Exploratory Studies On Prevention And Treatment Of Influenza Virus Infection

RNA interference (RNAi) is a process by which double-stranded RNA directssequence-specific degradation of homologous messenger RNA. Small interferingRNA(siRNA),short 21–25nt, is a powerful tool for sequence-specific,post-transcriptional gene silencing. RNAi can shut down or regulates gene expressionin mammalian cells and confers as a cellular defense mechanism against geneinvaders such as transposons and viruses. Many studies had shown that siRNA couldsignificantly suppress gene expression when delivered into mammalian cells invitro.Subsequently , a number of studies demonstrated that siRNA inhibited viral geneexpression and replication of RNA viruses in vitro , including HIV-1 virus ,hepatitisB virus , hepatitis C virus ,dengue fever poliovirus , foot- and mouth diseasevirus ,and serious acute respiration syndrome virus ,respiratory syncytial virus etc.Influenza A viruses belong to the Orthomyxoviridae family and are furtherdivided into subtypes according to their hemagglutinin(HA) and neuraminidase(NA)surface glycoproteins. Influenza virus infection has the potential to become a muchmore dangerous disease than it is at present because of easy transmission , antigenicshift and drift of the virus, and the limited efficacy of current vaccines andtherapy.Influenza virus genome consists of eight segmented RNAs encoding a total of11 proteins. Each protein is either an integral component of the viral structure orplays critical roles during the virus life cycle. Interfering with the production of anyone of them is likely to have severe consequence on viral replication.Firstly,several siRNAs specific for conserved regions of influenza virus matrixgene, were designed and inserted into pSilencer2.1-U6-hygro expression plasmid. Toprovide a reporting system for monitoring the effects of the siRNA, an eukaryoticexpression plasmids pGL-c-m , was constructed, in which m gene were fused to the 5'end of the luciferase coding sequence, and cotransfected with their specific siRNAexpression plasmids. So the inhibitory effects of the m-specific siRNAs on theexpression could be indirectly evaluated by the expression of luciferase in thetransfected cells. The results showed that the m-specific siRNAs M-25, M1-105, andM-306 could effectively down-regulate the expression of luciferase.To further evaluate for their abilities to inhibit influenza virus replication,MDCK cells were transfected with the siRNA expression plasmid, selected for stablyexpressing the siRNA with hygromycin and infected with influenza virus . Thesuppression effects were identified by Real-time PCR and Western bolt .All threesiRNAs have significant suppression effects on the level of M-mRNA. The inhibitionrate of M1-306 is about 60%. Western blot showed a reduction of NA proteinexpression. M2-25 and M1-105 have more significant suppression effects than M1-306. In conclusion, the constructed M-siRNAs could effectively inhibit thereplication of influenza virus .Secondly, as we known, chemically synthesized siRNAs represent the goldstandard for RNAi applications. They are of a uniform composition and can besynthesized at higher amounts and with a wider range of chemical modification thanby other methods.We ordered several chemically synthesized siRNAs which havebeen reported by others or assayed by us. We examined the effect of siRNA on theaccumulation of the corresponding mRNA. To follow transcription in a cohort ofsimultaneously infected cells, siRNA transfected MDCK cells were harvested for RNAisolation 24 h after infection . The viral mRNA was converted to cDNA by usingspecific primers . The level of each cDNA was then quantified by Real-time PCR. Incells transfected with M1-105, PA-2065 or NP-1453, the level of M- & NS- specificmRNA were reduced by 40-50%. In contrast, the levels of NS- & M-specific mRNAwere significantly reduced (70%) by the presence of Mixed (M1-105,PA-2065,NP-1453,PA496). The results shows that"cocktail therapy"have more significantsuppression effects.Thirdly, all siRNA target sites have suppression effects, but lower todesired .These correlations have been in-corporated into algorithms that are commonlyused to predict functional siRNAs. Despite their success at finding good siRNAs,manyeffective siRNA sequences are not predicted by current algorithms. An attractivealternative is to screen genome-based libraries of sequences for the most potentsiRNAs,without any bias for or against sequence features except for their presencewithin the target.So, we have done some research on siRNA libraries. To facilitatehigh-throughput screening ,we developed a selection scheme that used a selectivemarker gene for positive selection of efficient siRNA constructs in cells . The markergene is fusion gene of thymidine kinase and puromycin N-acetyltransferase. And then,we constructed a dual-promoter siRNA expression vector. dsRNA representing thegene of interest is digested by RNaseIII to produce 20–22bp siRNAs,which aresubsequently converted into corresponding DNA fragments through ligation ofadapters and reverse transcription-PCR (RT-PCR) . The resulting PCR products arecloned into the siRNA expression vector between opposing human U6 and H1promoters.In one word, our findings about the effectiveness of designed siRNAs ,provided a basis for further development of siRNA for prophylaxis and therapy ofinfluenza virus infection in human and animals. And the initial exploration ofRNaseIII-generated library is a promise for screening the most potent siRNAs.