title: Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Yeast 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo, 108-8641, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Tomo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Goto
authors.forename: Yuko
authors.initials: Y
authors.lastname: Morikawa
mesh_terms: D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D016325:HIV Long Terminal Repeat; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014158:Transcription, Genetic; D058425:Vaccines, Virus-Like Particle / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D015683:Gene Products, gag; D011994:Recombinant Proteins; D020718:Ribonucleoproteins, Small Nucleolar; D058425:Vaccines, Virus-Like Particle; C117377:ribonucleoprotein, U3 small nucleolar; D054322:tat Gene Products, Human Immunodeficiency Virus; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase