RNA structure interactions and ribonucleoprotein processes of the influenza A virus

Dawson, Wayne K; Lazniewski, Michal; Plewczynski, Dariusz

Kobayashi et al. [87] attempted to identify the actual RNA secondary structure (hairpins) in Segment 7 (the M Segment) and test their stability at different temperatures. (RNA secondary structure differs from protein secondary structure in that it shows the base pairing between diverse parts of the RNA sequence or between different RNA sequences; therefore, more topology information can be inferred from RNA secondary structure data.) In this analysis, the functionality of conserved stem-loop structures was directly tested using reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at three locations. These disruptions tended to attenuate the infectivity of the recombinant virus. The disruption of the stem-loop structure at nucleotide positions 219-240, 950-964 and 967-994 of Segment 7 (M Segment) showed a significant temperature dependence, which is characteristic of decrease in RNA stability in these regions. This suggests that the conserved secondary structures The abovementioned studies strongly suggest that base pairing in the vRNP may play a significant role in the mutual binding of the vRNP structures to one another. The data from Kobayashi et al. is stable. In Figure 3 , an actual scan of the free energy and variation in optimal and suboptimal structures of Segment 7 (Segment M) is shown for four different viral strains in the following order: A/Baltimore/0026/2016(H1N1), A/England/195/2009 (H1N1), A/udorn/1972(H3N2) and A/ruddy turnstone/DE/509531/ 2007(H5N1). Sequences of this segment were obtained from the influenza database at https://www.fludb.org [88] . The spectra represent scanning for RNA secondary structure over a window size of 200 nt and, based on the predicted suboptimal structures obtained in the scan, detecting regions where a particular secondary structure motif of RNA repeatedly appears in many (or most) of the optimal and suboptimal structures. The very dark gray/blue boxes represent regions of stable RNA secondary structure that are largely independent of the scanning window size, and segments of highly variable secondary structure are shown by their lack of any color. (The very short gray/magenta boxes indicate the center of the hairpin in the stable (gray/blue) regions of the spectra.) Covering the regions at loci 219-240, 950-964 and 967-994 of Segment 7 reported by Kobayashi et al. [87] , Figure 3 shows three long semi-opaque gray/green boxes (also marked by the dark arrows, bottom) overlapping these respective fragments. The scan was performed using vswindow; an expanded application of vs_subopt [89] (http://www.rna.it-chiba.ac.jp/vsfold/vs_ subopt/) with default settings (window size 200 nt, Kuhn length 4 nt), and the spectrum is generated using Genepoem [90] (http:// www.rna.it-chiba.ac.jp/vswindow/cgi-bin/index.cgi). Modifying the settings-such as changing the scanning window length or the Kuhn length (a measure of the stiffness of the RNA [89] )-generates similar results. As the RNP (RNA wrapped in NPs) is largely exposed [42] , the stable RNA secondary structure is also likely to appear in the RNP complex.