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.
CITATION STYLE
Kobayashi, M., Arias, C., Garabedian, A., Palmenberg, A. C., & Mohr, I. (2012). Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication. Journal of Virology, 86(19), 10686–10694. https://doi.org/10.1128/jvi.00896-12
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