The Role Of Specific Non-coding Region Of Section A Influenza Virus RNA Segments In Viral Replication

Influenza infection, which is caused principally by influenza A virus, is a major threat to human health and results in significant morbidity and mortality worldwide. The influenza A virus genome is composed of eight negative-sense RNA segments, each RNA segment contains one or two open reading frames (ORF) flanked by the5’and3’short non-coding regions(NCRs). The NCR of influenza A virus, as an important component of viral genome, contains the signals required for transcription, replication and packaging of the viral genes, but a systematic study of the role of the NCR in both transcription and translation has not previously been undertaken.In this study, we analyzed all available sequences of the noncoding region (NCR) of influenza A viruses in the NCBI Influenza Virus Resource and investigated the regulatory roles of these NCRs at the levels of viral RNA transcription, replication and translation in the RNP reconstitution systems. We further show that the segment-specific NCR of the influenza A virus PB1segment, contains a highly conserved suboptimal nucleotide at-3position of Kozak sequence that exceptionally down-regulates protein expression at the level of translation. A recombinant virus with an optimized Kozak sequence in PB1displays enhanced PB1expression at an early stage of infection and an accelerated single cycle replication, but is attenuated in multiple cycles of replication. Furthermore overexpression of PB1not only alters the RNA synthesis dynamics of the recombinant virus, but also leads to the induction of more type I interferon than in wild-type virus.In addition, we conducted a study to elucidate the function of the conservative nucleotide adjoining to viral promoter sequence in NCR. The results show that the base pairing at13:14’ position in NCR is essential for viral promoter activity. Meanwhile, we found the nucleotide at13-14position in3’NCR and nucleotide at14’-15’position in5’NCR may involve in other virological processes, such as genome packaging.Together, this study reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host anti-viral response through modulating a viral protein expression, and partially elucidated the biological significance of the conservative nucleotide adjoining to viral promoter sequence in NCR. These findings also provided insight into the regulating effect of NCR on the life cycle of influenza A virus.