Journal
RNA
Volume 19, Issue 12, Pages 1648-1658Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.040931.113
Keywords
Mason Pfizer monkey virus (MPMV); RNA dimerization; dimerization initiation site (DIS); RNA packaging signal; long-range interaction (LRI)
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Funding
- United Arab Emirates University [UAEU-NRF 26]
- College of Medicine and Health Sciences [NP 09/18]
- Centre National de la Recherche Scientifique (C.N.R.S.)
- Office of the Vice Provost for Research and Graduate Studies of the UAEU
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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.
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