Abstract
Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >10(7) PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to approximately 10(4) PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 10(2) PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.
Highlights
Murine hepatitis virus (MHV) is a member of the family Coronaviridae within the order Nidovirales
We have shown that murine hepatitis virus (MHV) has substantial flexibility in cleavage site and proteinase interactions around nsp1, nsp2, and nsp3 that can still allow virus replication in culture
While our previous work showed that cleavage site 1 (CS1) and even the entire nsp2 could be deleted in viable mutant viruses, the present study demonstrates that all cleavages mediated by PLP1 can be eliminated, as can the proteinase activity of PLP1
Summary
Murine hepatitis virus (MHV) is a member of the family Coronaviridae within the order Nidovirales. Group 2b and group 3 coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and infectious bronchitis virus (IBV), respectively, encode only one enzymatically active PLP in the position of PLP2. In addition to the PLP domains, the 210-kDa nsp contains other conserved domains with demonstrated or predicted functions, including an “Ac domain” highly enriched in acidic residues; zinc ribbon motifs within the PLP domains [23, 51]; an “X domain” with sequence homology to ADP ribose-1Љ monophosphate processing enzyme (Appr-1Љ-p), whose enzymatic activity has been shown in vitro for SARS-CoV and HCoV229E [33, 34]; and a C-terminal “Y domain” containing hydrophobic amino acid stretches that possibly confer membrane-spanning ability to this large protein. Nidoviruses, including coronaviruses, employ a complex RNA synthesis program that functions to generate both new genomic RNA as well as a nested set of subgenomic mRNAs that are the translational templates for ORFs 2 to 7
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