The yeast Saccharomyces cerevisiae has been used for many years as a model organism with which to study biological functions in higher eukaryotic cells. Such pioneering research has employed yeast genetics (e.g., gene-deletion mutant yeast) and molecular technologies (e.g., two-hybrid assay) and has uncovered fundamental cellular functions such as the cell cycle and mRNA turnover. Because of the accumulated knowledge of cell biology and systematic screening technologies, virologists have turned to the use of yeast as a model cell system to study the host factors required for the replication of higher eukaryotic viruses . For example, bromo mosaic virus, a positive-strand RNA virus, has been shown to replicate and encapsidate its genome into virus particles in yeast , and the human papillomavirus genome has been shown to replicate stably in yeast . The use of yeast genetic mutants has allowed to perform genome-wide screens to identify multiple host factors required for viral replication [4–6].
The applicability of yeast has been further expanded as cells for vaccine development, since yeast is recognized as generally safe and is utilized for the production of many pharmaceutical products. A good example is the hepatitis B surface antigen expressed in yeast, which is a safe and efficient vaccine used worldwide . Another is the human papillomavirus capsid protein expressed in yeast, which is currently available as a vaccine [8, 9]. Both viral proteins are self-assembled into virus-like particles (VLPs) in yeast expression systems, similar to mammalian and insect cell systems. Such VLPs are noninfectious but highly immunogenic because they mimic authentic viral particle structures. Consequently, VLPs represent new candidates for safe and efficacious vaccine components.
Human immunodeficiency virus type 1 (HIV-1), a member of the retrovirus family, is a causative agent for acquired immunodeficiency syndrome. The HIV-1 genomic RNA is reverse-transcribed into the cDNA and is integrated into the host cell chromosome. This cDNA form called proviral DNA is a template for transcription and replication of the HIV-1 genome. The proviral DNA has long terminal repeats (LTR) composed of unique region 3′ end (U3), repeat (R), and unique region 5′ end (U5) at both ends. These ends are important for viral transcription and replication: the U3 contains viral promoter and enhancer; the R contains a Tat-responsive region (TAR) and a poly A addition signal (pA); the U5 contains a primer binding site (PBS) . The U3-R junction is the transcription start site. The 5′ LTR is followed by stem-loop (SL) structure-enriched untranslated region. SL1 and SL3 are a dimerization initiation signal (DIS)  and an encapsidation signal Psi for the HIV-1 genome [12, 13], respectively, and both are absolutely required for HIV-1 genome packaging into viral particles. The three major genes, gag, pol, and env, encoding viral structural proteins, lie between the 5′ and 3′ LTRs. The gag gene encodes the viral capsid protein, Gag, which is essential for retroviral particle assembly. The pol and env genes encode viral specific enzymes and envelope proteins, both of which are necessary for multiple rounds of viral replication but are dispensable for viral particle production . The HIV-1 genome also contains the accessory genes, tat, rev, nef, vif, vpr, and vpu, all of which contribute to efficient viral replication. The tat gene encodes Tat protein, which binds to the TAR sequence and is obligatory for HIV-1 transcription, whereas the rev gene encodes Rev protein, which binds to a highly structured RNA region, termed the Rev-responsive element (RRE), within the env gene and exports unspliced and incompletely spliced HIV-1 RNAs to the cytoplasm . Thus, HIV-1 gene expression requires many RNA elements and the viral regulatory proteins Tat and Rev, whereas HIV-1 particle production requires only Gag.
Numerous protein expression systems, such as transfection with expression plasmids and infection with recombinant viral vectors, have shown that Gag protein expression alone in higher eukaryotic cells produces Gag VLPs, which are morphologically identical to the immature form of retroviral particles [16–19]. We previously showed that the expression of HIV-1 Gag protein in S. cerevisiae and the subsequent spheroplast formation produced Gag VLPs extracellularly . We also showed that the Gag VLPs encased in yeast cell membrane induced innate immune responses (e.g., cytokine production), suggesting that the yeast production system has practical applications such as vaccine development . Since the RNA elements required for HIV-1 genome packaging are well defined and distinct from the gag gene, it is possible that the addition of these RNA elements produces the HIV-1 VLPs containing the viral genome. In the present study, we tested this possibility and established trans-packaging of the HIV-1 genome into the Gag VLPs in a yeast cell system.