Mark Denison
Last active: 2/22/2016

A novel mutation in murine hepatitis virus nsp5, the viral 3C-like proteinase, causes temperature-sensitive defects in viral growth and protein processing.

Sparks JS, Donaldson EF, Lu X, Baric RS, Denison MR
J Virol. 2008 82 (12): 5999-6008

PMID: 18385240 · PMCID: PMC2395152 · DOI:10.1128/JVI.00203-08

Sequencing and reversion analysis of murine hepatitis virus (MHV) temperature-sensitive (ts) viruses has identified putative ts mutations in the replicase nonstructural proteins (nsp's) of these coronaviruses. In this study, reverse transcriptase PCR sequencing of the RNA genome of an isolate of the MHV ts virus Alb ts6, referred to as Alb/ts/nsp5/V148A, identified a putative ts mutation in nsp5 (T10651C, Val148Ala), the viral 3C-like proteinase (3CLpro). The introduction of the T10651C mutation into the infectious MHV clone resulted in the recovery of a mutant virus, the nsp5/V148A virus, that demonstrated reduced growth and nsp5 proteinase activity identical to that of Alb/ts/nsp5/V148A at the nonpermissive temperature. Sequence analysis of 40 degrees C revertants of Alb/ts/nsp5/V148A identified primary reversion to Ala148Val in nsp5, as well as two independent second-site mutations resulting in Ser133Asn and His134Tyr substitutions in nsp5. The introduction of the Ser133Asn or His134Tyr substitution into the cloned nsp5/V148A mutant virus background resulted in the recovery of viruses with increased growth fitness and the partial restoration of nsp5 activity at the nonpermissive temperature. Modeling of the nsp5 structure of Alb/ts/nsp5/V148A predicted that the Val148Ala mutation alters residue 148 interactions with residues of the substrate binding S1 subsite of the nsp5 active-site cavity. This study identifies novel residues in nsp5 that may be important for regulating substrate specificity and nsp5 proteinase activity.

MeSH Terms (19)

Alanine Amino Acid Motifs Amino Acid Sequence Amino Acid Substitution Animals Binding Sites Conserved Sequence Mice Models, Molecular Molecular Sequence Data Murine hepatitis virus Mutation Protein Binding Protein Processing, Post-Translational Protein Structure, Tertiary RNA, Viral Sequence Homology, Amino Acid Temperature Viral Proteins

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