Abstract
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 spatial and mechanistic dynamics of intersegment interactions remain unclear. To investigate this, we identified comprehensive intersegment interactions in infected cells using the customized ligation of interacting RNA and high-throughput sequencing (LIGR-seq). We found that intersegment interactions overlapping with those in virions were present under viral inclusion-promoting conditions. In contrast, such interactions were diminished in the absence of viral inclusions. Moreover, artificially increasing vRNP concentrations in the nucleus, where intersegment interactions typically do not occur, induced intersegment interaction patterns resembling those in virions. These findings suggest that intersegment interaction networks form in a vRNP concentration-dependent manner within viral inclusions, providing a spatiotemporal basis for segment bundling.