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Journal of Virology, February 2009, p. 1941-1951, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01752-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Leader-Induced Phosphorylation of Nucleoporins Correlates with Nuclear Trafficking Inhibition by Cardioviruses{triangledown}

Frederick W. Porter* and Ann C. Palmenberg

Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received 19 August 2008/ Accepted 3 December 2008

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 (C19A) 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.

* Corresponding author. Mailing address: Institute for Molecular Virology, Robert Bock Laboratories, University of Wisconsin-Madison, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 262-7228. Fax: (608) 262-6690. E-mail: fwporter{at}wisc.edu

{triangledown} Published ahead of print on 10 December 2008.

Journal of Virology, February 2009, p. 1941-1951, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01752-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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