Influenza A viruses cause annual influenza epidemics and occasional severe pandemics. Their genome is segmented into eight fragments, which offers evolutionary advantages but complicates genomic packaging. The existence of a selective packaging mechanism, in which one copy of each viral RNA is specifically packaged into each virion, is suspected, but its molecular details remain unknown. Here, we identified a direct intermolecular interaction between two viral genomic RNA segments of an avian influenza A virus using in vitro experiments. Using silent trans-complementary mutants, we then demonstrated that this interaction takes place in infected cells and is required for optimal viral replication. Disruption of this interaction did not affect the HA titer of the mutant viruses, suggesting that the same amount of viral particles was produced. However, it nonspecifically decreased the amount of viral RNA in the viral particles, resulting in an eightfold increase in empty viral particles. Competition experiments indicated that this interaction favored copackaging of the interacting viral RNA segments. The interaction we identified involves regions not previously designated as packaging signals and is not widely conserved among influenza A virus. Combined with previous studies, our experiments indicate that viral RNA segments can promote the selective packaging of the influenza A virus genome by forming a sequence-dependent supramolecular network of interactions. The lack of conservation of these interactions might limit genetic reassortment between divergent influenza A viruses.

The genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical ‘7 + 1’ arrangement.