Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA

Abstract

The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.

In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs

“…By applying RAPiD-MaPseq to living cells infected with IAV, we here define for the first time in vivo , as well as in vitro , IAV mRNA secondary structures. Although binding of NP packaging proteins to both vRNAs and cRNAs largely inhibits the formation of RNA secondary structures (59,60), we cannot completely rule out the possibility that some structural domains we have here described might protrude from the NP-cRNA complex; a possibility that is supported by recent works showing that vRNAs-bound NP proteins are non-uniformly distributed (6,61,62).…”

Selective Genome Packaging Mechanisms of Influenza A Viruses

“…Previously, it had been believed that NPs uniformly bind to the entire length of the singlestranded vRNA, where secondary and tertiary structures of the vRNA are completely melted by the binding of the NPs (Baudin et al 1994), although there is a report that a bacteriophage PP7 RNA tag inserted into the NA vRNA maintains the hairpin structure in the context of the vRNP (York et al 2013). Recently, Lakdawala and her colleagues performed a HITS-CLIP (high-throughput sequencing of RNA isolated by CLIP) analysis to determine the NP-binding sites of vRNAs within virion-derived vRNPs (Lee et al 2017;Le Sage et al 2018). They found that NPs associate with vRNAs in a nonuniform and nonrandom manner, in which NPs preferentially bind to guanine-rich and uracil-poor regions, and that there are NP-free regions on vRNAs.…”

Trans-Acting RNA–RNA Interactions in Segmented RNA Viruses

“…NP-bound regions were originally believed to be uniformly coated, with NP associating with the viral RNA in a consistent manner. However, recent studies have revealed that NP, which interacts with ~12 nt long segments of sequence, is spread unevenly on viral RNA, exhibiting regions of high and low density [10,11,12]. This unequal distribution is likely dictated primarily by RNA sequences and/or structures that influence NP binding, however the specific structural features guiding the differences in concentration remain to be defined.…”