The Role Of Host MiRNAs In Inhibiting The Replication Of Influenza A Virus

Influenza A virus is a kind of single negative-stranded RNA virus which belongsto the Orthomyxoviridae family. It can cause localized outbreak or worldwideepidemic in a short time for its great contagiosity, fast spread speed and a wide rangeof host.Since the19thcentury, there have been four global flu pandemia,"Spanish fluH1N1―pandemia (1918to1920), was the most serious and resulted in nearly50million people died which brought great harm to human health and the globaleconomy. At present, antiviral drugs about ion channel blockers or neuraminidaseinhibitors are used to prevent and treat influenza A virus infection. But these drugshave some side effects for the human nervous system, and because of the fastmutation rate of influenza A virus, it’s easy to produce drug-resistant strains with theextensive use of drugs. Therefore, research about the influenza virus drugs andantiviral target is extremely urgent.MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules with alength of about22nt, which are widespread in eukaryotic genomes. Gene location andsequences of miRNAs are very conservative. It functions in post-transcriptionalregulation of gene expression through complementary binding to the target mRNAs,and it’s becoming evident thar miRNAs are playing significant roles in many cellularprocesses, including cell proliferation, differentiation, apoptosis and tumor formation.Currently, studies have shown that host miRNAs can affect viral replication, infectionand pathogenic process through regulating host or viral gene expression during theinfection of hepatitis B virus, hepatitis C virus, human immunodeficiency virus orsome other viruses. In addition, because of the unique function pathway and soundeffects of miRNAs, they have become the new hot spot for gene medicine research ofthe treatment of viral diseases.The genome of influenza A virus consist of eight segments, the first threesegments encoding the polymerase proteins PB2, PB1and PA, which togetherly formthe RNA-dependent RNA polymerase protein complexes. The fifth segment encodingnucleoprotein NP which wrapped and protected the nucleic acid of virus. NP, viralRNA and three polymerase proteins togetherly form ribonucleoprotein complexes (RNPs) during the replication process of the influenza virus, which not only involvedin the transcription and translation of the viral genes, but also plays an importantfunction in the nuclear export of viral RNA and protein and in the viral aggregationprocess.In addition, there are a lot of conservative sites in this four gene segments. Soit’s essential to look for new drug targets within the four genes and develop stableantiviral drugs. Our study is committed to looking for host miRNAs thatbroad-spectrum regulating the replication of influenza A virus, whose targets are thepolymerase genes or the nucleoprotein gene. Our study not only provide a deeperunderstanding of host antiviral defense mechanisms, but also lay the foundation forlooking for new broad-spectrum antiviral gene drugs and target sites usingbioinformatics methods.In this study, we first used bioinformatics methods to predict and screenbroad-spectrum host miRNAs that targeting replication related genes of influenza Avirus. We designed broad-spectrum miRNA prediction screening process with thebase of miRNA target prediction software miRanda. Then we writed Perl code toextract useful information from the miRanda software output. By setting strictparameters and formulating detailed screening principle, the screened miRNA hadgood advantages of both broad-spectrum and high effectiveness of inhibiting viralreplication. For the PB2gene, we selected miR-188-3p and miR-345; for the PB1gene, we selected miR-3183; for the PA gene, we selected miR-15a*and miR-99b*;for the NP gene, we selected miR-769-3p; their broad-spectrum targeting rates ofinfluenza A virus were81.3%,95%,78.7%,83.1%,64.4%and95.3%, respectively.We then checked if the seasonal flu virus A/FM/1/47(H1N1) strain wasapplicable to miRNA functional verification. We first amplified the PB1, PB2, PA andNP gene and cloned them into T vector, which facilitated taking of gene in the future.By sequencing, we obtained sequences of the four genes. Prediction results ofmiRanda software showed that the screened6miRNA could target this strain withgood base pairs match, and the pattern of base pair match was representative whichmeans that A/FM/1/47(H1N1) was suitable for miRNA functional verification.We adopted a series of experimental methods to verify the validity of themiRNAs gradually.1. First, we used the dual luciferase reporter gene system for initial screening, theinhibition rate of relative luciferase value was used to determine the binding capacitybetween miRNA and potential targets. Our results showed all six miRNAs screened with bioinformatics methods have certain binding capacity with potential targets, butthe binding capacity of miR-15a*and miR-99b*targeting the PA gene was weak,relative luciferase inhibition rate was only28.82%and20.75%, respectively and theywould not continue to make follow-up experiments. Our preliminary view was thatthe remaining four miRNA had relatively strong binding capacity: miR-188-3p andmiR-345could bind to the PB2gene, miR-3183could bind to the PB1gene, theirrelative luciferase inhibition rate was about45%. miR-769-3p could bind to the NPgene with the relative luciferase inhibition rate of36.5%.2. We further validated the effects of miRNAs on the expression of influenza Avirus protein. We constructed eukaryotic expression vectors of the PB1, PB2, PA andNP proteins and they were co-transfected with miRNAs into HEK293T cells. Westernblot showed that four miRNAs could significantly inhibit the expression ofcorresponding viral protein. The inhibitory effect of miR-769-3p on the NP proteinexpression was the most significant. The inhibitory effect of miR-188-3p andmiR-345on PB2protein expression and miR-3183on PB1protein expression werecomparable to the inhibitory effect of positive control miRNA (miR-491) on the targetprotein expression.3. Finally, we observed that the inhibitory effect of miRNA on the replication ofinfluenza A virus. We first verified that A/FM/1/47(H1N1) strain could steadyproliferate and passage in MDCK cells and A549cell lines and studied the stepgrowth curve to determine the best experimental inoculation dose. We tested theinhibitory effect of miRNAs on viral replication Subsequent. Results showed thatmiR-188-3p could significantly inhibit the replication of influenza A viruses in A549cells. Compared with the control group, virulence reduced0.8TCID50titer48hourspost infection. miR-345effect Secondly, virulence reduced0.51TCID50titer48hourspost infection. They were both better than the positive control miR-491, whichreduced0.43TCID50titer48hours post infection.Based on the above studies, we screened an miRNA called miR-188-3p whichcould broad-spectrumly target PB2gene of influenza A virus, inhibit expression ofPB2protein, thereby inhibit the replication of influenza A virus through bioinformaticprediction and a series of experiments. Moreover, we found that miR-188-3p couldalso target H7N9avian influenza virus PB2gene which outbreaked recently, and wassuperior to its ability to bind to A/FM/1/47(H1N1). Our study provide a deeperunderstanding of host defense mechanisms of anti-influenza virus, propose a broad-spectrum miRNA screening method, and may lay a theoretical foundation fordeveloping miRNA gene medicine and new antiviral drug targets with the use ofbioinformatics methods.