Processing of the SARS-CoV pp1a/ab nsp7–10 region

Abstract

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/ transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease M pro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

Expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle

“…One would expect that these nsp's are cleaved in an order that reflects their associations with one another and other key proteins in line with the viral needs during the production of new viruses. Nsp10 and nsp9 have no reported structures involving either nsp7 or nsp8 ( Figure 1) this was also confirmed by by Krichel and coauthors [6] therefore it is not surprising that it is released from the polyprotein first as it requires no other partners to carry out its function. A spherical dodecameric structure has been reported that is composed of trimeric units of nsp10 alone [11] and a monomeric structure of nsp10 has also been reported [12].…”

“…Similarly, nsp9 has been shown to form dimeric structures that bind RNA [13,14] (Figure 1). Krichel and coauthors also confirmed the presence of nsp9 monomers and dimers in solution but no complexes of nsp9 with the other nsp's [6]. Therefore, it seems logical that nsp9 will be cleaved next rather than nsp7.…”

“…They have been proposed as interacting partners for nsp12, nsp14 (exoribonuclease, ExonN) and nsp16 (ribose 2 0 -O-MTase) as well as RNA [5], but their exact functions are not well characterized. Krichel and coauthors in their article [6] investigate the processing of the nsp7-10 polyprotein. Their results show for the first time the critical role protein conformational structure plays in the order in which the nsp's are released from the polyprotein.…”

“…It is likely that the need for so many key interactions is for distinguishing protein conformation of the polyprotein. Krichel and coauthors [6] indicated that conformation of the nsp7-10 polyprotein resulted in nsp's being cleaved from the polyprotein by M pro (PDB code 1UK3 [8]-purple) in order; nsp10 then nsp9 then nsp8 from nsp7. Structures available for the nsp's are depicted as ribbon structures visualized using Discovery Studio software version 19.1.0.18287 (Accelrys, San Diego, U.S.A.) in corresponding colors.…”

The COVID-19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control

“…Of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3CLpro (3C-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for RNA/DNA binding activity, (6) nsp12 is an RNA-dependent RNA polymerase (RdRp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3′-5′ exonuclease (ExoN), 9) nsp15 is a poly(U)-specific endoribonuclease (XendoU). The remaining nsps are involved in transcription and replication of the viral genome [13,46,47].…”