RESUMO
Like all viruses, HIV-1 requires cellular host factors for replication. The mechanisms for production of progeny virions involving these host factors, however, are not fully understood. To better understand these mechanisms, we used a yeast (Saccharomyces cerevisiae) genetic screen to identify mutant strains in which HIV-1 Gag targeting to the plasma membrane was aberrant. Of the 917 mutants identified, we selected 14 mutants whose missing genes had single orthologous counterparts in human and tested them for Gag-induced viruslike particle (VLP) release in yeast cells. We found that the Vps18 and Mon2 proteins were important for HIV-1 Gag-induced VLP release in yeast. In eukaryote cells, these host proteins are highly conserved and function in protein trafficking. Depletion of hVps18 or hMon2 reduced the efficient production of infectious HIV-1 virions in human cells. Our data suggest that these cellular factors play an important role in the efficient production of infectious HIV-1 virion particles.
Assuntos
HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Transporte Proteico , Saccharomyces cerevisiae/genética , Virossomos/metabolismo , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
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.
Assuntos
Produtos do Gene gag/metabolismo , HIV-1/fisiologia , Saccharomyces cerevisiae/genética , Esferoplastos/fisiologia , Virologia/métodos , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , HIV-1/genética , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Esferoplastos/genética , Virossomos/metabolismoRESUMO
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.