Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae
© Tomo et al.; licensee BioMed Central Ltd. 2013
Received: 23 October 2012
Accepted: 15 March 2013
Published: 26 March 2013
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.
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.
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.
KeywordsYeast HIV Virus-like particle Genome packaging
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.
Yeast did not support transcription or translation from HIV-1 LTR
We further replaced the U3 of the 5′ LTR (corresponding to the HIV-1 promoter) by the promoter for yeast GAP (referred to as PGAP-HIV-TGAP). Northern blotting revealed that this promoter rescued gag mRNA transcription (Figure 1B). The full-length gag mRNA was accompanied by some smaller RNA species, similar to the case with the transcription of proviral HIV-1 molecular clone pNL43  observed in mammalian cells. Nonetheless, Gag protein expression was not observed when the whole cell lysates were analyzed by Western blotting using anti-HIV-1 p24 antibody, suggesting that HIV-1 expression was also blocked at post-transcriptional steps in yeast (Figure 1C). HIV-1 Rev binds to the RRE residing in HIV-1 RNA and facilitates the nuclear export of gag mRNA, unspliced mRNA containing RRE, in mammalian cells. It has been reported that Rev similarly functions dependently of RRE in yeast [26, 27]. However, the co-expression of Rev from a pRS plasmid containing the rev gene with an expression cassette (the promoter and terminator for yeast GAP) (Figure 1D) did not support Gag protein expression (Figure 1C), suggesting, although not proving, that HIV-1 gag mRNA containing R-U5 was not efficiently translated in yeast.
The R-U5-SL region to the gag start ATG codon inhibited Gag protein expression in yeast
Total cellular RNA was isolated from these yeast transformants and was subjected to semi-quantitative RT-PCR analysis for HIV-1 gag mRNA and yeast actin mRNA. When the amplification kinetics were initially monitored with 300 ng of cellular RNA, we found that the RT-PCR products for both HIV-1 gag mRNA and yeast actin mRNA increased as the number of PCR cycles increased (up to 26 cycles) (Figure 2B, upper). Using a series of dilutions of the cellular RNA, the product yields at 20 PCR cycles indicated that all 5′ truncation constructs produce largely similar levels of HIV-1 gag mRNA (Figure 2B, lower). For the direct detection of RNA, a series of dilutions of the cellular RNA was slot-blotted on the membrane, followed by hybridization with the RNA probe for the HIV-1 pol gene. The results confirmed that the 5′ truncation constructs produced largely equivalent levels of HIV-1 gag mRNA (Figure 2C). Real-time RT-PCR analysis further confirmed these findings (Figure 2D). These data indicate that the HIV-1 R-U5-SL region that generates a highly structured RNA has an inhibitory effect on Gag translation in yeast, as reported in mammalian cells [35, 36].
Trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system
In mammalian cells, HIV-1 gag mRNA is also used as viral genomic RNA and is incorporated into viral particles. The genomic RNA/gag mRNA has the encapsidation signal Psi in the SL region, which is absolutely required for viral genome packaging into HIV-1 particles [37–39]. The SL region also includes a signal, termed dimerization initiation site (DIS), which is essential for the dimerization of HIV-1 genomic RNA and overlaps with a signal required for the packaging of the genomic RNA, suggesting that the dimerization and packaging processes are possibly coupled [40, 41]. Several studies have indicated that other RNA elements (e.g., TAR and PBS) are also involved in the efficiency of HIV-1 genome packaging [38, 42, 43]. From these studies, we considered that the use of the same construct for the synthesis of HIV-1 genomic RNA and Gag protein (i.e., cis-packaging) was a less effective method for the RNA packaging into Gag VLPs in the yeast cell system.
Electron microscopy confirmed no morphological differences in these Gag VLPs (Figure 3C). These data indicate that trans-packaging of HIV-1 genomic RNA into Gag VLPs is possible in a yeast cell system and is dependent on the presence of the R-U5-SL region, similar to the case with mammalian cell systems.
Selective packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system
Transgene expression by Gag VLPs in mammalian cells
Transcription of HIV-1 genome in yeast
The 5′ LTR of HIV-1 is composed of the U3-R-U5 and is followed by the SL region. The U3 region is a promoter and contains the binding sites for transcription factors, such as nuclear factor κB, Sp1, and AP2. However, HIV-1 transcription is regulated primarily by Tat protein. Tat binds to the TAR sequence within the R region of HIV-1 mRNA transcripts and recruits the host positive transcription elongation factor b (P-TEFb) complex containing cyclin-dependent kinase 9 and cyclin T1. The binding of the complex facilitates transcription of the provirus by cellular RNA polymerase II. In the absence of Tat, only short transcripts are generated because RNA polymerase II is readily dissociated from the DNA template . In this study, we found in S. cerevisiae that Tat protein overexpression did not support transcription from the LTR but that replacement of the U3 region by the promoter for yeast GAP did support the transcription (Figure 1), likely because the yeast promoter is Tat-independent as opposed to the HIV-1 promoter. It has also been suggested that cyclin T1 is a species-specific cofactor for HIV-1 transcription, since mouse cyclin T1 does not support HIV-1 transcription due to a single amino acid change [52, 53].
Translation of HIV-1 mRNA in yeast
The HIV-1 gag mRNA contains the R-U5 and SL regions upstream from the gag start ATG codon. This 5′ untranslated region (332 bases) has been suggested to form a highly folded RNA structure [29–31]. In cell-free systems and Xenopus oocytes, the 5′ untranslated region, especially the TAR region, inhibits cap-dependent translation of the gag gene [32–34, 36]. A recent study showed that in 293 cells the SL region, especially the Psi site, was the major determinant of translation inhibition . Consistent with these studies, we found that in yeast, (i) the 5′ untranslated region had an inhibitory effect on the Gag translation without reducing in the gag mRNA levels and (ii) no translation was observed when the untranslated region included the TAR region (Figure 2).
The 5′ untranslated region is required for packaging of HIV-1 genome into viral particles. Nevertheless, the 5′ untranslated region, if tested experimentally, inhibits its downstream Gag translation, which is required for virus particle production. To explain this discrepancy, some hypotheses have been proposed. One is the alternating structure model of the 5′ HIV-1 RNA, termed long-distance interaction (LDI) and branched multiple hairpin (BMH): the equilibrium between the two was hypothesized to regulate RNA dimerization, packaging, and translation [28, 54, 55]. However, mutations to alter the LDI-BMH equilibrium did not affect translation efficiency . Another possibility is internal ribosome entry site (IRES) activity within the 5′ untranslated region of the HIV-1 RNA. An earlier study did not identify putative IRES  and later studies found IRES activity in a lentiviral family [57, 58]. Interestingly, HIV-1 IRES functions only at the G2/M transition phase of the cell cycle , although IRES-dependent Gag translation is still controversial. In S. cerevisiae, IRES-mediated translation has been observed with endogenous yeast genes as well as the IRES elements of hepatitis C virus , but neither poliovirus nor encephalomyocarditis virus IRES can function [60, 61]. In the present study, deletion of the entire 5′ untranslated region (R-U5-SL) fully restored Gag translation. Thus, our study argues against the IRES-mediated Gag translation although we cannot rule out a failure of the IRES activity in yeast. It should be noted that in HIV-1 infected cells, the gag mRNA is efficiently translated to Gag protein even though it contains the 5′ untranslated region. It is possible that a host factor(s), for examples, to unwind the 5′ RNA structures is involved in gag mRNA translation in infected cells. We suggest that complementation of host factors, especially infection-induced host factors, to this yeast cell system would identify such factors for gag mRNA translation.
Genome packaging into Gag VLPs in yeast and the benefits of yeast-derived Gag VLPs encapsidating genes
Results from numerous mutagenesis studies indicate that each RNA element within the 5′ untranslated region is responsible for packaging of HIV-1 genome. For example, mutations at the SL region (the DIS and Psi sites and the gag start codon) severely impaired HIV-1 genome packaging [38, 39] and mutations at the TAR, pA, and PBS regions similarly impaired the genome packaging [40, 42], indicating that the entire 5′ untranslated region is involved in efficient packaging of HIV-1 genome. These studies also suggest that the structures, but not the specific sequences, of the 5′ untranslated region are important for packaging of HIV-1 genome. In our study, the TAR-gag construct that contained the full length of the R-U5-SL showed no Gag protein expression. When the 5′ untranslated region was progressively truncated, the Gag protein was expressed but the levels varied in the truncations. From these results, we suppose that the cis-packaging efficiencies of these 5′ truncated RNAs are difficult to evaluate in a yeast cell system.
In contrast, our study indicated that HIV-1 genomic RNA was preferentially incorporated into Gag VLPs in yeast when Gag was supplied in trans. Generally, the technology of trans-packaging of gene is used to produce replication-deficient viral vectors, which are much safer than replication-competent viral vectors produced by cis-packaging [44, 45]. In the field of vaccinology, various types of viral vaccines have been developed. Live attenuated viruses and recombinant viral vectors are the most effective vaccines and stimulate cellular as well as humoral immune responses. They replicate and express their antigens in cells, but safety concerns cannot be excluded. In contrast, viral protein components and peptides are safe but often lack the ability to induce cellular immunity. VLPs are highly assembled structures of viral protein components, mimicking the authentic virion without including viral genome. Thus, they are not infectious but often effective at stimulating cellular immunity . In fact, our previous study showed that the Gag VLPs encased in yeast cell membrane induced maturation and cross-presentation of dendritic cells . However, because VLPs do not contain genetic materials, they do not endogenously produce intracellular antigens that stimulates cellular immunity by the major histocompatibility complex I antigen presentation pathway. Also, they usually do not contain viral envelope glycoproteins that are major immunogens to elicit neutralizing antibodies. Since yeast does not trim the glycans to produce hyper-mannosylated glycans, it may not be readily available for production of VLPs presenting glycoproteins on the VLP membrane at present . Our data suggest the possibility that noninfectious VLPs encapsidating gene of interest can be produced in a yeast cell system and may provide clues to the development of yeast VLP vaccines that confer ensured safety and enhanced immunogenicity. For this purpose, we produced Gag VLPs packaging the bicistronic reporter gene cassettes (i.e., gagfl and egfp) in a yeast cell system and tested expression of the reporter genes transduced by Gag VLPs in HeLa and Raw264.7 cells. In this bicistronic construct, GagFL is translated in a cap-dependent fashion, whereas EGFP is in a cap-independent fashion. However, we did not see expression of GagFL or EGFP (Figure 5B, upper). We finally isolated the RNA from the Gag VLPs and transfected the RNA to HeLa cells but failed to see GagFL and EGFP expression (data not shown). These results suggested that the failure of the transgene expression was not due to target cell types or the uncoating ability of Gag VLPs. Rather, it is ascribable to extensive nicking of the RNA within Gag VLPs. Retroviral/lentiviral gene expression requires reverse transcription of viral RNA and subsequent integration of the cDNA into host cell chromosomes before its transcription and translation. The input viral RNA is not directly used as mRNA and viral protein expression occurs only from the integrated proviral cDNA. The reason is not known but it may be partly that the RNA in viral particles is randomly nicked [49, 50]. However, we believe that mRNA transduction without integration would be safer and more suitable for vaccine design than stable integration of transgene into chromosomes. Further studies are needed to develop the methods to incorporate intact mRNA into Gag VLPs (e.g., by using non-cognate packaging signals, by shortening transgene RNA, or by forming stable RNA-protein complex like mature Gag capsid).
Cells of the yeast Saccharomyces cerevisiae have been used to develop VLP vaccines (e.g., hepatitis B virus and human papillomavirus). Such VLPs are considered new candidates for safe and efficacious vaccine components because they are noninfectious and highly immunogenic. Yeast cell systems have also been used as model cell systems with which to study host factors required for the replication of higher eukaryotic viruses. Bromo mosaic virus, a positive-strand RNA virus, and human papillomavirus, a DNA virus, have been shown to replicate and encapsidate their genomes into virus particles in yeast. We previously demonstrated the production of Gag VLPs in yeast spheroplasts. Our present study established the trans-packaging of the HIV-1 genome into Gag VLPs in a yeast cell system. This study also revealed that the 5′ untranslated region of the HIV-1 genome (the R-U5-SL region) inhibited its downstream translation (Gag protein expression). This yeast system may be useful for the study of HIV-1 genome packaging and translation.
Plasmid construction and yeast expression
A full-length HIV-1 cDNA molecular clone, pNL43 , was used for DNA construction. A Kpn I-Nhe I fragment of the env gene (nucleotide positions 6343–7250) of pNL43 was initially deleted (for biosafety), and the 3′ LTR was replaced by the terminator for the yeast GAP gene. The chimeric HIV-1 DNA was cloned into yeast 2 μ plasmids containing the URA3 gene as a selective marker (referred to as HIV-TGAP). The U3 of the 5′ LTR (HIV-1 promoter) was further replaced by the constitutive promoter for the yeast GAP gene (referred to as PGAP-HIV-TGAP). The entire 5′ LTR and its downstream SL region, including the gag start codon, were also deleted in the context of PGAP-HIV-TGAP (referred to as PGAP-ΔHIV-TGAP). A series of truncations of the 5′ LTR and its downstream SL regions was carried out by PCR using relevant forward and reverse primers. For the expression of Gag protein in trans, the full-length gag gene was placed under the control of the GAP promoter, followed by the GAP terminator, and the expression cassette was cloned into yeast 2 μ plasmid pRS423 containing the HIS3 gene as a selective marker. For the expression of Tat and Rev proteins, the exons of the tat and rev genes were joined by overlapping PCR. The PCR fragments were similarly placed between the GAP promoter and the GAP terminator and were cloned into yeast 2 μ plasmid pRS423/424 containing the HIS3/TRP1 gene as a selective marker. S. cerevisiae strain RAY3A-D (MATa/ α ura3/ura3 his3/his3 leu2/leu2 trp1/trp1)  was transformed by the recombinant plasmids. The transformants were inoculated in the appropriate synthetic medim and grown at 30°C.
For protein expression in mammalian cells, the bicistronic IRES construct (the gag gene fused with a FLAG epitope tag sequence, the IRES sequence derived from encephalomyocarditis virus [Clontech], and the gene for egfp) was initially generated in the pNL43 derivative with deletions of the pol gene (nucleotide positions 2290–4553) and the Kpn I-Nhe I fragment of the env gene (nucleotide positions 6343–7250). The 3′ LTR and the U3 of the 5′ LTR were replaced by the terminator and the promoter for the yeast GAP gene, respectively, and the resultant construct was cloned into yeast 2 μ plasmid pKT10  (referred to as PGAP-GagFL-IRES-EGFP-TGAP). The pNL43 derivative expressing the Gag protein fused with a FLAG epitope tag and EGFP was similarly generated from the pNL43 derivative with deletions of the pol and env genes.
Preparation of yeast spheroplasts and production of Gag VLPs
The procedure for yeast spheroplast formation was described previously . Yeast transformants were grown at 30°C in synthetic defined medium (0.67% yeast nitrogen base, 2% glucose, and amino acid mixtures) without uracil, histidine, and/or tryptophane. Yeast cells were suspended in wash buffer (50 mM Tris [pH 7.5], 5 mM MgCl2, and 1 M sorbitol) containing 30 mM DTT and incubated at 30°C for 20 min with gentle shaking. The cells were resuspended in wash buffer containing 3 mM DTT and 0.4 mg/ml Zymolyase and were incubated at 30°C for 20 min for cell wall digestion. After being washed twice with 1 M sorbitol, spheroplasts were cultured in YPD (1% yeast extract, 2% peptone, and 2% glucose) medium containing 1 M sorbitol at 30°C overnight with gentle shaking (at 60 rpm).
Yeast-produced Gag VLPs were purified as described previously . Briefly, the culture medim of yeast spheroplasts was clarified and centrifuged through 30% (w/v) sucrose cushions in an SW28 rotor (Beckman Coulter) at 120,000 × g for 1.5 hr at 4°C. The VLP pellets were resuspended in phosphate-buffered saline and were centrifuged on 20-70% (w/v) sucrose gradients in an SW55 rotor (Beckman Coulter) at 120,000 × g overnight at 4°C.
Expression in mammalian cells
HeLa and Raw264.7 cells were grown in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum. Gag VLPs (equivalent to 1 μg RNA) were added to HeLa and Raw264.7 cells. Transfection with RNA isolated from Gag VLPs or plasmid DNA was carried out using Lipofectamine 2000 (Invitrogen).
Yeast cells (0.5 OD) were separated by SDS-PAGE. Western blotting was carried out using anti-HIV-1 p24CA, anti-HIV-1 Tat, and anti-HIV-1 Rev mouse monoclonal antibodies (Advanced Biotechnologies).
Semi-quantitative and quantitative RT-PCRs
Total cellular RNA and VLP RNA were isolated with the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Contaminant DNA was digested with DNase I during the isolation. Semi-quantitative RT-PCR was performed with the ReverTra Dash kit (Toyobo) according to the manufacturer’s instructions. For cDNA synthesis, RNA was mixed with random primers and the reaction was carried out at 42°C for 20 min. For amplification of the cDNA, aliquots of the RT reaction samples were mixed with 100 nM of each primer, and a three-step reaction (98°C for 10 sec, 60°C for 2 sec, and 74°C for 30 sec) was cycled. The following primer sets were used: 5′-ATGGGTGCGAGAGCGTCGGTATTAAGC-3′ and 5′-CAATAGGCCCTGCATGCACTGGATG-3′ for HIV-1 gag and 5′-GCCCCAGAAGAACACCCTGTTCTTT-3′ and 5′-TTAGAAACACTTGTGGTGAACGATA-3′ for yeast actin mRNAs.
Real-time RT-PCR was performed with PrimeScript RT reagent kit (Takara) and subsequently with SYBR Green Realtime PCR Master Mix (Toyobo). For cDNA synthesis, 1 μg of RNA was mixed with a mixture of oligo dT and random primers (supplied by the RT kit) and the reaction was carried out at 37°C for 15 min according to the manufacturer’s instruction. For amplification of the cDNA, 1/100th of aliquots of the reaction samples were mixed with 100 nM of each primer and two-step reaction (95°C for 5 sec and 60°C for 30 sec) was cycled. The following primer sets were used and produced single amplification products (confirmed by melting curve analysis): 5′-GCTTGCTGAAGCGCGCACGG-3′ and 5′-GACGCTCTCGCACCCATCTC-3′ for unspliced HIV-1 (nucleotide positions 701–806)  and 5′-ATAATCCACCTATCCCAGTAGGAGAAAT-3′ and 5′-TTTGGTCCTTGTCTTATGTCCAGAATGC-3′ for HIV-1 gag (nucleotide positions 1544–1658)  mRNAs. Relative quantification of HIV-1 RNA was performed in reference to a standard curve prepared by amplification of 10-fold serial dilutions (50–0.05 pg) of pNL43 .
Northern blotting and slot blotting
Minus-strand RNA probes were synthesized with Maxi script T7 kit (Ambion) according to the manufacturer′s instructions. The fragments of HIV-1 pol and yeast actin genes (nucleotide positions 3826–4160 and 579–1436, respectively) were cloned into pGEM3 vector (Promega) and were in vitro-transcribed with biotinylated UTP (Roche) at 37°C for 60 min. After digestion of the DNA templates with DNase I, RNA transcripts were purified using a Quick Spin Column (Roche).
For Northern blotting, RNA samples were denatured, electrophoresed in 0.8% agarose gels, and blotted onto Hybond N+ membrane (Amersham). Hybridization and detection were carried out with the Ultrahyb kit (Ambion) and the Biotin Luminescent Detection kit (Roche) according to the manufacturer’s instructions, respectively. Briefly, hybridization with RNA probes was performed at 68°C overnight and washing was performed first with 2×SSC buffer containing 0.1% SDS and then with 0.1×SSC buffer at 68°C. For slot-blot analysis, a series of dilutions of RNA samples was blotted onto Hybond N+ membrane by vacuuming and hybridization and detection were similarly carried out.
Yeast cells were fixed in 3.7% formalin in YPD at 30°C for 30 min. Following removal of the cell wall, spheroplasts were treated with 70% ethanol at 4°C for 5 min for membrane permeabilization. After blocking with 0.1% BSA, cells were incubated with anti-HIV-1 Tat or Rev mouse monoclonal antibodies and subsequently with anti-mouse IgG-Alexa Fluor 488 (Molecular Probes). Nuclei were stained with 4′, 6-diamidino-2-phenylindole dihydrocloride (DAPI).
HeLa nad Raw264.7 cells were fixed in 3.7% formalin in phosphate-buffered saline for 30 min and treated with 0.1% Triton-X 100 for 10 min for membrane permeabilization. After blocking with 0.1% BSA, cells were incubated with anti-FLAG mouse monoclonal antibody (Sigma) and subsequently with anti-mouse IgG-Alexa Fluor 568 (Molecular Probes). Nuclei were stained with DAPI. Cells were observed with a laser-scanning confocal microscope (TCS-SP5, Leica).
Purified Gag VLP pellets were fixed in 2% glutaraldehyde in 50 mM cacodylate buffer (pH 7.2) for 2 hr and postfixed with 1% osmium tetroxide for 1 hr. The pellets were embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with an electron microscope.
Dimerization initiation signal
Human immunodeficiency virus type 1
Long terminal repeats
Poly A addition signal
We thank S. Saegusa (Kitasato University, Japan) for RNA analysis. This work was supported by an AIDS grant from the Ministry of Health, Labor, and Welfare of Japan and a grant from the Ministry of Sciences, Sports, and Education of Japan.
- Galao RP, Scheller N, Alves-Rodrigues I, Breinig T, Meyerhans A, Diez J: Saccharomyces cerevisiae: a versatile eukaryotic system in virology. Microb Cell Fact. 2007, 6: 32- 10.1186/1475-2859-6-32.View ArticleGoogle Scholar
- Janda M, Ahlquist P: RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S. cerevisiae. Cell. 1993, 72: 961-970. 10.1016/0092-8674(93)90584-D.View ArticleGoogle Scholar
- Angeletti PC, Kim K, Fernandes FJ, Lambert PF: Stable replication of papillomavirus genomes in Saccharomyces cerevisiae. J Virol. 2002, 76: 3350-3358. 10.1128/JVI.76.7.3350-3358.2002.View ArticleGoogle Scholar
- Kushner DB, Lindenbach BD, Grdzelishvili VZ, Noueiry AO, Paul SM, Ahlquist P: Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc Natl Acad Sci USA. 2003, 100: 15764-15769. 10.1073/pnas.2536857100.View ArticleGoogle Scholar
- Panavas T, Serviene E, Brasher J, Nagy PD: Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses. Proc Natl Acad Sci USA. 2005, 102: 7326-7331. 10.1073/pnas.0502604102.View ArticleGoogle Scholar
- Jiang Y, Serviene E, Gal J, Panavas T, Nagy PD: Identification of essential host factors affecting tombusvirus RNA replication based on the yeast Tet promoters Hughes Collection. J Virol. 2006, 80: 7394-7404. 10.1128/JVI.02686-05.View ArticleGoogle Scholar
- Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD: Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature. 1982, 298: 347-350. 10.1038/298347a0.View ArticleGoogle Scholar
- Sasagawa T, Pushko P, Steers G, Gschmeissner SE, Hajibagheri MA, Finch J, Crawford L, Tommasino M: Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe. Virology. 1995, 206: 126-135. 10.1016/S0042-6822(95)80027-1.View ArticleGoogle Scholar
- Hofmann KJ, Neeper MP, Markus HZ, Brown DR, Muller M, Jansen KU: Sequence conservation within the major capsid protein of human papillomavirus (HPV) type 18 and formation of HPV-18 virus-like particles in Saccharomyces cerevisiae. J Gen Virol. 1996, 77: 465-468. 10.1099/0022-1317-77-3-465.View ArticleGoogle Scholar
- Vogt VM: Retrovial virions and genomes. In Retroviruses. Edited by: Coffin JM, Hughes SH, Varmus HE. 1997, New York: Cold Spring Harbor PressGoogle Scholar
- Mujeeb A, Clever JL, Billeci TM, James TL, Parslow TG: Structure of the dimer initiation complex of HIV-1 genomic RNA. Nature Struct Biol. 1998, 5: 432-436. 10.1038/nsb0698-432.View ArticleGoogle Scholar
- McBride MS, Panganiban AT: The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol. 1996, 70: 2963-2973.Google Scholar
- De Guzman RN, Wu ZR, Stalling CC, Pappalardo L, Borer PN, Summers MF: Structure of the HIV-1 nucleocapsid protein bound to the SL3-RNA recognition element. Science. 1998, 279: 384-388. 10.1126/science.279.5349.384.View ArticleGoogle Scholar
- Swanstrom R, Wills JW: Synthesis, assembly, and processing of viral proteins. In Retroviruses. Edited by: Coffin JM, Hughes SH, Varmus HE. 1997, New York: Cold Spring Harbor PressGoogle Scholar
- Emerman M, Malim MH: HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science. 1998, 280: 1880-1884. 10.1126/science.280.5371.1880.View ArticleGoogle Scholar
- Gheysen D, Jacobs E, de Foresta F, Thiriart C, Francotte M, Thines D, de Wilde M: Assembly and release of HIV-1 precursor Pr55 gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 1989, 59: 103-112.View ArticleGoogle Scholar
- Smith AJ, Srinivasakumar N, Hammarskjold ML, Rekosh D: Requirements for incorporation of Pr160 gag-pol from human immunodeficiency virus type 1 into virus-like particles. J Virol. 1993, 67: 2266-2275.Google Scholar
- Deml L, Speth C, Dierich MP, Wolf H, Wagner R: Recombinant HIV-1 Pr55gag virus-like particles: potent stimulators of innate and acquired immune responses. Mol Immunol. 2005, 42: 259-277. 10.1016/j.molimm.2004.06.028.View ArticleGoogle Scholar
- Hammonds J, Chen X, Zhang X, Lee F, Spearman P: Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines. Vaccine. 2007, 2547: 8036-8048.View ArticleGoogle Scholar
- Sakuragi S, Goto T, Sano K, Morikawa Y: HIV type 1 Gag virus-like particle budding from spheroplasts of Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2002, 99: 7956-7961. 10.1073/pnas.082281199.View ArticleGoogle Scholar
- Tsunetsugu-Yokota Y, Morikawa Y, Isogai M, Kawana-Tachikawa A, Odawara T, Nakamura T, Grassi F, Autran B, Iwamoto A: Yeast-derived human immunodeficiency virus type 1 p55gag virus-like particles activate dendritic cells (DCs) and induce perforin expression in Gag-specific CD8+ T cells by cross-presentation of DCs. J Virol. 2003, 77: 10250-10259. 10.1128/JVI.77.19.10250-10259.2003.View ArticleGoogle Scholar
- Tanaka K, Nakafuku M, Tamanoi F, Kaziro Y, Matsumoto K, Toh-e A: IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol Cell Biol. 1990, 10: 4303-4313.View ArticleGoogle Scholar
- Daviet L, Bois F, Battisti PL, Gatignol A: Identification of limiting steps for efficient trans-activation of HIV-1 promoter by Tat in Saccharomyces cerevisiae. J Biol Chem. 1998, 273: 28219-28228. 10.1074/jbc.273.43.28219.View ArticleGoogle Scholar
- Subramanian T, D’Sa-Eipper C, Elangovan B, Chinnadurai G: The activation region of the Tat protein of human immunodeficiency virus type-1 functions in yeast. Nucleic Acids Res. 1994, 22: 1496-1499. 10.1093/nar/22.8.1496.View ArticleGoogle Scholar
- Adachi A, Koenig S, Gendelman HE, Daugherty D, Gattoni-Celli S, Fauci AS, Martin MA: Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus. J Virol. 1987, 61: 209-213.Google Scholar
- Stutz F, Rosbash M: A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO J. 1994, 13: 4096-4104.Google Scholar
- Stutz F, Neville M, Rosbash M: Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 1995, 82: 498-506.View ArticleGoogle Scholar
- Huthoff H, Berkhout B: Two alternating structures of the HIV-1 leader RNA. RNA. 2001, 7: 143-157. 10.1017/S1355838201001881.View ArticleGoogle Scholar
- Clever JL, Miranda DJ, Parslow TG:RNA structure and packaging signals in the 5′leader region of the human immunodeficiency virus type 1 genome.J Virol. 2002, 76: 12381-12387.View ArticleGoogle Scholar
- Lu K, Heng X, Summers MF: Structural determinants and mechanism of HIV-1 genome packaging. J Mol Biol. 2011, 410: 609-633. 10.1016/j.jmb.2011.04.029.View ArticleGoogle Scholar
- Lu K, Heng X, Garyu L, Monti S, Garcia EL, Kharytonchyk S, Dorjsuren B, Kulandaivel G, Jones S, Hiremath A, Divakaruni SS, LaCotti C, Barton S, Tummillo D, Hosic A, Edme K, Albrecht S, Telesnitsky A, Summers MF: NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging. Science. 2011, 334: 242-245. 10.1126/science.1210460.View ArticleGoogle Scholar
- Parkin NT, Cohen EA, Darveau A, Rosen C, Haseltine W, Sonenberg N:Mutational analysis of the 5′non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation.EMBO J. 1988, 7: 2831-2837.Google Scholar
- Edery I, Petryshyn R, Sonenberg N: Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism. Cell. 1989, 56: 303-312. 10.1016/0092-8674(89)90904-5.View ArticleGoogle Scholar
- Geballe AP, Gray MK: Variable inhibition of cell-free translation by HIV-1 transcript leader sequences. Nucleic Acids Res. 1992, 20: 4291-4297. 10.1093/nar/20.16.4291.View ArticleGoogle Scholar
- Ka WH, Jeong YY, You JC:Identification of the HIV-1 packaging RNA sequence (ψ) as a major determinant for the translation inhibition conferred by the HIV-1 5′UTR.Biochem Biophys Res Commun. 2012, 417: 501-507.View ArticleGoogle Scholar
- Miele G, Mouland A, Harrison GP, Cohen E, Lever AM:The human immunodeficiency virus type 1 5′packaging signal structure affects translation but does not function as an internal ribosome entry site structure.J Virol. 1996, 70: 944-951.Google Scholar
- Luban J, Goff SP: Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994, 68: 3784-3793.Google Scholar
- Clever JL, Parslow TG: Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation. J Virol. 1997, 71: 3407-3414.Google Scholar
- McBride MS, Panganiban AT: Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo. J Virol. 1997, 71: 2050-2058.Google Scholar
- Sakuragi JI, Ueda S, Iwamoto A, Shioda T: Possible role of dimerization in human immunodeficiency virus Type-1 genome RNA packaging. J Virol. 2003, 77: 4060-4069. 10.1128/JVI.77.7.4060-4069.2003.View ArticleGoogle Scholar
- Russell RS, Liang C, Wainberg MA: Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?. Retrovirology. 2004, 1: 23- 10.1186/1742-4690-1-23.View ArticleGoogle Scholar
- McBride MS, Schwartz MD, Panganiban AT: Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J Virol. 1997, 71: 4544-4554.Google Scholar
- Das AT, Klaver B, Berkhout B:The 5′and 3′TAR elements of human immunodeficiency virus exert effects at several points in the virus life cycle.J Virol. 1998, 72: 9217-9223.Google Scholar
- Danos O: Construction of retro viral packaging cell lines. Methods Mol Biol. 1992, 8: 17-27.Google Scholar
- Lever AM: HIV RNA packaging and lentivirus-based vectors. Adv Pharmacol. 2000, 48: 1-28.View ArticleGoogle Scholar
- Baum C, Schambach A, Bohne J, Galla M: Retrovirus vectors: toward the plentivirus?. Mol Ther. 2006, 13: 1050-1063. 10.1016/j.ymthe.2006.03.007.View ArticleGoogle Scholar
- Dalba C, Bellier B, Kasahara N, Klatzmann D: Replication-competent vectors and empty virus-like particles: new retroviral vector designs for cancer gene therapy or vaccines. Mol Ther. 2007, 15: 457-466. 10.1038/sj.mt.6300054.View ArticleGoogle Scholar
- Morikawa Y, Goto T, Yasuoka D, Momose F, Matano T: Defect of Human Immunodeficiency Virus Type 2 Gag Assembly in Saccharomyces cerevisiae. J Virol. 2007, 81: 9911-9921. 10.1128/JVI.00027-07.View ArticleGoogle Scholar
- Lear AL, Haddrick M, Heaphy S: A study of the dimerization of Rous sarcoma virus RNA in vitro and in vivo. Virology. 1995, 212: 47-57. 10.1006/viro.1995.1452.View ArticleGoogle Scholar
- Sakuragi JI, Panganiban AT: Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer. J Virol. 1997, 71: 3250-3254.Google Scholar
- Rulli SJ, Hibbert CS, Mirro J, Pederson T, Biswal S, Rein A: Selective and nonselective packaging of cellular RNAs in retrovirus particles. J Virol. 2007, 81: 6623-6631. 10.1128/JVI.02833-06.View ArticleGoogle Scholar
- Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR: Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. EMBO J. 1998, 17: 7056-7065. 10.1093/emboj/17.23.7056.View ArticleGoogle Scholar
- Garber ME, Wei P, KewalRamani VN, Mayall TP, Herrmann CH, Rice AP, Littman DR, Jones KA: The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev. 1998, 12: 3512-3527. 10.1101/gad.12.22.3512.View ArticleGoogle Scholar
- Abbink TEM, Berkhout B: A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon. J Biol Chem. 2003, 278: 11601-11611. 10.1074/jbc.M210291200.View ArticleGoogle Scholar
- Ooms M, Huthoff H, Russell R, Liang C, Berkhout B: A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions. J Virol. 2004, 78: 10814-10819. 10.1128/JVI.78.19.10814-10819.2004.View ArticleGoogle Scholar
- Abbink TE, Ooms M, Haasnoot PC, Berkhout B: The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation. Biochemistry. 2005, 44: 9058-9066. 10.1021/bi0502588.View ArticleGoogle Scholar
- Brasey A, Lopez-Lastra M, Ohlmann T, Beerens N, Berkhout B, Darlix JL, Sonenberg N: The leader of human immunodeficiency virus type 1 genomic RNA harbors an internal ribosome entry segment that is active during the G2/M phase of the cell cycle. J Virol. 2003, 77: 3939-3949. 10.1128/JVI.77.7.3939-3949.2003.View ArticleGoogle Scholar
- Camerini V, Decimo D, Balvay L, Pistello M, Bendinelli M, Darlix JL, Ohlmann T: A dormant internal ribosome entry site controls translation of feline immunodeficiency virus. J Virol. 2008, 82: 3574-3583. 10.1128/JVI.02038-07.View ArticleGoogle Scholar
- Iizuka N, Najita L, Franzusoff A, Sarnow P: Cap-dependent and cap-independent translation by internal initiation of messenger-RNAs in cell-extracts prepared from Saccharomyces cerevisiae. Mol Cell Biol. 1994, 14: 7322-7330.View ArticleGoogle Scholar
- Coward P, Dasgupta A:Yeast-cells are incapable of translating RNAs containing the poliovirus 5′untranslated region: evidence for a translational inhibitor.J Virol. 1992, 66: 286-295.Google Scholar
- Evstafieva AG, Beletsky AV, Borovjagin AV, Bogdanov AA: Internal ribosome entry site of encephalomyocarditis virus RNA is unable to direct translation in Saccharomyces cerevisiae. FEBS Lett. 1993, 335: 273-276. 10.1016/0014-5793(93)80745-G.View ArticleGoogle Scholar
- Chackerian B: Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines. 2007, 6: 381-390. 10.1586/147605126.96.36.1991.View ArticleGoogle Scholar
- Hamilton SR, Gerngross TU: Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol. 2007, 18: 387-392. 10.1016/j.copbio.2007.09.001.View ArticleGoogle Scholar
- Ruggieri R, Tanaka K, Nakafuku M, Kaziro Y, Toh-e A, Matsumoto K: MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1989, 86: 8778-8782. 10.1073/pnas.86.22.8778.View ArticleGoogle Scholar
- Suyama M, Daikoku E, Goto T, Sano K, Morikawa Y: Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment. Rretovirology. 2009, 6: 63-10.1186/1742-4690-6-63. 10.1186/1742-4690-6-63.View ArticleGoogle Scholar
- Brussel A, Sonigo P:Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species.J Virol. 2004, 78: 11263-11271. 10.1128/JVI.78.20.11263-11271.2004View ArticleGoogle Scholar
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