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.