| Influenza A virus (IAV) is a popular pathogen causing respiratory disease in both animal and human.Outbreaks of influenza have resulted in huge economic loss in global husbandry industries and posed serious threaten to public health and safety. The control of IAV replication and spreading in livestock is important for animal industry and human health. With the rapid development of transgenic technology,genetic modification methods have been applied to improve livestock's disease resistance to IAV, and such applications in animal breeding may provide an efficient way to control outbreaks of influenza in husbandry industries. The untranslated regions (UTRs) at the 5' and 3' termini of IAV genomic segments are conserved, particularly the first 13 nucleotides at the 5' UTR are identical among all eight gene segments, while the first 12 nucleotides at the 3' UTR exhibit variation at only one position. The conserved UTRs contain necessary and sufficient cis-acting elements for RNA replication and transcription of IAV, which can be recognized by the viral polymerase, and play an important role in transcription, replication and packaging of the viral genome. Here, we developed an Influenza A Virus RNA Polymerase Decoy System (IVPDS), which could transcribe RNA flanked by the 5' and 3' UTR of the NP segment respectively. This RNA can serve as a competitor of viral RNA to suppress virus replication through combing with viral polymerase. Moreover, RNA polymerase of IAV could recognize the 3' and 5' UTR of vRNA-like sequence to drive the exogenous gene transcription, and the exogenous protein would activate the immune response of host, increasing the host resisitance to IAV.In this study, we constructed IVPDS plasmids containing reporter gene GFP under control of RNA polymerase I promoter, named pIVPDS-I-G. 293T and DF-1 cells transfected with pIVPDS-I-G or integrated with pIVPDS-I-G-Neo (referred to IVPDS-G cells) could transcribe vRNA-GFP(-), and expression of mRNA-GFP(+) and GFP protein was detected when cells were transfected with plasmids expressing polymerase and NP. The similar results were obtained in cells infected with IAV. However,compared to cells induced by plasmids expressing viral polymerase and NP, expression of mRNA(+)and GFP protein was significantly lower in cells induced by IAV. In DF-1 cells tansfected with pIVPDS-I-G, mRNA levels of type ? IFN and IFN-induced genes were up-regulated when cells were co-transfected with pIVPDS-I-C-G and polymerase and NP expressing plasmids, which indicated that dsRNA from pIVPDS-I-G might induce a type ? IFN response. After infected with IAV, expression of the IAV Matrix genes in vRNA-GFP(-)-transcribing cells was significantly lower than that in control cells, but there was no significant difference in the virus titers of cell culture between two groups.We also constructed IVPDS vectors containing human MxA or mouse Mx1 (referred to Mx collectively), named pIVPDS-I-Mx-Neo. Similarly, 293T or DF-1 cells transfected with pIVPDS-I-Mx-Neo could transcribe vRNA-Mx(-), and mRNA-Mx(+) and Mx protein were induced by co-transfection with plasmids expressing viral polymerase and NP. In infected DF-1, viral titers in supernatants of cells transfected with pIVPDS-I-C-MxA-Neo was significantly lower than that of control cells transfected with empty-vector at 24 and 36 hpi. However, there was no significant difference in the viral titers of cell culture between pIVPDS-I-Mxl-Neo-transfected cells and empty-vector-transfected cells.In summary, we developed the IVPDS system. Transcripts derived from vector of IVPDS, vRNA-like sequences, can be recognized by Influenza virus polymerase and NP and expressed exogenous proteins,achieving the goal of virus-induced expression of antiviral genes. In particular, cells transfected with pIVPDS-I-MxA-Neo were resistant to IAV. These results shed light on application of IVPDS to inhibit the replication of IAV in the future and provide an innovative strategy for genetically modified breeding of disease resistant animal. |
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