Non-infectious virus particle budding by yeast spheroplasts

我们之前已经发现，从表达人类免疫缺陷病毒(HIV)Gag的酵母中去除细胞壁，可以产生相当于高等真核细胞产生的VLP的Gag病毒样颗粒(VLP)，这表明酵母支持病毒颗粒的萌发。利用酵母突变体提高酵母VLP的产量，并分析VLP萌发所需的宿主因素1)表面携带病毒包膜蛋白的VLP的生产由于酵母对病毒包膜蛋白的高糖化，我们利用两个缺乏高糖基化的酵母突变体mnn9和och1/mnn1/mnn4，与Gag共表达HIV env。Western blotting显示，Env在细胞内被严重降解，虽然GagVLP已经萌发，但Env未能整合到VLP中。这些数据表明，尽管没有证明，Env可能无法插入到ER2的管腔中。2)确定负责GAG运输和随后VLP萌发的宿主因素在许多病毒中，肌动蛋白被认为参与了颗粒的产生。为了证明肌动蛋白的参与，我们使用了一系列肌动蛋白突变酵母，发现肌动蛋白亚域2上的突变阻碍了GAG向质膜的运输，导致VLP的生产效率低下。最近的研究还表明，许多病毒利用内体分选机器生产颗粒。为了获得内体途径负责GAG运输的线索，我们检测了缺乏内体分选蛋白的酵母突变株的VLP产生。而溶酶体的t-陷阱Vam3的缺陷并未改变VLP的产生水平。

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.