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Identification of Host Factors for Virus Particle Release Based on Yeast Genetics

基于酵母遗传学的病毒颗粒释放宿主因素的鉴定

基本信息

批准号：
15390152
负责人：
MORIKAWA Yuko
金额：
$ 9.34万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390152/
关键词：
VIirus Ywast Host Factor Trafficking Particle Assembly Budding

项目摘要

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 … More 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

我们以前已经证明酿酒酵母支持人类免疫缺陷病毒1型(HIV-1)Gag颗粒的组装，这表明酵母具有HIV-1 Gag颗粒所需的所有宿主因子，以确定负责HIV-1 Gag运输和随后的颗粒萌发的宿主因素/机制，1)利用最近研究提出的一系列酵母内体途径的遗传突变体，我们研究了Gag的转运途径；2)明确了HIV-2 Gag对酵母颗粒组装的负面影响；3)利用细胞陷阱双杂交技术，试图确定一个新的宿主因子(S)负责Gag的胞内转运。1)Gag转运的内体途径：利用一系列内体酵母基因突变体，我们发现早期和晚期内体t-陷阱的缺陷表现出严重的颗粒生成减少，但令人惊讶的是，E类空泡蛋白分选分子在很大程度上是可有可无的。定义T形陷阱在GA…中的作用更多的G贩运，我们耗尽/过度表达它们在人类细胞中的同源基因。2)HIV-2 Gag对酵母颗粒组装的负面影响：我们还利用不同的灵长类慢病毒Gag对我们先前的研究进行了扩展，发现酵母不支持HIV-2或猴免疫缺陷病毒(SIV)Mac Gag颗粒的产生。研究表明，1)HIV-2 MA的N端半部分的存在导致了酵母中颗粒萌发的失败；ii)Gag被运输到质膜；iii)在无盐的情况下，Gag很容易从膜上解离；iv)由于膜的褶皱结构，颗粒萌发被阻止了。综上所述，这些数据表明酵母可能缺乏HIV-2和SIVmac Gag VLP萌发所必需的因子(S)。3)负责Gag细胞内运输的新宿主因子(S)的分离：我们使用酵母细胞陷阱双杂交实验(一种蛋白质-蛋白质相互作用发生在质膜下的系统)并筛选了人cDNA的表达文库(目前有2-3个阳性)。较少