Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

Abstract

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 34 pandemic. Its ~30 kb RNA genome is translated into two large polyproteins subsequently cleaved 35 by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential 36 yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 37 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of 38 cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass 39 spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering 40 yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 41 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both 42 polyprotein conformation and junction accessibility determine the preference and order of 43 cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of 44 inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein 45 substrate.