RNA structure interactions and ribonucleoprotein processes of the influenza A virus

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

The vRNA is wrapped up in a scaffold composed of the NP proteins [42, 43] ; therefore, only a few examples of known RNA base-pair formation have been reported for the packaged Table 2 . List of viral proteins that enter the nucleus and importin factors that are used, adapted from [20] Protein Import co-proteins PB2 Importin a1, a3, a5, a7, b PB1 (PB1þPA)þRanBP5 PA NP Importin a1, a3, a5, a7, b vRNP Importin a1, a5, b the largely universally conserved segments reported in [87] are indicated by the long semitransparent gray/green boxes (also marked by dark arrows, bottom). The graph spectrum was generated using Genepoem [90] and calculated using vs_subopt/vswindow [89] using default settings (window size 200 nt, Kuhn length 4 nt). The very dark gray/blue boxes indicate regions of highly stable RNA secondary structure (not only hairpins but even a region of stability). The very short gray/magenta boxes indicate regions where a stable loop is present. The particular strains that were calculated are shown on the right side of each free energy spectrum. The sequences were obtained from the influenza database at https://www.fludb.org [88] . (A colour version of this figure is available online at: https://academic.oup.com/bfg) vRNPs. One observed interaction occurs at the terminal ends of each vRNA segment. In all eight vRNA gene segments, the terminal 13 and 12 nt fragments at the 5 0 -and 3 0 -most ends of the vRNA segments are conserved and known to form the vRNA promoter. This region also exhibits short hairpins of RNA secondary structure [45] . The second observed interactions were inferred from the fact that the majority of virions contain exactly one complete genome packed in the daisy chain structures [9, 46, 81, 83] . This daisy chain connectivity has long suggested that RNA/RNA interactions occur between the vRNPs that help bind them together. Recent observations have shown that there appear to be some distinct RNA/RNA interactions between different vRNPs [83, 84] . In particular, one study [84] revealed that a vRNA hairpin from Segment 2 (the PB1 Segment) and a vRNA hairpin from Segment 8 (the NP Segment) appear to mutually combine via the complementary base-pair sequences of the loops. (Two RNA hairpins bound together by complementary sequences found in their loops are called a 'kissing loop' [85, 86] .) The evidence for the kissing loop is supported by sequence alignment methods and in vitro experiments involving disrupting the complementary sequences and measuring the relevant fractions of products via native agarose gel electrophoresis [84] . Hence, the linking of the segments is most likely because of RNA/RNA interactions between adjacent segments. However, these interaction networks of kissing loops also appear to be straindependent [84] . This suggests that for a particular subtype of IAV to jump between species, not only does the successful subtype has to acquire specific mutations in HA, access effective nuclear import/export factors and produce proteins that successfully block host innate immunity but, additionally, the subtype requires a proper connectivity between vRNPs. This might partly explain why reassortment of various subtypes is not a common occurrence and how the strains manage to remain unique.