New Insights into the Nonconserved Noncoding Region of the Subtype-Determinant Hemagglutinin and Neuraminidase Segments of Influenza A Viruses

Abstract

The noncoding regions (NCRs) of the eight-segmented viral RNAs (vRNAs) of influenza A virus consist of the highly conserved promoter region and the nonconserved segment-specific NCRs at both the 3= and 5= ends. The roles of the segment-specific NCRs of the eight segments have been extensively studied. However, the diversities in the same region of the two subtype-determinant hemagglutinin (HA) and neuraminidase (NA) segments have received little attention. In this study, we bioinformatically analyzed all available NCRs of HA and NA vRNAs of influenza A viruses and found that nucleotides in the segment-specific NCRs of HA and NA vRNAs are subtype specific and vary significantly in sequence and length at both the 3= and 5= ends among different subtypes. We then systematically studied the biological significance of the HA subtype-specific NCRs (HA ssNCRs) of the common HA subtypes (H1 to H7 and H9) in the context of the WSN (H1N1) reverse genetics system. We found that the HA ssNCRs play a critical role in HA vRNA virion incorporation. Upon HA vRNA incorporation, the 3=-end HA ssNCR plays a more critical role than the 5=-end HA ssNCR, and no stringent compatibility between the two ends is required. Furthermore, our data imply that, in addition to a particular nucleotide(s), the length of the HA ssNCR is involved in regulating HA vRNA incorporation efficiency. These results provide new insights into the HA segment virion incorporation that is critical for the emergence of epidemic and pandemic influenza A virus strains. IMPORTANCEThe nonconserved noncoding regions (NCRs) of the vRNAs of influenza A virus have been extensively studied, whereas the diversities in the nonconserved NCRs of the two subtype-determinant segments hemagglutinin (HA) and neuraminidase (NA) have received little attention. In this study, we bioinformatically analyzed all available NCRs of HA and NA vRNAs and discovered that the HA and NA vRNAs contain key subtype signatures in the NCRs. Our functional studies of the HA subtype-specific NCRs (HA ssNCRs) of the common HA subtypes in the context of WSN virus (H1N1) demonstrated that the HA ssNCR modulates virus replication efficiency by influencing HA segment virion incorporation. Moreover, we revealed important features of the HA ssNCR in determining HA vRNA incorporation efficiency. These data not only show new genetic characteristics of influenza A viruses, but also provide further evidence for understanding the selective genome packaging of influenza virus required for the emergence of epidemic and pandemic influenza virus strains.

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

“…Interestingly, we found that among these HA subtype-specific NCRs, nucleotides CC at 3=-end positions 13 and 14 and GUG at 5=-end positions 14= to 16=, located in the extended duplex region of the viral RNA promoter, are absolutely conserved (4,15,20). In order to understand the biological significance of these HA segment-specific nucleotides, we conducted a mutagenic analysis on these HA segment-specific nucleotides and examined their effects on viral RNA synthesis and virion RNA packaging.…”

“…Unlike the single-stranded RNA viruses, the eight vRNA segments of influenza A virus contain different noncoding regions (NCRs) varying from 17 to 60 nucleotides at the two extremities of their open reading frames (ORFs) (1,2). The noncoding region of each segment consists of a highly conserved promoter region followed by the segment-specific NCR at the 3= and 5= ends (1,3,4). An evolutionary analysis of the influenza virus NCR has demonstrated that the segment-specific noncoding region has extremely low divergence and that its evolutionary rate is lower than that of the coding region (5).…”

Influenza A virus utilizes a suboptimal Kozak sequence to fine-tune virus replication and host response

“…The same bioinformatics analysis was performed as previously described (Zhao et al, 2014). The sequences of all segments (PB2, PB1, PA, H3, NP, N2, M and NS) were obtained from NCBI Influenza Virus Resource database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.…”

“…In order to obtain a full view for segment-specific NCRs of all influenza A viruses, we used bioinformatics to analyse all available NCR sequences of all the eight segments from the NCBI Influenza Virus Resource database as recently described (Zhao et al, 2014). We used H3 and N2 subtypes for the analysis of HA and NA segments.…”

“…It has previously been reported that the segmentspecific NCRs of influenza A virus play multiple roles in the virus life cycle. They not only act as cis-acting signals to regulate viral transcription, replication and protein expression (Bergmann & Muster, 1996;Park & Katze, 1995;Zheng et al, 1996), but also serve as part of the packaging signals during selective virus genome packaging (Gog et al, 2007;Hutchinson et al, 2010;Zhao et al, 2014).…”