Abstract
The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (M(pro)) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV M(pro) is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-β-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV M(pro) autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln↓(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of M(pro), resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative M(pro) autoprocessing sites has not been described previously for other nidovirus M(pro) domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales.
Highlights
The order Nidovirales comprises four profoundly separated clusters of plus-strand RNA viruses that have been assigned to three families called Coronaviridae, Arteriviridae, and Roniviridae [13, 20]
We decided to embark on a systematic characterization of the molecular biology of bafiniviruses, and we report here the results of a first set of experiments aimed to investigate the expression and autoprocessing of the White bream virus (WBV) main protease (Mpro), an enzyme predicted to play a key role in the proteolytic processing of the WBV ORF1a/1b-encoded polyproteins, pp1a and pp1ab, and in the formation and functional maturation of the viral replication-transcription complex, as shown previously for other nidoviruses
Based on comparative sequence analysis data (Fig. 4A and data not shown) and crystal structure information available for 3C and 3C-like proteases [2, 3, 7, 31, 32, 42], it is reasonable to suggest that the N-proximal two-thirds of the WBV Mpro have a chymotrypsin-like two--barrel fold that is extended at the C terminus by an extra domain
Summary
The order Nidovirales comprises four profoundly separated clusters of plus-strand RNA viruses that have been assigned to three families called Coronaviridae, Arteriviridae, and Roniviridae [13, 20]. Molecular studies of the biology of bafiniviruses may be expected to provide new insight into conserved and distinct biological features of the major clusters of nidoviruses and, possibly, help identify major driving forces involved in the evolution of this highly divergent group of RNA viruses. We decided to embark on a systematic characterization of the molecular biology of bafiniviruses, and we report here the results of a first set of experiments aimed to investigate the expression and autoprocessing of the WBV main protease (Mpro), an enzyme predicted to play a key role in the proteolytic processing of the WBV ORF1a/1b-encoded polyproteins, pp1a and pp1ab, and in the formation and functional maturation of the viral replication-transcription complex, as shown previously for other nidoviruses (reviewed in references 47 and 58). The present study provides initial insight into the autoprocessing characteristics and substrate specificity of the WBV Mpro using information derived from heterologous expression systems and WBV-infected cells and revealed conserved residues predicted to be involved in catalysis and substrate specificity
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