Abstract
BackgroundOne of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5’ end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5’ untranslated region (5’ UTR) and the beginning of gag.ResultsThe RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2’hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5’ region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5’ CGGCCG 3’) at the 5’ UTR and contains a canonical “GC” dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging.ConclusionsEmploying structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0096-6) contains supplementary material, which is available to authorized users.
Highlights
One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral genomic RNA (gRNA) in the form of a non-covalent RNA dimer by the assembling virions
The pal II deletion mutant (SA042) sequence was folded as dimer using the RNAstructure software, which predicted that in the absence of pal II, the mammary tumor virus (MMTV) RNA packaging signal could potentially dimerize via the two overlapping pals in the primer binding site (PBS) (5’ CAGCU GGCGCC 3’; the first pal is in italics and the second pal is in bold (Figure 4)
Results presented in this study validate the higher order structural features of the MMTV packaging signal RNA using the selective 2’hydroxyl acylation analyzed by primer extension (SHAPE) methodology, which includes Long-range interaction (LRI) involving complementary sequences from U5 and the gag (U5/Gag LRI), ssPurines, and four palindromic sequences (Figure 1)
Summary
One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. Recent retroviral RNA cross-packaging studies have shown that the specificity of retroviral gRNA packaging can be exchanged by substituting the packaging signals of genetically diverse retroviruses [12,13]. This has further been substantiated by the fact that heterodimers involving RNAs from two divergent retroviruses with no sequence homology can be packaged (reviewed in [2,11,14]). A number of RNA cross- and copackaging studies among diverse retroviruses suggest that the process of gRNA dimerization and packaging is likely to involve recognition of structural motifs rather than primary sequences [12,13,15,16,17,18,19]. Consistent with this, the packaging and dimerization sequences of almost all retroviruses have been shown to assume higher order structures comprising of various structural motifs that have been shown to mediate RNA-RNA and RNA-protein interactions during retroviral RNA dimerization and packaging
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