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.

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

“…Once in the nucleus, the hnRNP A1⅐Trn-1 complex is disassembled by RanGTP (67). Several viruses interfere with these processes via degradation of nucleoporins and a disruption of Trn-1 function (45, 68 -70) or by directly modulating Nup62 function (71,72). We found that, although the other FG repeat-containing Nup98 proteins were unaffected, the abundance of Nup62 was reproducibly decreased (Fig.…”

Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

“…Instead, Ran:L E binding is leveraged by consequent activation of a potent Nup phosphorylation cascade, the true cause of trafficking inhibition. This inhibition happens in infected cells even before the virus begins to replicate (6,26). The cascade involves Erk1/2 and p38 kinases and is absolutely dependent on Ran:L E interactions and also on the dual phosphorylation of L E itself, an activity that is a prerequisite, and not a consequence, of the Nup modifications (7,10,15,16,27).…”

“…Recombinant L E -GST (EMCV) and mutated derivatives were expressed and purified from Escherichia coli as previously described (6,15,24). GST-L M (Mengo) has also been previously described (14).…”

“…Ran, a member of the Ras superfamily of GTPases, normally alternates between nuclear GTP-and cytoplasmic GDP-bound conformers, acting as a molecular switch for the coordinated transport of large molecules back and forth through the nuclear pores (4). However, when L binds Ran, the perverted complex recruits and activates a specific cohort of cellular kinases responsible for the L-induced hyperphosphorylation of Phe/Gly-containing nuclear pore proteins (Nups) (5)(6)(7)(8). The consequence is rapid, potent inhibition of active nuclear-cytoplasmic trafficking.…”

HIV-1 remodels the nuclear pore complex

“…For instance, NPC destabilization may result from the association of Nups and integrated proviral cDNA or nuclear vRNA during transcription (Capelson et al, 2010;Hou and Corces, 2010;Strambio-De-Castillia et al, 2010). HIV-1 may disrupt NPCs to evade antiviral responses, as seen in other viruses (Gustin and Sarnow, 2002;Fontoura et al, 2005;Bardina et al, 2009;Porter and Palmenberg, 2009;Atasheva et al, 2010;Ren et al, 2010). Another possibility is that encapsidated Nup62 may be required for efficient delivery of the PIC into the nuclei of newly infected cells (Fig.…”