vRNA-vRNA interactions in influenza A virus HA vRNA packaging

The genome of the influenza A virus is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight different vRNAs are selectively packaged into progeny virions. This process likely involves specific interactions among vRNAs via segment-specific packaging signals located in the 3’ and 5’ terminal coding regions of vRNAs. To identify vRNA(s) that interact with hemagglutinin (HA) vRNA during genome packaging, we generated a mutant virus, HA 5m2, which possessed five silent mutations in the 5’ packaging signal region of HA vRNA. The HA 5m2 virus had a specific defect in HA vRNA incorporation, which reduced the viral replication efficiency. After serial passaging in cells, the virus acquired additional mutations in the 5’ terminal packaging signal regions of both HA and PB2 vRNAs. These mutations contributed to recovery of viral growth and packaging efficiency of HA vRNA. A direct RNA-RNA interaction between the 5’ ends of HA and PB2 vRNAs was confirmed in vitro . Our results indicate that direct interactions of HA vRNA with PB2 vRNA via their packaging signal regions are important for selective genome packaging and enhance our knowledge on the emergence of pandemic influenza viruses through genetic reassortment.