Research On Function Of The4^th Nucleotide At3’UTR Of H5N1Virus

Highly pathogenic avian influenza virus (HPAIV) H5N1, which can infect a wide range of hosts, such as wild birds, domestic poultry, human and some other mamals, posed a major therat to human health and social stabilization and caused severe economic lost. In1997, it was firstly proved that HPAIV H5N1can break through the species barriers to infect human directly, and even resulted death of patients. Since2003, more than600human infected with H5N1cases have been reported to WHO from15countries with a mortality rate of approximately60%. However, the molecular mechanism of H5N1virus breaking species restriction and the pathogenesis of virus on human were not fully understood.The genome of the influenza A virus (IAV) is composed of eight single-stranded RNA segments of negative polarity. Each genomic segment of IAV contains coding regions and untranslated regions (UTRs). UTRs comprise universally conversed sequences and segment-dependent sequences at both the5’and3’ends. And the first13nt at the5’ end and the first12nt at the3’end are highly conserved and segment-independent. Only one single natural variation, U or C at position4of the3’ end of the vRNA (U4or C4), was observed in the first12nt at the3’end of any influenza virus. These nucleotides are partially complementary and can form duplex structures, such as panhandle structures, which can be recognized by RdRp to initiate transcription and replication. However, no study has yet reported that the untranslated regions of H5N1make any contribution to its high pathogenicity.Herein, the substitution of U4and C4in genomic segments of influenza A viruses with UTR sequences, including H5N1influenza virus, was analyzed. The influence of the substitution in PB1, HA and NA on RNA-dependent RNA polymerase activity, reproduction, and pathogenicity of in A/Vietnam/1194/2004(H5N1) was evaluated.1. Substitution of the4th nucleotide at3’ end of UTR in IAV and H5N1virus.To facilitate identification of the nucleotide at the4th position from3’UTRs and comparison of the substitution of this nucleotide at this site, the genome sequences of the influenza A virus containing UTR information were downloaded from the Influenza Resource (IVR) at the National Center for Biotechnology Information (NCBI) on September30,2012. To guarantee the accuracy of these sequences, sequences containing ambiguous nucleotides, duplicate entries uploaded by different individuals, and entries from unknown origins were all eliminated. And sequences derived from the laboratory and vaccine strains, sequences containing minor insertions, deletions, and stop codons and sequences with any artificially adding sequences at the3’UTR were also excluded. Only8332sequences were selected from those obtained above and retained for the following analysis.To analyze substitution of U4and C4according to origins, subtypes, and gene segments, the sequences of the first12nucleotides from3’end UTR of each segment were evaluated. The first12nucleotides from3’end were found to be well conserved across all gene segments. The sequences were3’-UCG U/C UUUCGUCC. There was one single, natural variation at the4th position (either uracil or cytosine, here called U4and C4).The relative frequency of U4in the total influenza A virus or IAV with different origination is much higher than that of C4. As far as subtypes are concerned, the U4was still the most common of all the influenza viruses. Taking gene segments of influenza A virus into account shows there to be more C4in RdRp subunits (about40%) than in other five segments (less than15%).U4was found to outnumber C4in H5N1virus, which aggrees with the substitution of U4/C4in total influenza A virus. However, the substitution of this site in different gene segments of H5N1were different from that for all influenza A virus: in none-RdRp genes, percentage of U4>50%> percentage of C4, but in RdRp genes, percentage of C4>50%> percentage of U4. Taken origination and genomic segments of H5N1into account,There were more H5N1viruses with C4than with U4in PB2and PA among viruses isolated from avian hosts. There were more H5N1viruses with U4than with C4in PB2and PA among viruses isolated from human hosts.In general, U4in total influenza A virus and H5N1subtype is preferred under the presure of nature selection, indicating the important function of this single nucleotide. And the differential substitution of U4/C4in H5N1virus suggested that U4may be better-adapted to none-RdRp genes and C4may be better-adapted to RdRp genes. This kind of adaptation varies in view of hosts or subtypes.2. Association of Substitution of the4th nucleotide at3’end of UTR with viral pathogenicityTo determine the influence of U4/C4variation in PB1of VN1194virus on virulence, the reverse genetics system for H5N1virus and the plaque forming assay for titering recombinant virus were both constructed, and then the mortality and morbidity of these viruese were evaluated.Briefly, the recombinant viruses with all C4in eight gene segments (named as rVN-(C)) and single mutation (U4) in PB1, HA or NA on the basis of rVN-(C), which named as rVN-PB1(U), rVN-HA(U) and rVN-NA(U) were rescued. And Balb/C mice were inoculated with series doses of10,000,1000,100, and10PFU of rVN-(C), rVN-PB1(U), rVN-HA(U) and rVN-NA(U) by nasal dropping. Clinical symptoms, survival time and body weight were recorded daily.Mice infected with doses higher than100PFU of rVN-(C), rVN-PB1(U), rVN-HA(U) and rVN-NA(U) viruses were dead before day10after infection. However, after an intranasal inoculation of10PFU, more mice in the groups of recombinant virus with U4died than in other groups, and the average duration of survival of mice in these groups were less than that of the mice in the group of rVN-(C) group. The LD50of these four viruses reflected these differences:23.7PFU for rVN-(C),13.6PFU for rVN-PB1(U) and less than10PFU for rVN-HA(U) and rVN-NA(U).The infected mice showed significant decreases in body weight before death on days5and6after inoculation (P<0.05) between groups of U4viruses and C4virus. Although infection with1000PFU was too high for mice to endure, differences of body weight were significant between rVN-HA(U) or rVN-NA(U) group and rVN-(C) group (P＜0.05, P＜0.01, P＜0.0001). At an inoculation dose of100PFU, mice infected with U4virus started losing weight on day2and continued losing until they died. The rVN-(C) virus caused delayed and milder decreases in weight from5to8days post-infection. In this way, the weight loss of mice infected with rVN-(C), rVN-PB1(U), rVN-HA(U) and rVN-NA(U) was found to differ significantly (P<0.05).In summary, recombinant viruses with U4were found to be more virulent to mice than that with C4.3. Influence of the4th nucleotide at3’ end of UTR on virus reproductionTo determine the relationship of U4/C4with viral reproduction, the characteristics of growth kinetics of four recombinant viruses were detected. MDCK cells in24-well plates were infected at an MOI of0.01at33℃,37℃, and39℃. The supernatants were harvested at0,12,24, and48h after infection and then titers were determined by a plaque formation assay in MDCK cells. The growth properties of recombinant viruses with U4and C4viruses showed somewhat different patterns for different temperatures. At33℃, both mutant viruses replicated at equal rates and showed similar trends. Both viral titers increased continuously until the detection period ended. However, rVN-PB1(U), rVN-HA(U) and rVN-NA(U) replicated more efficiently than rVN-(C), and their growth curves reached a plateau at24h at37℃, which indicates that U4confers a growth advantage to the virus at37℃. At39℃, during the early stage of viral reproduction, the titers of U4viruses were found to be significantly higher than that of rVN-(C), but the latter virus caught up at24h pi (Fig.3C). Taking together, these data suggests that U4confers a growth advantage than C4to the H5N1virus in cells across a wide range of temperatures.4. Effect of the4th nucleotide at3’end of UTR on viral RNA polymerase activityIn order to determine the effect of the4th nucleotide at3’end of UTR on viral RNA polymerase activity and regulation of viral genomic RNA expression, dual-luciferase reporter system assay for RNA polymerase activity and tag-primed real-time RT-PCR assay for mRNA, cRNA and vRNA were constructed.The reporter plasmid was constructed and named as PB1-LUC(U4), PB1-LUC(C4), HA-LUC(U4), HA-LUC(C4), NA-LUC(U4) and NA-LUC(C4). Then luciferase activity was measured and the significant differences in RdRp activity contributed by PB1(U4) and PB1(C4), or HA(U4) and HA(C4), or NA(U4) and NA(C4) were confirmed:compared with that of PB1(U4)-LUC, the transcription activity of PB1(C4)-LUC was40.60%+3.53%(33℃),36.91%±2.43%(37℃) and29.70±3.52%(39℃). Similar statistically significant results were found for HA-LUC,52.91±2.52%(33℃),54.86±5.66%(37℃) and41.21±1.73%(39℃) and for NA-LUC,40.60±3.53%(33℃),36.91±2.43%(37℃) and29.70±3.52%(39℃).Tag-primed real-time RT-PCR assay was established to quantifying mRNA, cRNA and vRNA precisely and separately during MDCK cells infection with rVN-(C), rVN-PB1(U), rVN-HA(U) and rVN-NA(U) viruses at an MOI of0.01at33℃,37℃and39℃. At three different temperatures, the mRNA, cRNA and vRNA copies of both viruses varied time-dependently. More PB1mRNA was produced by rVN-PB1(U) than rVN-PB1(C), while less cRNA and vRNA of PB1were synthesized by rVN-PB1(U) than rVN-PB1(C). And the enhancement or inhibiton was temperature-independent.Taken together, present of U4promotes greater transcription activity of RdRp across a range of temperatures. These results indicate that the variation of U4and C4could differentially regulate viral transcription and replication in the H5N1background. Moreover, the similar patterns can also be found at lower temperature (33℃) and higher temperature (39℃), which imply that acquisition of U4from C4could up-regulate transcription and down-regulate replication.In summary, this research foucsed on the differential substitutions of the4th nucleotide at3’end of UTR, indicating the important function of this site under pressure of evolution and the possible association of U4/C4with species barriers of H5N1virus. The U4/C4polymorphism at the3’end of UTRs in PB1, HA or NA segment of H5N1influenza virus promotes changes in RdRp activity, regulates RNA expression level differentially, contributes on viral proliferation and pathogenicity.