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.

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 longrange 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 transcomplementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5 ′ -CGGCCG-3 ′ ) functions as DIS.

High-risk human papillomaviruses (HPV) can be present and cooperate with Epstein–Barr virus (EBV) to promote the onset and/or progression of various cancers including cervical, breast, head and neck as well as colorectal. In this investigation, we explored the co-prevalence of high-risk HPV and EBV in 74 breast cancer tissues from Qatari women using polymerase chain reaction. We found that high-risk HPV and EBV are present in 48/74 (65%) and 36/74 (49%) of the cases, respectively. While we noted that the presence of HPV presence is associated with triple-negative breast cancer (TNBC) ( p = .008), however, the presence of EBV did not correlate with any breast cancer subgroup. Moreover, our data revealed that high-risk HPV and EBV are co-present in 35/74 (47%) of the samples and their co-presence is significantly associated with tumor grade ( p = .04) and tumor stage ( p = .04). These data indicate that HPV and EBV are commonly co-present in breast cancer and their association could be linked with a more aggressive tumor phenotype. Thus, further investigations are essential to understand the underlying mechanisms of HPV and EBV cooperation in breast carcinogenesis.

Background: Human papillomaviruses (HPVs) and Epstein-Barr virus (EBV), known oncoviruses, can be co-present and cooperate in the initiation and/or progression of human carcinomas, including head and neck. Based on this fact, we recently reported the prevalence of both HPVs and EBV in cervical and breast cancers. Methods: We herein explore for the first time the co-prevalence of high-risk HPVs and EBV in 98 head and neck (HN) squamous cell carcinoma (SCC) tissues from Bosnian patients using polymerase chain reaction (PCR) and immunohistochemistry (IHC) analysis, as well as tissue microarray methodology. Results: The majority of these cancer tissue cases were from the oral cavity (68%). We found that high-risk HPVs and EBV are co-present in 34.7% of the SCC samples; with a significant correlation between the various HPV types and EBV coincidence (p = 0.03). Our data showed that 30.8% of oral SCCs are positive for E6 oncoprotein of high-risk HPVs and 44.6% are positive for LMP1 of EBV. The most commonly expressed HPVs in our HNSCC samples include HPV types 16, 18, 45 and 58. Additionally, 37.5% of oral SCCs are positive for both HPVs and EBV, with statistically significant association between high-risk HPV types and EBV (p < 0.05). More importantly, our data revealed that the co-presence of HPV and EBV is strongly correlated with advanced tumor stage (p = 0.035). Conclusion: In this study we show that HPV and EBV oncoviruses are co-present in HNSCC, particularly in oral cancer, where they can cooperate in the initiation and/or progression of this cancer. Thus, further studies are necessary to elucidate the mechanism of this cooperation.

Colorectal cancer (CRC) is a common malignancy with a high mortality rate worldwide. It is a complex, multifactorial disease that is strongly impacted by both hereditary and environmental factors. The role of microbes (e.g., viruses) in the pathogenesis of CRC is poorly understood. In the current study, we explored the status of high-risk human papillomaviruses (HPV) and Epstein–Barr virus (EBV) in a well-defined CRC cohort using immunohistochemistry and polymerase chain reaction assays. Our data showed that high-risk HPVs were common (~80%) and EBV had a low presence (14–25%) in the CRC samples. The most common high-risk HPVs are HPV16, 31, 18, 51, 52 and 45 genotypes. The co-presence of high-risk HPV and EBV was observed in ~16% of the sample population without any significant association with the clinicopathological variables. We conclude that high-risk HPVs are very prevalent in CRC samples while EBV positivity is relatively low. The co-expression of the two viruses was observed in a minority of cases and without any correlation with the studied parameters. Further studies are necessary to confirm the clinical relevance and potential therapeutic (preventive) effects of the observations reported herein.