Studies On The Biochemical And Functional Interaction Between NS1 Of AIV And PARP10 Of Host Cell

The NS1 protein of avian influenza virus is not a structural component of the virion, so called non-structural protein, and extensively expressed in infected cells. The expressed NS1 is mainly localized in the nucleus. The NS1 protein of avian influenza virus can block the export of host mRNA from the nucleus. It can also cut the poly(A) sequences of host mRNAs which were then used as primers of viral RNA synthesis, promoting the viral RNA replication and protein expression. By binding to dsRNA, NS1 also can inhibit the activation of PKR and Jun/AP-1, blocking IFN response, and then antagonizing the host antiviral defense of IFN response. All these may contribute to efficient virus replication and virulence during infection.For further studying the function of NS1 correlated to H5N1 subtype avian influenza virus replication and pathogenesis, the yeast hybrid experiments were performed with NS1 as the bait protein to screen its interactor. Eight positive clones were identified, and one of them was homo sapiens PARP10. The specific interaction between PARP10 and NS1 was confirmed by yeast hybrid experiments again and the NS1 of H3N2 subtype avian influenza virus interacting with PARP10 was also identified by the same experiments.PARP10 is a new member of the protein family of poly(ADP-ribose) polymerases, possessing catalytic activity of poly(ADP-ribosyl)ation. It can transfer multiple ADP-ribose units from NAD+ to substrate proteins, resulting a linear and multibranched polymer, and conferring the substrate protein with massive negative charges. The interaction between PARP10 and c-myc can affect the cell propagation. Researches showed that overexpression and down-expression of PARP10 both affected the cell viability. The PARP10 can also modify core histones, with potential functions of chromatin damage repair and duplication. After phosphorylation by CDK in late G1 phase, PARP10 can interact with RNA polymerase I, and so regulate construction and reconstitution of the rDNA and transcription of the rRNA.To study the interaction between PARP10 and NS1 and the effect of PARP10 on the replication of H5N1 avian influenza virus, total RNA were extracted from cell lines A549 and used as template for obtaining parp10 cDNA. Two fragments of parp10 were obtained by RT-PCR with the parp10 specific primers, and the full length cDNA of parp10 gene was amplified through overlap extension. The sequence containing 3078 base pairs was confirmed by matching with GenBank database.Second, murine single strand cDNA library of different tissues was prepared by extraction of total RNA from tissues and RT-PCR amplification with universal oligo(dT) primer. The expression of PARP10 in tissues was assayed by semi-quantitative PCR with parp10 specific primers. The expression profile showed that PARP10 protein was expressed in brain, lung, kidney, muscle and testis, and lower-expressed in heart, liver and spleen tissues.The biological functions of PARP10 were also tentatively investigated in this study. The A549 and 293FT cells were transfected with pCMV-3myc/PARP10 plasmid for transient expression of Myc-PARP10 fusion protein. Theβ-actin protein was identified as the interacting protein of PARP10 by the co-immunoprecipitation from the cell lysate, suggesting that PARP10 protein could interact withβ-actin in cells. Moreover, the expression of PARP10 could be increased by UV irradiation following treatment with 0.5 or 1.0 J UV, suggesting PARP10 may involved in irritated responsiveness of DNA damage and indicated a positive correlation with radiation dose, similar to other PARP family members. In addition, PARP10 harbors a RNA-interacting motif and a nuclear exporting signal (NES) indicating a potential function of RNA binding and transporting. Enrichment of mRNA was showed up in the nucleus of A549 cells when ectopic expression of GFP-PARP10 and CY-3 tagged oligo (dT) in situ hybrization were performed, which indicated that PARP10 could also regulate mRNA transporting process in a piggyback manner.Third, GST-NS1 fusion protein was successfully expressed in E.coli BL21, and soluble form of GST-NS1 fusion protein was harvested and purified with GST affinity chromatography. Myc-PARP10 fusion protein was expressed in cell line A549. GST-pulldown experiments showed that NS1 protein could precipitate PARP10 in vitro. Myc-PARP10 and Flag-NS1 were co-transfected and expressed in cell line A549. Co-immunoprecipitation experiments with anti-Myc and anti-Flag antibody respectively showed that PARP10 and NS1 could precipitate each other, demonstrating existence of in vivo interaction between these two proteins. Expression of fragments of PARP10 and immunoprecipitation showed that NS1-interacting sites of PARP10 lied in its C-terminus, namely PAR catalytic domain and glutamate-rich region. Co-expression of GFP-PARP10 and RFP-NS1 in cell line A549 and DAPI staining of cell nucleus showed that they were co-localized in cell nucleus. Fouth, three specific RNAi target sites of parp10 were designed using siRNA Target Finder provided by Applied Biosystems website and two effective RNAi target sites were selected by dual-luciferase reporter assay system. RNAi efficiency of one target was up to 80%. Western blotting analysis showed that the siRNA could interfere with the exogenous and endogenous PARP10 expression efficiently.The biological functions of PARP10 and NS1 were studied. Overexpression of NS1 protein in host cells could inhibit PARP10 expression on the level of transcription and translation. MTT assays showed that overexpression of NS1 protein or down-expression of PARP10 by RNAi could inhibit the cell proliferation. Moreover, NS1 could inhibit the cell proliferation markedly when the expression of PARP10 protein was interfered by RNAi. Meanwhile, the cell cycle of A549 cells was blocked to G2-M phase with NS1 overexpression and PARP10 down-expression, And the cell cycle get recovery when the expression of PARP10 was increased.Based on the physiological association of PARP10 and NS1, we further analyzed their effect on the replication of H5N1 avian influenza virus in BHK21. Cells and supernatants collected from the cells infected with the virus at different time were used to detect the virus with anti-M1 antibody. The amount of virus in the supernatants reached the maximum at 48h. In addition, the amount of virus reduced when PARP10 was overexpressed in cells, but increased when the expression of PARP10 was suppressed. TCID50 analysis confirmed that overexpression of PARP10 could inhibit the production of virus in BHK21 cells.The interaction between NS1 and PARP10 was confirmed in the mammalian cells in this project. As little biological function of PARP10 was known at present, some experiments to discover the biological fuction of PARP10 was performed. The results indicated that PARP10 could interact withβ-actin in the cytoplasm, and cell injury caused by ultraviolet ray could induce PARP10 stress reaction. Overexpression of PARP10 in A549 cells could make the ploy(A) RNA enrich in the nucleus. At the same time, the biological meanings of this interaction were also studied. In these researches, we discovered that overexpresson of NS1 protein in host cells could inhibit PARP10 expression. When the expression of PARP10 was down-regulated in mammalian cells, cell proliferation was inhibited and cell cycle was blocked to G2-M phase. In host cells, H5N1 subtype avian influenza virus replication was inhibited as PARP10 overexpression.In a word, our studies revealed that avian influenza virus facilitated the replication of itself through down-regulating PARP10 by NS1 overexpression. These works provided us a good foundation for further studying the biological function of NS1 interacting with the host cells and the replication of H5N1 subtype avian influenza virus in the host cells. Antiviral drugs will be an important initial defence against rapidly emerging novel strains of influenza A virus. Given the numerous roles of NS1 during virus replication, one potential target for anti-influenza drug design may be to disrupt interactions of NS1 with PARP10. This may be particularly relevant for prophylaxis and treatment in the event of an emerging influenza A virus outbreak.