Dual Roles of the Hemagglutinin Segment-Specific Noncoding Nucleotides in the Extended Duplex Region of the Influenza A Virus RNA Promoter

We recently reported that the segment-specific noncoding regions (NCRs) of the hemagglutinin (HA) and neuraminidase (NA) segments are subtype specific, varying significantly in sequence and length at both the 3' and 5' ends. Interestingly, we found that nucleotides CC at positions 13 and 14 at the 3' end and GUG at positions 14 to 16 at the 5' end (termed 14' and 16' to distinguish them from 3' positions) are absolutely conserved among all HA subtype-specific NCRs. These HA segment-specific NCR nucleotides are located in the extended duplex region of the viral RNA promoter. In order to understand the significance of these highly conserved HA segment-specific NCR nucleotides in the virus life cycle, we performed extensive mutagenesis on the HA segment-specific NCR nucleotides and studied their functional significance in regulating influenza A virus replication in the context of the HA segment with both RNP reconstitution and virus infection systems. We found that the base pairing of the 3' -end 13 position with the 5' -end 14' position ((3')13-(5')14') position is critical for RNA promoter activity while the identity of the base pair is critical in determining HA segment packaging. Moreover, the identity of the residue at the 3'-end 14 position is functionally more important in regulating virus genome packaging than in regulating viral RNA synthesis. Taken together, these results demonstrated that the HA segment-specific NCR nucleotides in the extended duplex region of the promoter not only form part of the promoter but also play a key role in controlling virus selective genome packaging. IMPORTANCE The segment-specific complementary nucleotides (13 to 15 in the 3' end and 14' to 16' in the 5' end) in the extended duplex region of the influenza virus RNA promoter vary significantly among different segments and have rarely been studied. Here, we performed mutagenesis analysis of the highly conserved HA segment-specific nucleotides in the extended duplex region and examined their effects on virus replication in the context of the influenza A/ WSN/ 33 (WSN) virus infection. We found that these HA segment-specific nucleotides not only act as a part of the RNA promoter but also play a critical role in HA segment packaging. Therefore, we showed experimentally, for the first time, the requirement of the nucleotides in the extended duplex region for the RNA promoter and also identified specific noncoding residues in regulating HA segment packaging. This work has implications for the development of attenuated vaccine strains and for elucidation the mechanisms of the virus genome packaging.