A novel interaction between the 5' untranslated region of the Chikungunya virus genome and Musashi RNA binding protein is essential for efficient virus genome replication.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes—causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In the current study, we identify host cell Musashi RNA binding protein-2 (MSI-2) as a proviral factor. MSI-2 depletion and small molecule inhibition assays, demonstrated that MSI-2 is required for efficient CHIKV genome. Depletion of both MSI-2 and MSI-1 homologues resulted in a synergistic increase in CHIKV inhibition, suggesting redundancy in their proviral function. EMSA competition studies demonstrated that MSI-2 interacts specifically with an RNA binding motif within the 5’ untranslated region (5’UTR) of CHIKV and reverse genetic analysis showed that mutation of the binding motif inhibited genome replication and blocked rescue of mutant virus. For the first time, this study identifies the proviral role of MSI RNA binding proteins in the replication of the CHIKV genome, providing important new insight into mechanisms controlling replication of this significant human pathogen and offers the potential of a new therapeutic target

Statistical properties of thermodynamically predicted RNA secondary structures in viral genomes

By performing a comprehensive study on 1832 segments of 1212 complete genomes of viruses, we show that in viral genomes the hairpin structures of thermodynamically predicted RNA secondary structures are more abundant than expected under a simple random null hypothesis. The detected hairpin structures of RNA secondary structures are present both in coding and in noncoding regions for the four groups of viruses categorized as dsDNA, dsRNA, ssDNA and ssRNA. For all groups hairpin structures of RNA secondary structures are detected more frequently than expected for a random null hypothesis in noncoding rather than in coding regions. However, potential RNA secondary structures are also present in coding regions of dsDNA group. In fact we detect evolutionary conserved RNA secondary structures in conserved coding and noncoding regions of a large set of complete genomes of dsDNA herpesviruses.Comment: 9 pages, 2 figure

Structural and phenotypic analysis of Chikungunya virus RNA replication elements

Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with single-molecule resolution. The conformation of biomolecules, such as RNA, is highly dependent on the electrolyte composition, but solid-state nanopore systems often require high salt concentration to operate, precluding analysis of macromolecular conformations under physiologically relevant conditions. Here, we report the implementation of a polymer–electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer–electrolyte bath governs the translocation dynamics of the analyte which correlates with the physical properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer–electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiologically relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer–electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomolecules under physiologically relevant conditions

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core- RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly

Fathers and Sons: <i>Ecclesiazusae</i> 644-45

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The aerodynamic performance of a range of FIFA-approved footballs

This article was published in the journal, Proceedings of the Institution of Mechanical Engineers, Part P: Journal of Sports Engineering and Technology [© Sage] and is available at: http://dx.doi.org/10.1177/1754337111415768Much discussion surrounds the flight of a football especially that perceived as irregular and is typically done so with little understanding of the aerodynamic effects or substantive evidence of the path taken. This work establishes that for a range of FIFA approved balls there is a significant variation in aerodynamic performance. This paper describes the methods used for mounting stationary and spinning footballs in a wind tunnel enabling accurate force data to be obtained, and the analysis techniques used. The approach has been to investigate a number of scenarios: Non-spinning Reynolds Sweep, Unsteady Loads, Orientation Sensitivity (Yaw Sweep) and Spinning Reynolds Sweep. The techniques are applied to a number of footballs with differing constructions and the results reported. To put the aerodynamic data into context the results are applied in a flight model to predict the potential differences in the behaviour of each ball in the air. The paper concludes that although the drag characteristics are different for the different balls tested the simulation suggests that this has only a limited effect on the flight of the ball. It is also shown that the unsteadiness of the aerodynamic loads is unlikely to be responsible for unpredictable behaviour. However, it is also shown that there are significant differences in the lateral aerodynamic forces for a range of FIFA approved match balls, and that these aerodynamic differences have a significant effect on the flight path for both spinning and for slowly rotating balls