Conserved serine and proline residues in SARS-CoV-2 spike cleavage sites modulate fusogenicity and infectivity

ABSTRACT

Background and Objectives: * The spike (S) protein of SARS-CoV-2 virus binds to the host cell receptor which facilitates the virus entry. This interaction is primed by host cell proteases like furin and TMPRSS2 acting at S1/S2 and S2' cleavage sites, respectively. * Both the cleavage sites have Serine and Proline residues conversed in all the coronaviruses. It has been speculated that mutations at these conserved residues may provide a gain-offunction, easing the SARS-CoV-2 entry into the host cell and cellto- cell spread, thus modulating the virulence and pathogenicity. * Unravelling the effects of these conserved residues in the S protein cleavage site in virus entry and transmission might facilitate development of novel therapeutics. Material(s) and Method(s) * This study employed a lentivirus based pseudovirus (PSV) system, where P and S residues at S1/S2 site of Spike gene, present in an expression vector, were mutated to Alanine (Fig A). * We then assessed the expression of the SARS-CoV-2-S variants in HEK293T cells and tested the infectivity and fusogenicity of mutant PSV and spike, respectively in the presence or absence of S1/S2 and S2' protease inhibitors. Results and Conclusion(s) * Conserved Serine residue mutation (S2SA) at S2' cleavage site resulted in complete loss of spike cleavage by furin and cathepsins (Fig B). * TMPRSS2 protease treatment was not able to rescue loss of spike cleavage and fusogenicity (Fig C & D). * S2SA mutant showed no significant response against E-64d and TMPRSS2 inhibitor. * Serine at S2' site in spike protein provides an ideal site to be further evaluated for the therapeutic purpose against SARS-CoV- 2.