title: Leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses.
abstract: Picornaviruses disrupt nucleocytoplasmic trafficking pathways during infection. Poliovirus and rhinovirus inhibit nuclear protein import/export through a series of 2A protease-dependent cleavages within nuclear pore proteins (nucleoporins [Nups]), including Nup62, Nup98, and Nup153. Cardioviruses lack the same protease and instead affect trafficking inhibition through an activity mapped to their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide with no known enzymatic activity. We have shown that L from encephalomyocarditis virus (EMCV) binds and inhibits the activity of Ran-GTPase, a key regulator of nucleocytoplasmic transport. We now report that recombinant EMCV L triggers the unregulated efflux of protein cargo from preloaded HeLa cell nuclei in cell-free reactions dependent upon Xenopus egg cytosol or HeLa cell-derived cytosol. Recombinant L was the only viral protein necessary for this activity or for nuclear protein import inhibition. Mutational disruption of the L protein zinc finger domain (C(19)A) abrogated the inhibitory activity for both import and efflux in cell extracts, but mutations in the C-terminal acidic domain of L (aa 37 to 61) did not. Notably, HeLa cell nuclei treated with L, or those from EMCV-infected cells, showed reproducibly altered patterns of nucleoporin phosphorylation. Nup62, Nup153, and Nup214 each became hyperphosphorylated in an L-dependent manner. Staurosporine, a broad-spectrum kinase inhibitor, blocked this phosphorylation and rescued nuclear import/export activity from L-dependent inhibition. Therefore, cardioviruses target the same group of nucleoporins as enteroviruses, but the effector mechanism triggered by L (or L-Ran complexes) involves a unique cytosol-dependent phosphorylation cascade rather than proteolysis.
authors.affiliation: Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, 53706, USA. fwporter@wisc.edu
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Porter
authors.forename: Ann C
authors.initials: AC
authors.lastname: Palmenberg
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004686:Encephalomyocarditis virus / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D010063:Ovum; D010766:Phosphorylation; D021381:Protein Transport; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D028861:Nuclear Pore Complex Proteins; D014764:Viral Proteins