Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non-shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi-permeabilized infected cells con®rmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor±cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirusinfected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus.

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.Picornaviruses are small, nonenveloped viruses that contain RNA genomes of positive polarity. The genomes of all picornaviruses are organized in a similar fashion, with a long 5Ј untranslated region (UTR), an open reading frame encoding the viral polyprotein, and a 3Ј UTR (reviewed in reference 55). The 5Ј UTR contains sequences that are important for replication of the viral genome, as well as an internal ribosomal entry site (IRES), which is required for translation of the viral polyprotein (5,6,45). The viral polyprotein is translated from a single large open reading frame and is co-and posttranslationally processed to produce the individual viral gene products (reviewed in reference 55). The 3Ј UTR contains a high degree of secondary structure as well as conserved sequences important for viral replication (49,53,56,57).Numerous interactions between poliovirus and the host cell have been described. For example, during poliovirus infection, the translation initiation factors eIF4GI and -II are cleaved, and translation of capped cellular mRNAs is inhibited (15, 19). Likewise, alterations in cellular transcription rates have been attributed to cleavage of components of the transcriptional apparatus (12,13,(70)(71)(72). In addition, poliovirus infection results in the inhibition of host cell secretion (14) and the induction and subsequent inhibition of apoptosis (3, 65). Recently, we demonstrated that poliovirus infection of HeLa cells results in a dramatic inhibition of n...

Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2Apro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.Poliovirus is a positive-strand RNA virus belonging to the family Picornaviridae. Poliovirus, like other members of this family, encodes a single large polyprotein that is co-and posttranslationally processed by virus-encoded proteases to produce the individual viral gene products (reviewed in reference 40). After the production of viral proteins, RNA synthesis ensues on virus-induced vesicles in the cell cytoplasm. Despite the fact that viral translation, RNA synthesis, and assembly occur in the cytoplasm, a number of host nuclear proteins have been attributed roles in the viral life cycle (3,6,27,32,33,51). Consistent with their having a role in the replication of poliovirus in the cytoplasm, several host nuclear factors have been shown to redistribute to the cytoplasm after infection (3,5,32,33,51).For nuclear factors to move from the nucleus to the cytoplasm they must transit the nuclear pore complex (NPC), a large protein channel found embedded in the nuclear envelope. The vertebrate NPC has a molecular mass of 125 MDa and is composed of multiple copies of roughly 30 different proteins that are collectively called nucleoporins (Nups; reviewed in reference 18). A number of findings have suggested that the redistribution of host nuclear factors to the cytoplasm in poliovirus-infected cells is due to alterations of the NPC and a disruption in nucleocytoplasmic trafficking. Previous work has shown that two NPC proteins, Nup153 and Nup62, are targeted for degradation in ...

Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.