title: Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral 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.
authors: Department of Microbiology & NYU Cancer Institute, New York University School of Medicine, New York, New York, USA.
authors: Mariko
authors: M
authors: Kobayashi
authors: Carolina
authors: C
authors: Arias
authors: Alexandra
authors: A
authors: Garabedian
authors: Ann C
authors: AC
authors: Palmenberg
authors: Ian
authors: I
authors: Mohr
mesh_terms: D000086042:3C Viral Proteases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004686:Encephalomyocarditis virus / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D039102:Poly(A)-Binding Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases