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

我们之前的研究表明，酿酒酵母支持人类免疫缺陷病毒1型（HIV-1） Gag颗粒的组装，表明酵母具有HIV-1 Gag颗粒所需的所有宿主因子。为了确定负责HIV-1 Gag运输和随后的颗粒出芽的宿主因子/机制，1)利用最近研究提出的一系列酵母内体基因突变体，我们研究了Gag运输途径；2)明确了HIV-2 Gag对酵母颗粒组装的负面影响；3)利用CytoTrap双杂交试验，试图确定一个负责Gag细胞内运输的新宿主因子。1)Gag运输的内体途径：利用一系列酵母内体基因突变体，我们发现早期和晚期内体t-SNAREs缺陷显示颗粒产生严重减少，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替代性的。为了确定t-SNAREs在Ga…More g运输中的作用，我们在人类细胞中减少或过表达它们的同源物。结果表明t-SNAREs是HIV-1 Gag转运的阳性因子。2) HIV-2 Gag对酵母颗粒组装的负面影响：我们还扩展了我们之前使用多种灵长类慢病毒Gag的研究，结果表明酵母不支持HIV-2或猴免疫缺陷病毒（SIV） mac Gag颗粒的生产。研究表明，i) HIV-2 MA的n端一半的存在导致酵母颗粒出芽失败；ii) Gag被运送到质膜；iii)在没有盐的情况下，Gag很容易与膜分离；Iv)结果，膜褶结构阻止了颗粒出芽。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)分离一种负责Gag细胞内转运的新型宿主因子：我们使用酵母CytoTrap双杂交试验（一种蛋白-蛋白相互作用发生在质膜下的系统），筛选了一个人类cdna表达文库（目前有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