Search

Integration, an indispensable step for retrovirus replication, is executed by integrase (IN), which is expressed as a part of a Gag-Pol precursor. Although mechanistic detail of the IN-catalyzed integration reaction is well defined, numerous evidence have demonstrated that IN is involved in multiple steps of retrovirus replication other than integration. In this study, Huwe1, a HECT-type E3 ubiquitin ligase, was identified as a new cellular interactor of human immunodeficiency virus type 1 (HIV-1) IN. The interaction was mediated through the catalytic core domain of IN and a wide-range region of Huwe1. Interestingly, although depletion of Huwe1 in target cells did not affect the early phase of HIV-1 infection in a human T cell line, we found that infectivity of HIV-1 released from the Huwe1 knockdown cells was significantly augmented more than that of virus produced from control cells. The increase in infectivity occurred in proviral DNA synthesis. Further analysis revealed that Huwe1 interacted with HIV-1 Gag-Pol precursor protein through an IN domain. Our results suggest that Huwe1 in HIV-1 producer cells has a negative impact on early post-entry events during the next round of virus infection via association with an IN region of Gag-Pol.

We developed a unique monoclonal antibody, mAb61A5, using the nucleoprotein (NP) of influenza virus A/Puerto Rico/8/34 (PR8) strain. Truncation and alanine substitution experiments showed that mAb61A5 recognized the NP fragment with residues 17 to 123 in which a conformational epitope formed by the beta1 sheet and the linker region between the alpha1 and alpha2 helices. Variations in the epitope or nearby can partly account for the poor mAb61A5 reactivity with the NP of A/Aichi/2/68 or A/duck/Pennsylvania/10128/84 strains. Interestingly, immunoprecipitation analysis revealed that mAb61A5 preferentially interacted with viral ribonucleoprotein complexes, composed of RNA polymerase, negative/positive sense RNA and NP, rather than exogenously added NP. Immunofluorescence microscopy using mAb61A5 showed a punctate staining in the cytoplasm during the late phase of infection. The punctate NPs accumulated at the microtubule organizing center and co-localized with microtubules. The treatment with leptomycin B to block a CRM1-dependent nuclear export failed to produce the punctate NP. The treatment with nocodazole, a microtubule-depolymerizing agent, showed random distribution of the punctate NP in the cytoplasm. These results suggest that microtubule networks, although were not required for the formation of punctate structures, were responsible for the polarized distribution of the punctate NP antigens, most likely viral progeny ribonucleoprotein complexes.

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.

The genome of influenza type A virus consists of single-stranded RNAs of negative polarity. Progeny viral RNA (vRNA) replicated in the nucleus is nuclear-exported, and finally transported to the budding site beneath the plasma membrane. However, the precise process of the membrane targeting of vRNA is unclear, although viral proteins and cytoskeleton are thought to play roles. Here, we have visualized the translocation process of progeny vRNA using fluorescence in situ hybridization method. Our results provide an evidence of the involvement of vesicular trafficking in membrane targeting of progeny vRNA independent of that of viral membrane proteins.

Dendritic cells (DCs) are professional antigen-presenting cells that possess a unique capacity to cross-present exogenous antigens efficiently to CD8 + T cells. We previously demonstrated that monocyte-derived DCs (MDDCs) pulsed with yeast-derived HIV-1 Gag virus-like particles (VLPs) were able to activate Gag-specific CD8 + T cells from HIV-1-infected individuals. Yeast VLPs are abundantly mannosylated (high-mannose type: HmVLPs) and are highly immunogenic. Because lectin receptors are shown to negatively regulate Th1 responses, we investigated the relationship between VLP mannosylation level and MDDC cross-presentation activity. Poorly mannosylated VLPs (low-mannose type: LmVLPs) were prepared using a yeast mnn9 mutant strain that lacks a core mannosylation enzyme. We found that MDDCs pulsed with LmVLPs activated Gag-specific T cells more strongly than those pulsed with HmVLPs. However, MDDCs showed similar antigen uptake and intracellular transport of both types of VLPs. Interestingly, LmVLPs induced IL-12 production slightly more than HmVLPs (yet statistically significant). Furthermore, the level of LPS-induced IL-10 production was enhanced by pulsing with HmVLPs, but not with LmVLPs. These results indicate that lectin receptors recognizing mannose may influence the Th1/Th2 balance of the immune response, resulting in reduced efficiency of CD8 + T cell activation by a heavily mannosylated antigen presented by DCs.