Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication.

TitleSite-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication.
Publication TypeJournal Article
Year of Publication2012
AuthorsKobayashi M, Arias C, Garabedian A, Palmenberg AC, Mohr I
JournalJ Virol
Volume86
Issue19
Pagination10686-94
Date Published2012 Oct
ISSN1098-5514
KeywordsCell Line, Cysteine Endopeptidases, Encephalomyocarditis virus, Escherichia coli, Fibroblasts, Gene Expression Regulation, Viral, HEK293 Cells, HeLa Cells, Humans, Plasmids, Poly(A)-Binding Proteins, Polyribosomes, Protein Structure, Tertiary, Recombinant Proteins, RNA, Viral, Viral Proteins, Virus Replication
Abstract

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.

DOI10.1128/JVI.00896-12
Alternate JournalJ. Virol.
PubMed ID22837200
PubMed Central IDPMC3457283
Grant ListT32 AI007647 / AI / NIAID NIH HHS / United States
U19 AI070503 / AI / NIAID NIH HHS / United States
R01 GM056927 / GM / NIGMS NIH HHS / United States
T32AI007647 / AI / NIAID NIH HHS / United States
R01 AI017331 / AI / NIAID NIH HHS / United States
AI017331 / AI / NIAID NIH HHS / United States
T32 AI007180 / AI / NIAID NIH HHS / United States
T32AI07180 / AI / NIAID NIH HHS / United States
AI073898 / AI / NIAID NIH HHS / United States
GM056927 / GM / NIGMS NIH HHS / United States
R01 AI073898 / AI / NIAID NIH HHS / United States
R56 AI017331 / AI / NIAID NIH HHS / United States