Roles Of Influenza A Virus Genome RNA Sequence And Structural Characteristics On Virus Replication

Influenza A viruses(IAVs)are major infectious respiratory tract diseases and cause annual epidemic and sporadic pandemic with significant morbidity and mortality in humans.The genome of IAV consists of eight single-stranded,negative-sense viral RNA(vRNA)segments.Each vRNA segment contains the internal open reading frame(ORF)and flanked by the 3’ and 5’ noncoding regions(NCRs).The nonconserved noncoding regions(NCRs)at the 3’ and 5’ termini of each segment appear as segment-specific among the eight segments and also subtype-specific among different subtype-determinant HA and NA segments.The viral genomes are replicated in the form of viral ribonucleoprotein complexes(vRNPs)in the nucleus of infected cells,followed by transportation to the plasma membrane and selective packaging into progeny virions.Viral genomic RNA fold to form complex structures and acts as an essential regulatory hub to coordinate both virus and host molecular to fulfill these functions.Therefore,investigation on the role that RNA structure of virus genome plays in these regulatory processes will deepen our understanding of the fine regulation mechanisms of virus life cycle.In this study,by using "in vivo click selective 2-hydroxyl acylation and profiling experiments"(icSHAPE),we resolved the vRNA structures of the WSN strain both in vivo and in virio.We also investigated vRNA long range RNA-RNA interactions by using"psoralen analysis of RNA interactions and structures"(PARIS)in the two conditions.We noted that there is a similarity between the in vivo and in virio viral RNA icSHAPE data,while the reactivity score tended to be higher in the in vivo dataset relative to the in virio dataset,which may be due to the closure of intracellular RNA binding proteins.A large number of RNA-RNA interactions have been found in vivo and in virio,while the RNARNA interaction map shows significant differences in viral genomic RNA interactions between the two conditions.The intrasegment interactions clustered into 832 duplexes in vivo and into 738 duplexes in virio;the intersegmental interactions clustered into 41 duplexes in vivo and 2077 duplexes in virio.In addition,our in-depth functional analyses reveal the first specific long-range intrasegment RNA structure in PA segment that plays a direct role on packaging efficiency of this segment into progeny virions.More importantly,our animal experiment further confirmed the essentiality of this RNA structure for the virus pathogenicity.This work presented here provides important insights into the RNA structure of influenza A virus genome and packaging process,and provides a platform for the design of antiviral oligonucleotides.In addition,in order to acquire in-depth knowledge on the sequence characteristics of the segment-specific or subtype-specific NCRs(ssNCRs)in the influenza virus replication cycle,we designed a virus Random Nucleotide Selection Assay(vRNSA)in which we provide randomly mutated PHW2000-HA rescue plasmids with random nucleotides at each grouped nucleotide position in 3’ and 5’ H1-ssNCR in the context of WSN(H1N1)reverse genetics system,followed by virus rescue,serial passages and deep sequencing.The results showed that,in the process of continuous passages,the 3’-terminal sequences of H1-ssNCR,in general,stabilize more quickly than the 5’-terminal sequences.Furthermore,the nucleotides close to the highly conserved 3’ and 5’ promoter region showed higher requirements for the sequence stability.For the nucleotides distant from the promoter,they displayed a higher requirement of the proportion of A and U bases than the actual sequences.In addition,we found that a point mutation 3’ A19U severely impairs transcription and replication of HA vRNA and thus attenuated the virus.After serial passages,an adjacent mutation 3’ U23G appeared and restored the HA RNAs level and virus titres,indicating a functional coordination among these nucleotides.These results reveal,for the first time,the distinctions of sequence requirements between 3’ and 5’ H1ssNCR and also inside the nucleotides with different locations of 3’ or 5’ H1-ssNCR upon regulating virus propagation.