Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV)

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.

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

“…We have recently shown that the entire 5ʹ UTR and the first 120 nt of gag are required for efficient MMTV gRNA packaging and propagation [23,24]. Furthermore, these sequences were predicted to fold into higher order structures comprising of several structural motifs, which were validated by SHAPE (selective 2ʹ-hydroxyl acylation analyzed by primer extension [25]. …”

“…A distinguishing feature of the SHAPE-validated structure of the MMTV packaging signal RNA is the presence of a bifurcated stem loop 4 (SL4) containing a palindromic sequence (pal II; 5ʹ CUGCAG 3ʹ) and a 9 nt stretch (5ʹGGAGAAGAG 3ʹ) of single-stranded purines (ssPurines) in adjacent apical loops [25]. Both pal II and ssPurines are phylogenetically conserved in eight different strains of MMTV at the sequence as well as the secondary structural levels [25].…”

“…Both pal II and ssPurines are phylogenetically conserved in eight different strains of MMTV at the sequence as well as the secondary structural levels [25]. Pal II (5ʹ CUGCAG 3ʹ) has been shown to act as the dimerization initiation site (DIS) for MMTV gRNA from amongst four other palindromes within the 5ʹ UTR of the viral genome [25].…”

“…Both pal II and ssPurines are phylogenetically conserved in eight different strains of MMTV at the sequence as well as the secondary structural levels [25]. Pal II (5ʹ CUGCAG 3ʹ) has been shown to act as the dimerization initiation site (DIS) for MMTV gRNA from amongst four other palindromes within the 5ʹ UTR of the viral genome [25]. 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].…”

“…The role of the sequences as well as the higher order structure of the MMTV SL4 region have not been tested genetically, although they offer a logical and perhaps mechanistic explanation for their central role in MMTV gRNA dimerization and packaging [24,25]. Therefore, to establish the biological significance of the sequences and structural components of SL4 and to further provide functional evidence for the role of pal II and ssPurines in MMTV RNA packaging, a series of mutations were introduced that included deletions, substitutions, and compensatory mutations in SL4.…”