Impact of the segment-specific region of the 3'-untranslated region of the influenza A virus PB1 segment on protein expression.

The 12 and 13 terminal nucleotides in the 3'- and 5'-untranslated regions (UTRs) of the influenza A virus genome, respectively, are important for the transcription of the viral RNA and the translation of mRNA. However, the functions of the segment-specific regions of the UTRs are not well known. We utilized an enhanced green fluorescent protein (eGFP) flanked at both ends by different UTRs (from the eight segments of H1N1 PR8/34) as a reporter gene to evaluate the effects of these UTRs on protein expression in vitro. The results showed that the protein expression levels of NP-eGFP, NS-eGFP, and HA-eGFP were higher than those of the other reporters and that the protein level of PB1-eGFP remained at a relatively low amount 48-h post-transfection. The results revealed that the UTRs of all segments differently affected the protein expression levels and that the effect of the UTRs of PB1 segment on protein expression was significant. The deletion of "UAAA" and "UAAACU" motifs in the PB1-3'-UTR significantly increased the protein expression level by 49.8 and 142.6%, respectively. This finding suggests that the "UAAACU" motif in the PB1-3'-UTR is at least partly responsible for the low protein expression level. By introducing the "UAAACU" motif into other 3'-UTRs (PA, NS, NP, and HA) at similar locations, the eGFP expression was reduced as expected by 56, 61, 22, and 22%, respectively. This result further confirmed that the "UAAACU" motif of the PB1-3'-UTR can inhibit protein expression. Our findings suggest that the segment-specific regions in the UTRs and not just the conserved regions of the UTRs play an important role in the viral protein expression. Additionally, the reported findings may also shed light on novel regulatory mechanism for the influenza A virus genome.