a critical role for fusion (Madu et al, 2009a). The small sub-domain formed by the cysteine pair form a loop structure, a situation that is reminiscent to the avian sarcoma/leukosis virus subtype A in which a pair of cysteines flank the internal fusion peptide of the Env glycoprotein .…”

Dos terapias conocidas podrían ser efectivas como adyuvantes en el paciente crítico infectado por COVID-19

“…El coronavirus en su envoltura es rico en cisteína, y sus residuos deben estar intactos para su actividad viral. La cisteína contiene un grupo tiol o sulfhidrilo (-SH); muchos virus, entre ellos el coronavirus, requieren de estos grupos sulfhidrilo reducidos para la fusión y entrada celular 36 . Los grupos sulfhidrilo son vulnerables a la oxidación, y por tanto susceptibles al ozono dado su poder oxidante.…”

“…En el paciente afectado por COVID-19 suele haber hipoxia, de modo que este beneficio oxigenante será muy beneficioso. Se ha sugerido que, en los leucocitos, puede mejorar la actividad fagocítica de los neutrófilos, dentro de los monocitos y linfocitos, el peróxido de hidrógeno es reconocido como un compuesto señalizador intracelular, capaz de activar una tirosín quinasa que fosforila al NF-B con la consecuente síntesis de diferentes proteínas 36 . El NF-B juega un papel clave en la regulación de la respuesta inmune debida a la infección y en la respuesta inflamatoria 37,38 .…”