Introduction Norovirus is the most common cause of viral gastroenteritis in man. It affects approximately 267 million people/annum and, although usually self-limiting, infection is still associated with around 200,000 deaths/annum. Norovirus infection has a significant, detrimental impact on societal infrastructure; it is the leading pathogen responsible for forced-ward closures in the NHS. There are no specific treatments available; the most promising target for antiviral therapy is noroviral 3 C protease (3 CLpro) which processes the polyprotein essential for the production of viral proteins. Inhibiting 3 CLpro would stop viral replication. This study focused on the function of 3 CLpro, especially the cleavage sites from its precursor.
Methods The wild-type (WT) 3 CLpro sequence was altered with a cysteine to alanine base substitution to create a 3 CLpro mutant with greatly reduced catalytic activity. The mutant was expressed in E.coli and purified using ion exchange and size-filtration chromatography. Protein expression was confirmed by gel electrophoresis and Western blotting. The specificity of 3 CLpro was studied using a spectrophotometric assay and the rates of reaction of mutant and WT 3 CLpro with substrate were compared. The chemical composition of mutant and WT 3 CLpro were examined using mass spectroscopy.
Results Western blot analysis showed multiple bands indicating that both WT and mutant 3 CLpro appeared to be cleaved out of their precursor. Enzyme kinetic studies, however, confirmed that mutant 3 CLpro had negligible catalytic activity; the WT 3 CLpro’s turnover rate of catalytic activity was 25 times faster than that of mutant 3 CLpro. Mass spectrometry of mutant 3 CLpro generated a mass spectrum and transformed to a protein mass of 18,747.5. This confirmed the identity of noroviral 3 CLpro, which is 19 kDA.
Conclusion Mutant 3 CLpro was still cleaved out of its precursor despite the fact that it has negligible catalytic activity. The most likely explanation is that the cleavage was effected by an E.coli protease, possibly a metalloprotease, acting at the upstream and downstream boundaries thereby releasing the processed mutant 3 CLpro. The fact that mutant 3 CLpro can be cleaved out of its precursor by host-cell proteases raises the possibility that WT 3 CLpro could also be processed by exogenous proteases, rather than cleaving itself from its precursor. If this action by host cell proteases occurs in vivo, then it might indicate that the norovirus 3 CLpro is only needed for the cleavage of polyproteins within the newly formed virions, which do not have access to host cell proteases. This has significant implications for future research.
Disclosure of Interest None Declared
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