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.

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5 ′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. Keywords: retroviruses; Mason-Pfizer monkey virus (MPMV); RNA secondary structure; RNA packaging and dimerization; long-range interactions (LRI); SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension)

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5ʹ UTR) to~120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.