Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus-cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely determine how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.coronavirus | virus entry | receptor | protease E nveloped viruses deposit their genomes into host cells by coalescing their membranes with the cell. These functions are executed by virion envelope-anchored glycoprotein trimers termed "membrane-fusion proteins." In virus-infected cells, these proteins are synthesized as inactive forms, structured such that they can maintain their membrane-fusion potential throughout their residence on extracellular virus particles. The proteins then transit into fusion-competent forms during virus-cell entry. Various environmental stimuli control these cell entry-related structural transitions. Proteolysis is central, as fusion proteins cleaved by host proteases are frequently liberated to undergo transitions into fusion-competent forms (1-3). Knowledge of the proteolytic cleavages and host proteases regulating virus infections can be used to predict viral tropism and pathogenesis (4, 5), and can also reveal antiviral strategies (6).Coronaviruses (CoVs) are enveloped, positive-stranded RNA viruses in the order Nidovirales. These viruses infect mammals and birds, and are mainly associated with respiratory and enteric tract disorders (7). Of six known human CoVs, the severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV are the most recent to have emerged from zoonotic reservoirs, which inc...

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

“…The present study indicates that SARS-CoV-2 spread also depends on TMPRSS2 activity, although we note that SARS-CoV-2 infection of Calu-3 cells was inhibited but not abrogated by camostat mesylate, likely reflecting residual S protein priming by CatB/L. One can speculate that furin-mediated precleavage at the S1/S2 site in infected cells might promote subsequent TMPRSS2-dependent entry into target cells, as reported for MERS-CoV (Kleine-Weber et al, 2018;Park et al, 2016). Collectively, our present findings and previous work highlight TMPRSS2 as a host cell factor that is critical for spread of several clinically relevant viruses, including influenza A viruses and coronaviruses (Gierer et al, 2013;Glowacka et al, 2011;Iwata-Yoshikawa et al, 2019;Kawase et al, 2012;Matsuyama et al, 2010;Shulla et al, 2011;Zhou et al, 2015).…”

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

“…S comprises two functional subunits responsible for binding to the host cell receptor (S 1 subunit) and fusion of the viral and cellular membranes (S 2 subunit). For many CoVs, S is cleaved at the boundary between the S 1 and S 2 subunits, which remain non-covalently bound in the prefusion conformation Bosch et al, 2003;Burkard et al, 2015;Park et al, 2016;Walls et al, 2016a). The distal S 1 subunit comprises the receptor-binding domain(s) and contributes to stabilization of the prefusion state of the membrane-anchored S 2 subunit that contains the fusion machinery (Gui et al, 2017;Kirchdoerfer et al, 2016;Pallesen et al, 2017;Song et al, 2018;Walls et al, 2016a;Walls et al, 2017b;Yuan et al, 2017).…”

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

“…Proteolytic processing and receptor-binding act in synergy to induce large-scale S conformational changes promoting coronavirus entry. Priming involves S cleavage by host proteases at the boundary between the S 1 and S 2 subunits (S 1 /S 2 ), in a subset of coronaviruses, and at a conserved site upstream of the fusion peptide (S 2 ') in all known coronaviruses (Belouzard et al, 2009;Burkard et al, 2014;Millet and Whittaker, 2014;Park et al, 2016). The latter site is believed to be the most important for membrane fusion activation.…”