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Non-infectious virus particle budding by yeast spheroplasts

酵母原生质球出芽的非感染性病毒颗粒

基本信息

批准号：
13670307
负责人：
MORIKAWA Yuko
金额：
$ 2.3万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670307/
关键词：
Yeast Virus Particle Spheroplast H IV Host Factor ウイルス粒子形成細胞内輸送

项目摘要

We have previously found that removal of the cell wall from yeast expressing human immunodeficiency virus (HIV) Gag allows production of Gag virus-like particle (VLP) equivalent to VLP produced by higher eukaryotic cells, indicating yeast supports viral particle budding. By using yeast mutants we attempted to improve VLP produced by yeast and analyzed host factors required for VLP budding.1) Production of VLP carrying viral Env protein on its surfaceAs yeast confers hyperglycosylation of viral envelope protein, we used two yeast mutants deficient in hyperglycosylation, mnn9 and och1/mnn1/mnn4, and co-expressed HIV Env with Gag. Western blotting revealed that Env was severely degradated in the cells and that although GagVLP was budded, Env failed to be incorporated into the VLP. These data suggest, although do not prove, Env might fail to be inserted into the lumen of ER.2) Identification of host factors responsible for Gag transport and subsequent VLP buddingIn many viruses, actin has been suggested to be involved in particle production. To prove actin involvement, we used a series of actin mutant yeast and found that mutations on the subdomain 2 of actin impair Gag transport to the plasma membrane, leading to inefficient VLP production.Recent studies also suggest that many viruses make use of endosomal sorting machinery for particle production. To get a clue which endosomal pathways are responsible for Gag transport, VLP production was examined in yeast mutants deficient in endosomal sorting protein. The defect of Pep12, the t-SNARE of late endosome, showed reduction in VLP production but, in contrast, that of Vam3, the t-SNARE of lysosome, did not alter the level of VLP production.

我们先前已经发现，从表达人类免疫缺陷病毒（HIV）Gag的酵母中去除细胞壁允许产生与高等真核细胞产生的VLP等同的Gag病毒样颗粒（VLP），表明酵母支持病毒颗粒出芽。利用酵母突变体，我们试图改进酵母产生的VLP，并分析VLP出芽所需的宿主因素。1）在其表面上携带病毒Env蛋白的VLP的产生由于酵母赋予病毒包膜蛋白高糖基化，我们使用两种高糖基化缺陷的酵母突变体mnn 9和och 1/mnn 1/mnn 4，并与Gag共表达HIV Env。Western blotting显示Env在细胞中严重降解，并且尽管GagVLP出芽，但Env未能掺入VLP中。这些数据表明，虽然没有证明，Env可能无法插入到ER的内腔。2）鉴定负责Gag运输和随后的VLP出芽的宿主因子在许多病毒中，肌动蛋白被认为参与颗粒的产生。为了证明肌动蛋白参与，我们使用了一系列肌动蛋白突变酵母，发现肌动蛋白亚结构域2上的突变损害了Gag向质膜的转运，导致VLP生产效率低下。最近的研究还表明，许多病毒利用内体分选机制生产颗粒。为了得到内体途径负责Gag转运的线索，在内体分选蛋白缺陷的酵母突变体中检查VLP产生。Pep 12的缺陷，晚期内体的t-SNARE，显示VLP生产的减少，但相反，Vam 3的缺陷，溶酶体的t-SNARE，并没有改变VLP生产的水平。