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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 Mpro 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.
Highlights
The discovery of severe acute respiratory syndrome coronavirus (SARS-CoV) as the causative agent of the SARS epidemic in 2003 brought the world’s attention to the human pathogenic potential of zoonotic infections by coronaviruses [1,2]
FPS4–5 served as a positive control analogous to the highly efficient N-terminal auto-cleavage site nsp4–5 of SARS-CoV main chymotrypsin-like protease (Mpro)
We analyzed the processing of the SARS-CoV polyprotein region nsp7–10 in vitro
Summary
The discovery of severe acute respiratory syndrome coronavirus (SARS-CoV) as the causative agent of the SARS epidemic in 2003 brought the world’s attention to the human pathogenic potential of zoonotic infections by coronaviruses [1,2]. Two-thirds of their single-strand (+)-sense RNA genome exhibit the overlapping replicase genes ORF 1a and ORF 1ab, and the other one third encode for a set of subgenomic mRNAs which are required for accessory proteins as well as for the structural proteins. ORF 1a and ORF 1ab are directly translated into either replicase polyprotein pp1a (nsp1–11) or pp1ab (nsp1–16), respectively, depending on a ribosomal (-1)-frameshift [3]. The nsp’s take part in forming the replication/transcription complex (RTC), a membrane-anchored, highly dynamic protein–RNA complex facilitating the replicative processes [4,5,6,7]. Two CoV proteases facilitate the processing of the polyprotein, the papain-like protease (PLpro; nsp3) between nsp and main chymotrypsin-like protease (Mpro; 3CLpro, nsp5) between nsp4–11/
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