Non-infectious virus particle budding by yeast spheroplasts

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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.