SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

Abstract

Earlier genetic and structural prediction analyses revealed that the packaging determinants of Mason Pfizer monkey virus (MPMV) include two discontinuous core regions at the 5 ′ end of its genomic RNA. RNA secondary structure predictions suggested that these packaging determinants fold into several stem-loops (SLs). To experimentally validate this structural model, we employed selective 2 ′ hydroxyl acylation analyzed by primer extension (SHAPE), which examines the flexibility of the RNA backbone at each nucleotide position. Our SHAPE data validated several predicted structural motifs, including U5/Gag longrange interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive G-C-rich palindromic (pal) SL. Minimum free-energy structure predictions, phylogenetic, and in silico modeling analyses of different MPMV strains revealed that the U5 and gag sequences involved in the LRIs differ minimally within strains and maintain a very high degree of complementarity. Since the pal SL forms a helix loop containing a canonical "GC" dyad, it may act as a RNA dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Analyses of wild-type and pal mutant RNAs revealed that disruption of pal sequence strongly affected RNA dimerization. However, when in vitro transcribed transcomplementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5 ′ -CGGCCG-3 ′ ) functions as DIS.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

“…The specificity for gRNA during packaging, thus, has been ascribed to high-affinity interactions between the NC domain of Gag and the packaging signal [5–13]. We have made a similar observation in the case of MPMV, where genetic and structure-prediction analyses have suggested that either ssPurines (Region A) or its partially repeated but base-paired sequence (Region B) plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33]. …”

“…Several riboswitch models have been proposed for HIV-1 [50–53], and in the case of FIV, the existence of dual structures has been validated by SHAPE and RNA dimerization assays [54]. Flexibility in retroviral gRNA is created via long-range interactions that have now been observed in several retroviruses, from HIV-1, FIV, MLV, to MPMV and MMTV [6,25,26,33,55–59]. The DIS in one conformation is occluded by base pairing and not available for dimerization, thus earmarking this RNA for translation, while the DIS in the other conformation is available as an apical loop on a stem loop, thus allowing dimerization and packaging of the dimeric RNA [54].…”

“…hSHAPE experiments [64–66] were performed on in vitro transcribed RNAs corresponding to nucleotides 1 to 713 using benzoyl cyanide (BzCN) and following the protocol described previously [25,33,57]. Briefly, RNAs were modified in a buffer favoring gRNA dimerization (50 mM sodium cacodylate (pH 7.5), 300 mM KCl and 5 mM MgCl 2 ) in the presence of 2 µg total yeast tRNA (Sigma Aldrich).…”

“…The presence of a stretch of purines in the packaging sequences on retroviral gRNA has been proposed to facilitate RNA packaging by functioning as a potential NC binding site [26–31]. It is also interesting to note that the positioning of the purine-rich loop in MMTV adjacent to the pal II loop is reminiscent of the situation found in Mason-Pfizer monkey virus (MPMV) [32,33]. In the case of MPMV, genetic and structure-prediction analyses have suggested that either ssPurines or its partially-repeated base-paired sequence in an adjacent region plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33].…”

“…It is also interesting to note that the positioning of the purine-rich loop in MMTV adjacent to the pal II loop is reminiscent of the situation found in Mason-Pfizer monkey virus (MPMV) [32,33]. In the case of MPMV, genetic and structure-prediction analyses have suggested that either ssPurines or its partially-repeated base-paired sequence in an adjacent region plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33]. Therefore, it is reasonable to propose that the bifurcated SL4 structure in MMTV functions as a major dimerization and packaging determinant during MMTV gRNA packaging process with pal II initiating the process of dimerization and ssPurines facilitating the encapsidation of the dimerized RNA into the virus particles by functioning as a potential Gag binding site.…”