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

我们先前已经表明酿酒酵母支持人类免疫缺陷病毒1型（HIV-1）Gag颗粒组装，表明酵母具有HIV-1 Gag颗粒所需的所有宿主因子，为了鉴定负责HIV-1 Gag转运和随后的颗粒出芽的宿主因子/机制，1）使用最近研究提出的一系列内体途径的酵母遗传突变体，我们检查了Gag运输途径; 2）确定了HIV-2Gag对酵母中颗粒组装的负面影响;和3）通过使用CytoTrap双杂交测定，试图鉴定负责Gag细胞内运输的新宿主因子。1）Gag运输的内体途径：使用一系列酵母内体遗传突变体，我们发现早期和晚期内体t-SNARE的缺陷显示出颗粒产生的严重减少，但令人惊讶的是，E类液泡蛋白分选分子在很大程度上被破坏。定义Ga中t-SNARE的角色 关于我们 g贩运，我们在人细胞中耗尽/过度表达它们的直系同源物。结果表明，t-SNARE是HIV-1 Gag转运的积极因素。2）HIV-2 Gag对酵母中颗粒组装的负面影响：我们还扩展了我们先前使用不同灵长类慢病毒Gag的研究，并表明酵母不支持HIV-2或猿猴免疫缺陷病毒（SIV）mac Gag颗粒的产生。研究表明，i）HIV-2 MA的N-末端一半的存在导致酵母中颗粒出芽失败; ii）Gag被转运至质膜; iii）在不存在盐的情况下，Gag容易从膜上解离; iv）因此，颗粒出芽被膜皱褶结构阻止。总之，这些数据表明酵母可能缺乏HIV-2和SIVmac Gag VLP出芽所必需的因子。3）负责Gag细胞内转运的新宿主因子的分离：我们使用酵母CytoTrap双杂交测定（其中蛋白质-蛋白质相互作用发生在质膜下的系统）并筛选人cDNA的表达文库（目前2-3个阳性）。少

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