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 long-range 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 trans-complementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5'-CGGCCG-3') functions as DIS.

Keywords: Mason Pfizer monkey virus (MPMV); RNA dimerization; RNA packaging signal; dimerization initiation site (DIS); long-range interaction (LRI).

FIGURE 1.

Minimal free-energy and SHAPE (selective 2′ hydroxyl acylation analyzed by primer extension)-validated models of the MPMV packaging signal RNA. The region used for analysis by Mfold and SHAPE included sequences from R up to 120 nt of gag. (A) MPMV packaging signal RNA secondary structure predicted earlier (Jaballah et al. 2010) using Mfold (Mathews et al. 1999; Zuker 2003). Sequences in orange, green, red, and blue represent the primer binding site (PBS), regions “A” and “B” (that have been shown to be important in gRNA packaging), and pal sequences, respectively. Boxed areas in purple show the predicted LRIs between U5 and gag. (B) SHAPE-constrained RNAstructure (Reuter and Mathews 2010) model of MPMV packaging signal. Nucleotides are color annotated as per the SHAPE reactivities key. SD indicates splice donor.

FIGURE 2.

Sequences in pal SL augments MPMV gRNA dimerization by functioning as DIS. (A) Table showing the 6-nt wild-type pal sequence (in red) and the different mutations introduced (in blue). (B) Gel shift assays of the MPMV wild-type and pal mutants. The upper panel shows native 1% TBM (50 mM Tris base, 45 mM boric acid, 0.1 mM MgCl₂) gel run at 4°C, and the lower panel shows semi-native 1% TB (50 mM Tris base, 45 mM boric acid) gel run at room temperature. M indicates monomer lane or monomer conformer; D, dimer lane or dimer conformer for each sample. (C) Relative dimerization of the MPMV wild-type and pal mutants RNAs. Following the gel shift assays, dimerization abilities of the RNAs in each lane were calculated, and the dimerization data were then represented as relative to the wild-type dimerization. (D) Structure prediction of the MPMV wild-type RNA +1–388 dimer structure. The site of interaction between the two gRNAs involving the pal sequence is enlarged for the sake of clarity. The central 6 nt of the pal are shown in red.

FIGURE 3.

In vitro heterodimerization can be mediated by trans-complementary sequences on two RNAs. (A) Table showing the 6-nt wild-type pal sequence (in red) and the two complementary substitution mutations (in blue). (B) In vitro dimerization assay of the MPMV wild-type and pal mutants. The upper panel shows native 1% TBM gel run at 4°C, and the lower panel shows semi-native 1% TB gel run at room temperature. M indicates monomer lane or monomer conformer; D, dimer lane or dimer conformer for each sample. (C) Relative dimerization of the complementary mutants to the wild-type RNAs. Dimerization abilities of the RNAs in each lane were calculated, and the dimerization data were represented as relative to the wild-type dimerization. (D,E) Structure predictions of the mutant RCR005 (D) and RCR006 (E) MPMV RNAs +1–388 using RNAstructure. (F) Schematic representation of the expected point of heterodimerization between RCR005 and RCR006 MPMV RNAs. (G) Predicted heterodimer structure of RCR005 and RCR006 RNAs.

FIGURE 4.

Mfold structural predictions (A–E) and ClustalW (F) sequence alignment of the 5′ end genomes of different MPMV strains. (A–E) The U5/Gag LRIs and the pal sequence are highlighted by red and green boxes, respectively. (F) The aligned sequences pertaining to major structural motifs are highlighted by different colors and boxed. The accession nos. for MPMV6/A, SRV1, SRV2, SRV4, and SRV5 are M12349.1 (Sonigo et al. 1986), M11841.1 (Power et al. 1986), AF126467.1 (Marracci et al. 1995), FJ979638.1 (Zao et al. 2010), and AB611707.1 (Takano et al. 2013), respectively.