Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition.

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 confirmed 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 poliovirus-infected 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.

Encapsidation of viral DNA requires the adenovirus L1 52/55-kilodalton protein.

Previous work demonstrated that the adenovirus L1 52/55-kDa protein is required for assembly of viral particles, although its exact role in the assembly process is unclear. The 52/55-kDa protein's early expression, however, suggests that it might have other roles at earlier times during infection. To uncover any role the 52/55-kDa protein might have at early times and to better characterize its role in assembly, a mutant adenovirus incapable of expressing the 52/55-kDa protein was constructed (H5pm8001). Analysis of the onset and extent of DNA replication and late protein synthesis revealed that H5pm8001-infected 293 cells entered the late stage of infection at the same time as did adenovirus type 5 (Ad5)-infected cells. Interestingly, H5pm8001-infected cells displayed slightly lower levels of replicated viral DNA and late proteins, suggesting that although not required, the 52/55-kDa protein does augment these activities during infection. Analysis of transcripts produced from the major late and IVa2 promoters indicated a slight reduction in H5pm8001-infected compared to Ad5-infected cells at 18 h postinfection that was not apparent at later times. Analysis of particles formed in H5pm8001 cells revealed that empty capsids could form, suggesting that the 52/55-kDa protein does not function as a scaffolding protein. Subsequent characterization of these particles demonstrated that they lacked any associated viral DNA. These findings indicate that the 52/55 kDa-protein is required to mediate stable association between the viral DNA and empty capsid and suggest that it functions in the DNA encapsidation process.

Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection.

The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of adenovirus infection. A potential role for the IVa2-52/55-kDa protein interaction in the regulation of transcription from the major late promoter and in viral assembly is discussed.