2005 Fiscal Year Final Research Report Summary
Identification of Host Factors for Virus Particle Release Based on Yeast Genetics
| Project/Area Number |
15390152
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Virology
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| Research Institution | Kitasato University |
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Principal Investigator |
MORIKAWA Yuko Kitasato University, Kitasato Institute for Life Sciences, Professor, 北里生命科学研究所, 教授 (20191017)
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| Co-Investigator(Kenkyū-buntansha) |
MOMOSE Fumitaka Kitasato University, Kitasato Institute for Life Sciences, Research Associate, 北里生命科学研究所, 助手 (90332204)
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| Project Period (FY) |
2003 – 2005
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| Keywords | VIirus / Ywast / Host Factor / Trafficking / Particle Assembly / Budding |
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| Research Abstract |
We have previously shown that Saccharomyces cerevisiae supports human immunodeficiency virus type 1 (HIV-1) Gag particle assembly, indicating that yeast has all the host factors necessary for HIV-1 Gag particle, To identify the host factors/machinery responsible for HIV-1 Gag transport and subsequent particle budding, 1)using a series of yeast genetic mutants of endosomal pathways suggested by recent studies, we examined Gag trafficking pathways ; 2)defined the negative effect of HIV-2 Gag on particle assembly in yeast ; and 3)by use of CytoTrap two-hybrid assay, attempted to identify a novel host factor(s) responsible for intracellular transport of Gag.
1)Endosomal pathways for Gag trafficking : Using a series of yeast genetic mutants of endosomal, we found that the defect of early and late endosomal t-SNAREs showed severe reduction in particle production but surprisingly, the class E vacuolar protein sorting molecules were largely dispensable. To define the roles of the t-SNAREs in Ga
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g trafficking, we depleted/over-expressed their orthologues in human cells. Results indicated that the t-SNAREs are positive factors for HIV-1 Gag transport.
2)Negative effect of HIV-2 Gag on particle assembly in yeast : We also expanded our previous study using diverse primate lentiviral Gags and showed that yeast did not support production of HIV-2 or simian immunodeficiency virus (SIV) mac Gag particles. Studies revealed that, i)the presence of the N-terminal half of HIV-2 MA resulted in a failure of particle budding in yeast ; ii)the Gag was transported to the plasma membrane ; iii)the Gag was easily dissociated from the membrane in the absence of salt ; iv)as a result, particle budding was arrested with membrane ruffling structures. Together, these data suggest that yeast may lack a factor(s) necessary for HIV-2 and SIVmac Gag VLP budding.
3)Isolation of a novel host factor(s) responsible for intracellular transport of Gag : We used yeast CytoTrap two-hybrid assay (a system in which protein-protein interactions occur underneath the plasma membrane) and screened an expression library of human cDNAs (2-3 positives at present). Less
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Research Products
(9 results)
