SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

SARS-CoV-2 Spike-Mediated Entry and Its Regulation by Host Innate Immunity.

The constantly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) fuel the worldwide coronavirus disease (COVID-19) pandemic. The spike protein is essential for the SARS-CoV-2 viral entry and thus has been extensively targeted by therapeutic antibodies. However, mutations along the spike in SARS-CoV-2 VOC and Omicron subvariants have caused more rapid spread and strong antigenic drifts, rendering most of the current antibodies ineffective. Hence, understanding and targeting the molecular mechanism of spike activation is of great interest in curbing the spread and development of new therapeutic approaches. In this review, we summarize the conserved features of spike-mediated viral entry in various SARS-CoV-2 VOC and highlight the converging proteolytic processes involved in priming and activating the spike. We also summarize the roles of innate immune factors in preventing spike-driven membrane fusion and provide outlines for the identification of novel therapeutics against coronavirus infections.

Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Spike Protein Proteolytic Processing.

The coronavirus spike envelope glycoprotein is an essential viral component that mediates virus entry events. Biochemical assessment of the spike protein is critical for understanding structure-function relationships and the roles of the protein in the viral life cycle. Coronavirus spike proteins are typically proteolytically processed and activated by host cell enzymes such as trypsin-like proteases, cathepsins, or proprotein-convertases. Analysis of coronavirus spike proteins by western blot allows the visualization and assessment of proteolytic processing by endogenous or exogenous proteases. Here, we present a method based on western blot analysis to investigate spike protein proteolytic cleavage by transient transfection of HEK-293 T cells allowing expression of the spike protein of the highly pathogenic Middle East respiratory syndrome coronavirus in the presence or absence of a cellular trypsin-like transmembrane serine protease, matriptase. Such analysis enables the characterization of cleavage patterns produced by a host protease on a coronavirus spike glycoprotein.