Selective and Nonselective Packaging of Cellular RNAs in Retrovirus Particles

HIV Drug Resistance Program, National Cancer Institute-Frederick, P.O. Box B, Frederick, MD 21702-1201, USA.
Journal of Virology (Impact Factor: 4.44). 06/2007; 81(12):6623-31. DOI: 10.1128/JVI.02833-06
Source: PubMed


Assembly of retrovirus particles normally entails the selective encapsidation of viral genomic RNA. However, in the absence
of packageable viral RNA, assembly is still efficient, and the released virus-like particles (termed “Ψ−” particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace the genome in Ψ− particles. We have now analyzed the mRNA content of Ψ− and Ψ+ murine leukemia virus (MLV) particles using both microarray analysis and real-time reverse transcription-PCR. The majority
of mRNA species present in the virus-producing cells were also detected in Ψ− particles. Remarkably, nearly all of them were packaged nonselectively; that is, their representation in the particles was
simply proportional to their representation in the cells. However, a small number of low-abundance mRNAs were greatly enriched
in the particles. In fact, one mRNA species was enriched to the same degree as Ψ+ genomic RNA. Similar results were obtained with particles formed from the human immunodeficiency virus type 1 (HIV-1) Gag
protein, and the same mRNAs were enriched in MLV and HIV-1 particles. The levels of individual cellular mRNAs were ∼5- to
10-fold higher in Ψ− than in Ψ+ MLV particles, in agreement with the idea that they are replacing viral RNA in the former. In contrast, signal recognition
particle RNA was present at the same level in Ψ− and Ψ+ particles; a minor fraction of this RNA was weakly associated with genomic RNA in Ψ+ MLV particles.

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    • "For some retroviruses , such as murine leukemia virus (MLV) and Rous sarcoma virus, the minimal ψ packaging element is well defined (Lu et al. 2011b, and references therein), but in HIV-1, in addition to SL1–SL4, other elements in the 5 ′ UTR contribute either directly or indirectly to packaging (for review, see Lu et al. 2011b) (Skripkin et al. 1994; Russell et al. 2003; Parkash et al. 2012). Some specific cellular RNAs that lack ψ are also packaged into virions (Houzet et al. 2007; Kleiman et al. 2010; Keene and Telesnitsky 2012), and in the absence of ψ-containing RNAs, selectively packaged cellular RNA levels increase while additional cellular RNAs are also abundantly and nonselectively incorporated into virions (Muriaux et al. 2001; Rulli et al. 2007). Aside from the ψ stem–loops, the 5 ′ UTR of the HIV-1 genome also contains the transactivation response (TAR) stem– loop critical for viral RNA transcription, the polyadenylation site-containing PolyA stem–loop (PolyA), and the primer binding site (PBS), which harbors the sequence that anneals to primer tRNA Lys3 for reverse transcription initiation (Fig. 1A). "
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    ABSTRACT: Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.
    Full-text · Article · Jun 2013 · RNA
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    • "The RNA content of retroviral particles can be quite diverse. It includes specifically recruited RNAs bearing the packaging signal (psi) and a wide variety of captured, small cytoplasmic RNAs [7-10]. Any mRNA can be packaged into retroviral particles provided that a canonical psi sequence is cloned into the transcript [11-13]. "
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    ABSTRACT: Background Deliberate cellular reprogramming is becoming a realistic objective in the clinic. While the origin of the target cells is critical, delivery of bioactive molecules to trigger a shift in cell-fate remains the major hurdle. To date, several strategies based either on non-integrative vectors, protein transfer or mRNA delivery have been investigated. In a recent study, a unique modification in the retroviral genome was shown to enable RNA transfer and its expression. Results Here, we used the retroviral mRNA delivery approach to study the impact of modifying gene-flanking sequences on RNA transfer. We designed modified mRNAs for retroviral packaging and used the quantitative luciferase assay to compare mRNA expression following viral transduction of cells. Cloning the untranslated regions of the vimentin or non-muscular myosin heavy chain within transcripts improved expression and stability of the reporter gene while slightly modifying reporter-RNA retroviral delivery. We also observed that while the modified retroviral platform was the most effective for retroviral mRNA packaging, the highest expression in target cells was achieved by the addition of a non-viral UTR to mRNAs containing the packaging signal. Conclusions Through molecular engineering we have assayed a series of constructs to improve retroviral mRNA transfer. We showed that an authentic RNA retroviral genomic platform was most efficiently transferred but that adding UTR sequences from highly expressed genes could improve expression upon transfection while having only a slight effect on expression from transferred RNA. Together, these data should contribute to the optimisation of retroviral mRNA-delivery systems that test combinations of UTRs and packaging platforms.
    Full-text · Article · Apr 2013 · BMC Biotechnology
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    • "In contrast, when the RNA dilutions were slot-blotted and probed with the minus-strand RNA probe for yeast actin mRNA, no preferential incorporation into Gag VLPs was observed (Figure 4C). This is consistent with a previous study, in which the majority of cellular RNAs were nonselectively incorporated into retrovirus particles [51]. Together, these data indicate selective packaging of HIV-1 genomic RNA into Gag VLP in a yeast cell system. "
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    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.
    Full-text · Article · Mar 2013 · Microbial Cell Factories
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