Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two... Show moreCoronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1(pro)) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1(pro) domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2(pro), a downstream paralog that is conserved in the nsp3's of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1(pro) with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1(pro) was shown to cleave a peptide mimicking the cognate nsp2 vertical bar nsp3 cleavage site. Like its PL2(pro) paralogs from several coronaviruses, TGEV PL1(pro) was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2(pro) from other alphacoronaviruses, e. g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PL(pro)s with overlapping specificities toward both viral and cellular substrates. Show less
The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses... Show moreThe gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenomelength templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle. Show less
