Article

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: 4.65). 06/2007; 81(12):6623-31. DOI: 10.1128/JVI.02833-06
Source: PubMed

ABSTRACT 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 "Psi-" particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace the genome in Psi- particles. We have now analyzed the mRNA content of Psi- and Psi+ 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 Psi- 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 Psi+ 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 approximately 5- to 10-fold higher in Psi- than in Psi+ 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 Psi- and Psi+ particles; a minor fraction of this RNA was weakly associated with genomic RNA in Psi+ 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.
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    • "Recombinant capsids produced in homologous or heterologous systems are known to incorporate cellular RNAs, with the level of each RNA species in the VLPs representative of the level of that RNA in the cellular pool (Lokesh et al., 2002; Rulli et al., 2007), with one of the most abundant RNAs in cells being 16S rRNA. In retrovirus particles, cellular RNAs are thought to be packaged to compensate for a deficiency in packaging viral genomic RNAs (Rulli et al., 2007). Therefore, we performed experiments to determine if cellular RNAs were encapsidated in MRFV VLPs by using primers corresponding to the E. coli 16S ribosomal RNAs. "
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    • "Through this interaction, the cellular trafficking of viral RNA and Gag polyprotein precursors, as a ribonucleoprotein complex, are likely to be intimately linked; however, the precise cellular location at which Gag-RNA association begins is poorly understood and may differ between retroviruses. It has been well demonstrated that interactions between Gag and RNA, through the NC domain, are required for assembly of retrovirus particles (Campbell and Rein, 1999; Campbell and Vogt, 1995; Muriaux et al., 2001; Rulli et al., 2007). It is not yet clear why lentiviral NCs have two zinc (Zn) fingers, since most NC functions apparently rely on the proximal Zn finger. "
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