SHAPE Validated Secondary Structures of Mason-Pfizer Monkey Virus (MPMV) and Mouse Mammary Tumor Virus (MMTV) Packaging Signal RNAs Reveal Pal Helix Loops Functioning as Dimerization Initiation Sites (DIS) Controlling their Genomic RNA (gRNA) Packaging

The “diploid” genome of retroviruses consists of two strands of RNA that are non-covalently linked as a “dimer” at their 5’ ends. The ubiquitous presence of a dimeric genome among retroviruses suggests that dimerization plays a crucial role in gRNA packaging during viral life-cycle. For almost all retroviruses, determinants of gRNA dimerization and packaging, which are for the most part physically and genetically indistinguishable, reside at the 5’ end of the gRNAs and have been shown to assume higher order structures. Employing a combination of genetic and structural prediction approaches, we have earlier shown that Mason-Pfizer monkey virus (MPMV) and mouse mammary tumor virus (MMTV) packaging determinants comprise sequences at the 5’ end of the genome, starting from R and extending into the beginning of Gag. Sequences encompassing these regions were predicted to fold into stable RNA secondary structures comprising several structural motifs. In an attempt to establish structure-function relationship of the higher order features of MPMV and MMTV packaging signal RNAs, we first validated their predicted structures employing a novel chemo-enzymatic probing strategy, selective 2’hydroxyl acylation primer extension (SHAPE). The SHAPE-analyzed structures of MPMV and MMTV packaging signal RNAs validated the major structural motifs, including U5/Gag long range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive GC-rich palindromic (pal) helix loop. Minimum free-energy structure predictions, phylogenetic, and in silico analyses of different MPMV and MMTV strains further suggested the existence of these major structural motifs. viii To test the importance of the pal sequences in MPMV and MMTV gRNA dimerization and packaging, we introduced a series of mutations in the pal helix loops. Tests of these mutations employing in vitro and in vivo complementary approaches, phylogenetic, and structure prediction analyses revealed pal