Takizawa, N. | Search bioRxiv

bioRxiv2024

Concentration-dependent formation of intersegment interactions in viral droplets of influenza A virus infected cells

The influenza A virus genome consists of eight RNA segments, each incorporated into a virion. It has been proposed that intersegment interactions bundle these segments, and viral inclusions, which contain viral ribonucleoproteins (vRNPs) and Rab11, facilitate this process. However, the locations and mechanisms of intersegment interaction formation remain unclear. To investigate this, we identified comprehensive intersegment interactions in infected cells using customized LIGR-seq. Our results revealed that intersegment interactions in infected cells overlapped with those in the virion and were partially formed through direct RNA-RNA interactions. Additionally, we found that the formation of these interactions was delayed in cells expressing a Rab11 dominant-negative. Furthermore, artificially increasing vRNP concentrations in the nucleus, where intersegment interactions typically do not occur, triggered the formation of these interactions, which again overlapped with those in the virion. These results suggest that intersegment interactions are formed in viral inclusions where progeny vRNPs accumulate.

microbiology↗

bioRxiv2020

A functional RNA structure in the influenza A virus ribonucleoprotein complex for segment bundling

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.

microbiology↗