Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network.

Selective assembly of influenza virus segments into virions is proposed to be mediated through intersegmental RNA-RNA interactions. Here, we developed a method called 2CIMPL that includes proximity ligation under native conditions to identify genome-wide RNA duplexes. Interactions between all eight segments were observed at multiple sites along a given segment and are concentrated at hotspots. Furthermore, synonymous nucleotide changes in a hotspot decreased the formation of RNA-RNA interactions at this site and resulted in a genome-wide rearrangement without a loss in replicative fitness. These results indicate that the viral RNA interaction network is flexible to account for nucleotide evolution. Moreover, comparative analysis of RNA-RNA interaction sites with viral nucleoprotein (NP) binding to the genome revealed that RNA junctions can also occur adjacent to NP peaks, suggesting that NP association does not exclude RNA duplex formation. Overall, 2CIMPL is a versatile technique to map in vivo RNA-RNA interactions.

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

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.

Genome-wide analysis of influenza viral RNA and nucleoprotein association.

Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of 'beads on a string'. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP-vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.