Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase

Abstract

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1 positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more ␤-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.The outbreak of a severe atypical pneumonia in early 2003 has caused 8422 cases and 916 related deaths. The World Health Organization has designated the illness as severe acute respiratory syndrome (SARS).1 A novel form of coronavirus has been identified as the major cause of SARS (1, 2). The genome of SARS coronavirus has been sequenced within a short period of time after confirmation of the virus (3, 4). Currently, 23 genome sequences of different variations of SARS coronavirus have been released at the NCBI web site (www.ncbi.nlm.nih. gov/). Coronaviruses are members of positive-stranded RNA viruses featuring the largest viral RNA genomes up to date. The SARS coronavirus replicase gene encompasses two overlapping translation products, polyproteins 1a (ϳ450 kDa) and 1ab (ϳ750 kDa), which are conserved both in length and amino acid sequence to other coronavirus replicase proteins. Polyproteins 1a and 1ab are cleaved by the internally encoded 3C-like proteinase to release functional proteins necessary for virus replication. The SARS 3C-like proteinase is fully conserved among all of the released SARS coronavirus genome sequences and is highly homologous with other coronavirus 3C-like proteinase.Two crystal structures of coronavirus 3C-like proteinase from transmissible gastroenteritis virus (TGEV) (5) and human coronavirus (hCoV) 229E have been solved (6). The structure of coronavirus 3C-like proteinase contains three domains. The first two domains form ...

Enzymatic activity of the SARS coronavirus main proteinase dimer

“…The amino acid sequence Ala-ArgLeu-Gln is similar to sequences recognized by the SARS CoV main proteinase at cleavage sites in the replicase polyprotein [17,18]; cleavage occurs after Gln. We also cloned the gene for the SARS CoV main proteinase, expressed the gene in Escherichia coli without purification tags, and purified the enzyme.…”

“…The least sensitive and most laborious assay for the SARS CoV main proteinase monitors cleavage of peptides by reverse phase HPLC [18,21,5]. A continuous, chromogenic enzyme assay has been developed using p-nitroanilide as the chromophore [22].…”

Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization

“…Surface Plasmon Resonance (SPR) Biosensor Analysis-A 6-amino acid peptide (TSAVLQ) representing the N-terminal P6 -P1 sequence of a reported best peptide substrate (TSAVLQSGFRK) for SARS 3CL pro (13) was synthesized as a model substrate. The binding affinities of the full-length and N-terminal deleted SARS 3CL pro s to the substrate peptide were determined using the SPR biosensor technology (21).…”

“…The critical role of domain III in dimerization and enzymatic activity of SARS 3CL pro has been presented by a recent study (12). In addition, Lai and co-workers (13) reported that SARS 3CL pro exists as a monomer/dimer mixture at a relatively high protein concentration (ϳ4 mg/ml) and is exclusively monomer at a lower protein concentration (ϳ0.2 mg/ml) in solution, and only the dimer was the active form of the proteinase.…”

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation

“…These precursors undergo proteolytic processing via two virally encoded proteinases to generate 16 mature proteins, nsp1 through nsp16. Probably most of the gene 1 proteins are important for viral RNA synthesis (5,(7)(8)(9)(10)(11). Some gene 1 proteins are predicted to have biological functions related to RNA synthesis (11), and some have demonstrated functions that appear to be involved in RNA synthesis (7,8,10,12), whereas some gene 1 proteins may have functions other than viral RNA synthesis (13)(14)(15)(16).…”