Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae

Microbial Cell Factories

Research

Yuko Morikawa¹  Naoki Tomo²  Toshiyuki Goto³ 

[1] Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato University, Shirokane, 5-9-1, Minato-ku, 108-8641, Tokyo, Japan;Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato University, Shirokane, 5-9-1, Minato-ku, 108-8641, Tokyo, Japan;Life Technologies Japan Ltd, Shibaura 4-2-8, Mita Twin Bldg, 108-0023, Minato-ku, Tokyo, Japan;School of Health Science, Faculty of Medicine, Kyoto University, ShogoinKawahara-cho 53, Sakyo-ku, 606-8507, Kyoto, Japan;

关键词: Yeast;    HIV;    Virus-like particle;    Genome packaging;   

DOI  :  10.1186/1475-2859-12-28

 received in 2012-10-23, accepted in 2013-03-15,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundYeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system.ResultsThe nearly full-length HIV-1 genome containing the entire 5′ long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells.ConclusionsOur data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.

【 授权许可】

Unknown   
© Tomo et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.