Prediction and biochemical analysis of putative cleavage sites of the 3C-like protease of Middle East respiratory syndrome coronavirus

Abstract

Coronavirus 3C-like protease (3CLpro) is responsible for the cleavage of coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature non-structural proteins (nsps) of nsp4-16. The nsp5 of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as 3CLpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp1a/1ab awaits further analysis. Here, we proposed a method for predicting coronaviral 3CLpro cleavage sites which balances the prediction accuracy and false positive outcomes. By applying this method to MERS-CoV, the 11 canonical cleavage sites were readily identified and verified by the biochemical assays. The Michaelis constant of the canonical cleavage sites of MERS-CoV showed that the substrate specificity of MERS-CoV 3CLpro is relatively conserved. Interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by MERS-CoV nsp5. These results pave the way for identification and functional characterization of new nsp products of coronaviruses.

Middle East respiratory syndrome coronavirus: a comprehensive review

“…The third approach involves in vitro inhibition of S protein to block virus entry into host cells using designed antiviral peptides targeting the HR2 domain of the S2 subunit of the MERS-CoV and preventing the interaction between the HR1 and HR2 domains required for the formation of the heterologous six-helix bundle in viral fusion core formation [22,23]. Other drugs that act as inhibitors for viral proteases and helicase to suppress MERS-CoV infection were tested [141][142][143][144][145]. Other investigators studied inhibition of MERS-CoV infection by competitive inhibition of DPP4 cell receptor using compounds such as sitagliptin, vildagliptin, and saxagliptin [19,146].…”

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

“…This was shown in detail for the processing order of the nsp7-10 region, where domain deletions or switching as well as cleavage site mutations were lethal to the virus replication [19]. In general, the order of processing is directly dependent on the substrate specificity of M pro , which has been tested in peptide-based biochemical assays [20][21][22]. However, peptide assays interrogate merely the substrate specificity for the isolated amino-acid sequence but disregard the polyprotein's structural layout, i.e.…”

High-Throughput Screening for Inhibitors of the SARS-CoV-2 Protease Using a FRET-Biosensor

“…Testing of 10,000 compounds with this probe identified seven hits, with ebselen being the strongest inhibitor. However, the specialised nature and cost of chemically synthesised probes makes them inaccessible to use in high-throughput screens for many facilities [ 10 ]. In contrast, protein-based biosensors can readily be prepared using equipment available in most molecular biology labs.…”