The structure of the influenza A virus genome

Abstract

Influenza A viruses (IAVs) are segmented single-stranded negative sense RNA 12 viruses that constitute a major threat to human health. The IAV genome 13 consists of eight RNA segments contained in separate viral ribonucleoprotein 14 complexes (vRNPs) that are packaged together into a single virus particle 1,2 . 15While IAVs are generally considered to have an unstructured single-stranded 16 genome, it has also been suggested that secondary RNA structures are 17 required for selective packaging of the eight vRNPs into each virus particle 3,4 . 18Here, we employ high-throughput sequencing approaches to map both the . Although 43 not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/236620 doi: bioRxiv preprint first posted online Dec. 21, 2017; 3 cryo-EM studies revealed the overall architecture and organisation of vRNPs, the 44 resolution of currently available structures is not sufficiently high to provide 45 information about the conformation of the vRNA 8,9 . It is thought that, through 46 specific RNA-RNA interactions, exposed regions of vRNA in the vRNP mediate 47 segment-specific interactions during virion assembly, ensuring that the correct set 48 of eight vRNPs is selected 3,4 . However, the identities of these interacting regions as 49 well as the overall structure of vRNA in vRNPs are currently unknown. 50 51To better understand the vRNA structure in the context of vRNPs, we employed 52 SHAPE-MaP (Selective 2'-Hydroxyl Acylation Analysed by Primer Extension and 53Mutational Profiling), which probes the conformational flexibility of each vRNA 54 nucleotide both ex virio and in virio (Extended Data Fig. 1) 10,11 . For the ex virio 55 experiments, the eight vRNA segments were individually transcribed from plasmid 56 DNA using T7 RNA polymerase (ivtRNA) or "naked" vRNA was purified from 57 deproteinated influenza A/WSN/1933 (H1N1) (WSN) particles (nkvRNA). For the in 58 virio experiments, vRNA was probed in the context of vRNPs directly inside purified 59 virions (Extended Data Fig. 1a). 60 61Three biological replicates of the in virio SHAPE-MaP experiments resulted in highly 62 reproducible SHAPE reactivity profiles suggesting that there is an underlying 63 structure of vRNA in the context of vRNP (Extended Data Fig. 2a). We found that 64 the eight different vRNPs in virio have unique vRNA conformations, as 65 demonstrated by the different SHAPE reactivity profiles (Fig. 1a). Regions of 66 extensive low SHAPE reactivities in virio (where the median-SHAPE value is below 67 zero, Fig. 1a) indicate that the vRNA in the context of vRNPs is capable of 68 accommodating secondary RNA structures with extensive base-pairing. However, 69 comparison of in virio and ex virio SHAPE profiles shows a significant shift in the 70 distribution of SHAPE reactivities in virio, consistent with an overall more open (less 71 structured) RNA conformation (Fig. 1b). In addition, the vRNA forms...

Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions

“…2K). Indeed, a recent biochemical study revealed that most vRNA segments have multiple RNA contact sites with other genome segments, thereby creating a redundant web of RNA-RNA interactions (29). Importantly, the individual disruption of some of these interaction sites by synonymous mutation did not provoke obvious genome packaging defects (29).…”

“…Indeed, a recent biochemical study revealed that most vRNA segments have multiple RNA contact sites with other genome segments, thereby creating a redundant web of RNA-RNA interactions (29). Importantly, the individual disruption of some of these interaction sites by synonymous mutation did not provoke obvious genome packaging defects (29). In line with this concept, we further speculate that such a redundant set of packaging sequences would allow multiple interaction "solutions" to the specific incorporation of the eight genome segments into virions.…”

“…However, their combined mutation caused packaging defects characterized by the preferential absence of distinct vRNP subsets from the virions. We speculate that this mutational robustness of the vRNP network is inherently coupled to its redundant architecture, especially through the involvement of a large number of possible RNA-RNA interactions between the eight viral genome segments (29). Such complex systems are not only known to be robust to genetic perturbations but also highly plastic and adaptable (37)(38)(39), features which would greatly facilitate the acquisition of new viral genome segments during genetic reassortment.…”

A Comprehensive Review on the Interaction Between the Host GTPase Rab11 and Influenza A Virus

“…Seminal work established the requirement of cis -acting and intersegment RNA–RNA interactions in the formation of the eight-vRNP viral genome [for reviews check (Ferhadian et al, 2018)]. In addition, cryo-tomography of budding virions captured images of inter-connected segments (Fournier et al, 2012a,b; Noda and Kawaoka, 2012; Gavazzi et al, 2013a) and recent preprint data from Fodor Lab using PAR-CLIP (Dadonaite et al, 2017) found biochemical evidence in support of existing inter-segment ligations in isolated virions. Together, these studies push forward the notion that IAV genome actually forms a complex with strong interactions among segments.…”

Replication competent, 10-segmented influenza viruses as antiviral therapeutics

“…The UTRs, as well as the proximal portions of the coding regions, form "packaging signals" that are both necessary and sufficient for incorporation of each vRNA into progeny virions [6][7][8][9][10][11]. Although the underlying mechanisms that control packaging are incompletely understood, it has been hypothesized that segments may potentially interact with one another via vRNA-vRNA interactions across genome segments [12][13][14][15][16].…”