H5N1亚型禽流感病毒NS1蛋白与宿主蛋白PARP10相互作用研究
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摘要
禽流感病毒的NS1蛋白是病毒感染宿主细胞后大量表达的蛋白,表达后主要分布在细胞核内,在成熟的病毒内并不含有此蛋白,因此NS1被称为非结构蛋白。禽流感病毒的非结构蛋白NS1能够阻止宿主细胞内mRNA的核输出,同时将宿主mRNA多聚腺苷酸帽子切下作为合成病毒RNA的引物,促进自身RNA的复制和蛋白的表达。另外,NS1通过和dsRNA相结合,抑制PKR、Jun (Jun N-terminal Kinase)/AP-1的激活,阻止干扰素的生成,进而拮抗干扰素所构成的病毒防线,这些对病毒感染和复制是有很大帮助的。
为了进一步研究NS1蛋白在禽流感病毒复制及致病过程中的作用,我们采用酵母双杂交技术以H5N1型禽流感病毒NS1蛋白为诱饵蛋白寻找更多的与NS1相互作用的蛋白质。结果我们获得了一系列克隆。其中一个克隆的插入片段来自parp10 cDNA。我们进一步在酵母细胞中通过共转化实验证明了这种相互作用的特异性,同时也验证了H3N2型禽流感病毒的NS1蛋白与其的作用。
PARP10是多聚腺苷酸二磷酸核糖基聚合酶(PARP)家族中一个新成员,拥有多聚腺苷酸二磷酸核糖基(PAR)催化活性,以NAD+为底物,将ADP核糖单体转移到底物蛋白上,形成直线状和多枝状的不均一聚合物,使被修饰蛋白带有大量负电荷。PARP10能够和c-myc相互作用影响细胞的增殖,有研究表明,该蛋白的表达水平过高或过低都会影响细胞的活性。PARP10还能够对核心组蛋白进行修饰,在调控染色质损伤修复及复制方面具有潜在的功能。PARP10在G1晚期被CDK磷酸化后和RNA聚合酶Ⅰ相互作用,调节rDNA的结构和重建,影响rRNA的转录。
为了探究PARP10和NS1之间的相互作用以及PARP10对病毒复制的影响,我们首先提取A549细胞的总RNA,以其为模板通过RT-PCR获得了parp10 cDNA的两个片段。进一步通过重叠延伸PCR获得了parp10全长cDNA,总长度为3078bp,与GenBank公布的序列一致。
其次,通过半定量PCR检测了PARP10在小鼠各组织中的转录水平,发现该蛋白在心、脑、肺、肾、肝、脾、肌肉和睾丸内都有表达,属于广泛性表达的蛋白,丰度较低,在心、肝和脾中的表达水平相对其它组织更低。
在A549和293FT细胞内瞬时表达Myc-PARP10蛋白,将裂解液用Myc抗体进行免疫沉淀,发现β-actin被沉淀,利用β-actin抗体进行反向沉淀,在沉淀产物内检测到PARP10蛋白,说明瞬时表达的PARP10与β-actin共沉淀。鉴于PARP家族对UV和烷化剂造成的细胞损伤有应激反应,为此我们检测了PARP10对UV的反应,当用0.5J和1.0J的照射量进行细胞损伤后,通过抗PARP10的抗体检测到细胞内的PARP10表达量升高,而且PARP10的表达量与紫外照射的剂量呈正相关。PARP10含有RNA结合序列和核输出信号,可能参与mRNA的转运,我们在A549细胞内瞬时表达GFP-PARP10,利用CY-3标记的oligo(dT)探针进行原位杂交,发现在表达PARP10的细胞内,poly(A)RNA出现核聚集。
第三,在大肠杆菌中表达了GST-NS1融合蛋白,用纯化的GST-NS1蛋白对瞬时表达PARP10的A549细胞裂解液进行pull-down,发现在细胞外NS1蛋白能够将PARP10蛋白沉淀下来。将Myc-PARP10和Flag-NS1蛋白共转染到A549细胞内进行表达,将裂解产物分别用Myc和Flag抗体进行免疫沉淀,发现PARP10和NS1能够将彼此沉淀,说明瞬时表达的PARP10和NS1在细胞内存在相互作用;通过对PARP10进行分段表达并进行免疫共沉淀实验,发现NS1作用于PARP10 C端的PAR催化结构域和谷氨酸富集区。在A549细胞内共表达GFP-NS1和RFP-PARP10,发现瞬时表达的PARP10和NS1共定位在细胞核内。
第四,通过Applied Biosystems网站提供的siRNA Target Finder设计软件设计了3个parp10特异性RNA干扰片段,利用荧光蛋白酶报告基因筛选到两个有效干涉片段,其中起始于parp10 cDNA第617位核苷酸的片段抑制效率达80%以上,该片段能有效抑制瞬时表达的和内源的PARP10。
在瞬时表达NS1蛋白的细胞内检测PARP10的蛋白水平和mRNA水平,发现NS1的过表达能够降低宿主细胞内PARP10的表达。MTT染色实验发现,瞬时表达的NS1和抑制PARP10的表达都能降低细胞活性,在PARP10表达被抑制时,瞬时表达NS1对细胞活性的影响更加明显。通过流式细胞检测发现在PARP10表达抑制的情况下,NS1能够改变A549的细胞周期,引起细胞在G2到M期的停滞,当增加PARP10蛋白表达量时,G2到M期的细胞含量又回复到正常水平。
我们进一步研究了PARP10对H5N1亚型禽流感病毒在BHK21细胞中的复制的影响。用病毒感染瞬时表达PARP10的细胞、PARP10表达被抑制的细胞,用抗M1抗体进行检测发现,PARP10高表达时M1蛋白的量降低,而表达抑制时,M1蛋白的量增加;测定半数细胞培养物感染量(TCID50)发现PARP10高表达时病毒的量降低,而表达抑制时,病毒的量增加,与用M1抗体检测的结果是一致的,即PARP10抑制禽流感病毒在细胞中的复制。
本研究首次证明了NS1蛋白与PARP10蛋白在哺乳动物细胞中的相互作用,初步探索了PARP10的功能,发现PARP10与β-actin有相互作用;对紫外照射产生应激反应;瞬时表达的PARP10引起poly(A)RNA的核聚集。同时探讨了NS1与PARP10相互作用的生理功能,发现瞬时表达的NS1能够降低宿主细胞内PARP10的表达;PARP10表达抑制时,NS1能够降低细胞的增殖活性,改变A549的细胞周期,引起该细胞在G2到M期的停滞;PARP10对禽流感病毒在宿主细胞内的复制具有抑制作用。
总之,我们的研究揭示了禽流感病毒可能通过其非结构蛋白NS1对PARP10蛋白的抑制而促进自身的增殖。我们的工作为了解NS1蛋白的功能提供了更多的实验数据,对进一步了解禽流感病毒在细胞内的复制提供了帮助。抗病毒药物对新流感病毒的爆发将会有很好的防御作用,基于NS1蛋白能够和宿主蛋白PARP10相互作用,在病毒复制过程中担当的多种角色,将其作为抗病毒药物设计的靶位,将会扰乱NS1和细胞及病毒蛋白的相互作用,为病毒的防治提供保障手段。
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.
为了进一步研究NS1蛋白在禽流感病毒复制及致病过程中的作用,我们采用酵母双杂交技术以H5N1型禽流感病毒NS1蛋白为诱饵蛋白寻找更多的与NS1相互作用的蛋白质。结果我们获得了一系列克隆。其中一个克隆的插入片段来自parp10 cDNA。我们进一步在酵母细胞中通过共转化实验证明了这种相互作用的特异性,同时也验证了H3N2型禽流感病毒的NS1蛋白与其的作用。
PARP10是多聚腺苷酸二磷酸核糖基聚合酶(PARP)家族中一个新成员,拥有多聚腺苷酸二磷酸核糖基(PAR)催化活性,以NAD+为底物,将ADP核糖单体转移到底物蛋白上,形成直线状和多枝状的不均一聚合物,使被修饰蛋白带有大量负电荷。PARP10能够和c-myc相互作用影响细胞的增殖,有研究表明,该蛋白的表达水平过高或过低都会影响细胞的活性。PARP10还能够对核心组蛋白进行修饰,在调控染色质损伤修复及复制方面具有潜在的功能。PARP10在G1晚期被CDK磷酸化后和RNA聚合酶Ⅰ相互作用,调节rDNA的结构和重建,影响rRNA的转录。
为了探究PARP10和NS1之间的相互作用以及PARP10对病毒复制的影响,我们首先提取A549细胞的总RNA,以其为模板通过RT-PCR获得了parp10 cDNA的两个片段。进一步通过重叠延伸PCR获得了parp10全长cDNA,总长度为3078bp,与GenBank公布的序列一致。
其次,通过半定量PCR检测了PARP10在小鼠各组织中的转录水平,发现该蛋白在心、脑、肺、肾、肝、脾、肌肉和睾丸内都有表达,属于广泛性表达的蛋白,丰度较低,在心、肝和脾中的表达水平相对其它组织更低。
在A549和293FT细胞内瞬时表达Myc-PARP10蛋白,将裂解液用Myc抗体进行免疫沉淀,发现β-actin被沉淀,利用β-actin抗体进行反向沉淀,在沉淀产物内检测到PARP10蛋白,说明瞬时表达的PARP10与β-actin共沉淀。鉴于PARP家族对UV和烷化剂造成的细胞损伤有应激反应,为此我们检测了PARP10对UV的反应,当用0.5J和1.0J的照射量进行细胞损伤后,通过抗PARP10的抗体检测到细胞内的PARP10表达量升高,而且PARP10的表达量与紫外照射的剂量呈正相关。PARP10含有RNA结合序列和核输出信号,可能参与mRNA的转运,我们在A549细胞内瞬时表达GFP-PARP10,利用CY-3标记的oligo(dT)探针进行原位杂交,发现在表达PARP10的细胞内,poly(A)RNA出现核聚集。
第三,在大肠杆菌中表达了GST-NS1融合蛋白,用纯化的GST-NS1蛋白对瞬时表达PARP10的A549细胞裂解液进行pull-down,发现在细胞外NS1蛋白能够将PARP10蛋白沉淀下来。将Myc-PARP10和Flag-NS1蛋白共转染到A549细胞内进行表达,将裂解产物分别用Myc和Flag抗体进行免疫沉淀,发现PARP10和NS1能够将彼此沉淀,说明瞬时表达的PARP10和NS1在细胞内存在相互作用;通过对PARP10进行分段表达并进行免疫共沉淀实验,发现NS1作用于PARP10 C端的PAR催化结构域和谷氨酸富集区。在A549细胞内共表达GFP-NS1和RFP-PARP10,发现瞬时表达的PARP10和NS1共定位在细胞核内。
第四,通过Applied Biosystems网站提供的siRNA Target Finder设计软件设计了3个parp10特异性RNA干扰片段,利用荧光蛋白酶报告基因筛选到两个有效干涉片段,其中起始于parp10 cDNA第617位核苷酸的片段抑制效率达80%以上,该片段能有效抑制瞬时表达的和内源的PARP10。
在瞬时表达NS1蛋白的细胞内检测PARP10的蛋白水平和mRNA水平,发现NS1的过表达能够降低宿主细胞内PARP10的表达。MTT染色实验发现,瞬时表达的NS1和抑制PARP10的表达都能降低细胞活性,在PARP10表达被抑制时,瞬时表达NS1对细胞活性的影响更加明显。通过流式细胞检测发现在PARP10表达抑制的情况下,NS1能够改变A549的细胞周期,引起细胞在G2到M期的停滞,当增加PARP10蛋白表达量时,G2到M期的细胞含量又回复到正常水平。
我们进一步研究了PARP10对H5N1亚型禽流感病毒在BHK21细胞中的复制的影响。用病毒感染瞬时表达PARP10的细胞、PARP10表达被抑制的细胞,用抗M1抗体进行检测发现,PARP10高表达时M1蛋白的量降低,而表达抑制时,M1蛋白的量增加;测定半数细胞培养物感染量(TCID50)发现PARP10高表达时病毒的量降低,而表达抑制时,病毒的量增加,与用M1抗体检测的结果是一致的,即PARP10抑制禽流感病毒在细胞中的复制。
本研究首次证明了NS1蛋白与PARP10蛋白在哺乳动物细胞中的相互作用,初步探索了PARP10的功能,发现PARP10与β-actin有相互作用;对紫外照射产生应激反应;瞬时表达的PARP10引起poly(A)RNA的核聚集。同时探讨了NS1与PARP10相互作用的生理功能,发现瞬时表达的NS1能够降低宿主细胞内PARP10的表达;PARP10表达抑制时,NS1能够降低细胞的增殖活性,改变A549的细胞周期,引起该细胞在G2到M期的停滞;PARP10对禽流感病毒在宿主细胞内的复制具有抑制作用。
总之,我们的研究揭示了禽流感病毒可能通过其非结构蛋白NS1对PARP10蛋白的抑制而促进自身的增殖。我们的工作为了解NS1蛋白的功能提供了更多的实验数据,对进一步了解禽流感病毒在细胞内的复制提供了帮助。抗病毒药物对新流感病毒的爆发将会有很好的防御作用,基于NS1蛋白能够和宿主蛋白PARP10相互作用,在病毒复制过程中担当的多种角色,将其作为抗病毒药物设计的靶位,将会扰乱NS1和细胞及病毒蛋白的相互作用,为病毒的防治提供保障手段。
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.
引文
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[30] Mok C K P, Lee D C W, Cheung C Y, et al. Differential onset of apoptosis in influenza A virus H5N1- and H1N1-infected human blood Macrophages [J]. J Gen Virol, 2007, 88: 1275-1280
[31] Mattiussi1 S, Tempera1 I, Matusali G. Inhibition of Poly(ADP-ribose)polymerase impairs Epstein Barr Virus lytic cycle progression [J]. Infect Agent Cancer, 2007, 2(18): 1-9
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[33] Beneke S, Burkle A. Poly(ADP-ribosyl)ation in mammalian ageing [J]. Nucleic Acids Res, 2007, 35(22): 7456-7465
[34] Piskunova T S, Yurova M N, Ovsyannikov A I, et al. Deficiency in Poly(ADP-ribose) Polymerase-1 (PARP-1) Accelerates Aging and Spontaneous Carcinogenesis in Mice [J]. Curr Gerontol Geriatr Res, 2008, 2008: 1-10
[35] Ratnam K, Low J. Current Development of Clinical Inhibitors of Poly(ADP-Ribose) Polymerase in Oncology [J]. Clin Cancer Res, 2007, 13(5):1383-1388
[36] Kanai M, Tong W M, Sugihara E, et al. Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function [J]. Mol. Cell. Biol, 2003, 23(7): 2451-2462
[37] Cepeda V, Fuertes M A, Castilla J, et al. Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors in Cancer Chemotherapy [J]. Recent Patents on Anti-Cancer Drug Discovery, 2006, 1(1):39-53
[38] Albert J M, Cao C, Kim K W, et al. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Cell Death and Improves Tumor Growth Delay in Irradiated Lung Cancer Models [J]. Clin Cancer Res, 2007, 13(10): 3033-3042
[1] Petrucco S. Sensing DNA damage by PARP-like fingers. Nucleic Acids Res, 2003, 31(23): 6689-6699
[2] Woodhouse B C, Dianov G L. Poly ADP-ribose polymerase-1: An international molecule of mystery. DNA Repair (Amst), 2008, 7(7): 1077-1086
[3] De Rycker M, Venkatesan R N, Wei C, et al. Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization. Biochem J, 2003, 372(pt 1): 87-96
[4] Christophe Ame J, Spenlehauer C, De Murcia G. The PARP superfamily. BioEssays, 2004, 26(8): 882-893
[5] Chou H Y E, Chou H T, Lee S C. CDK-dependent Activation of Poly(ADP-ribose) Polymerase Member 10 (PARP10). J Biol Chem, 2006, 281(22): 15201-15207
[6] Yu M, Schreek S, Cerni C, et al. PARP-10, a novel Myc- interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation. Oncogene, 2005, 24: 1982-1993
[7] Fisher A E O, Hochegger H, Takeda S, et al. Poly(ADP-Ribose) Polymerase 1 Accelerates Single-Strand Break Repair in Concert with Poly(ADP-Ribose) Glycohydrolase. Mol Cell Biol, 2007, 27(15):5597-5605
[8] Piskunova T S, Iurova M N, Zabezhinski? M A, et al. Poly(ADP-ribosa)polymerase-the relationships with life span and carcinogenesis. Adv Gerontol, 2007, 20(2):82-90
[9] Guastafierro T, Cecchinelli B, Zampieri M, et al. CTCF activates PARP-1 affecting DNA methylation machinery. J Biol Chem, 2008, 283(32):21873-21880
[10] Wacker D A, Ruhl D D, Balagamwala E H, et al. The DNA Binding and Catalytic Domains of Poly(ADP-Ribose) Polymerase 1 Cooperate in the Regulation of Chromatin Structure and Transcription. Mol Cell Biol, 2007, 27(21):7475-7485
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[12] Augustin A, Spenlehauer C, Dumond H, et al. PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. J Cell Sci, 2003, 116(8): 1551-1562
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[17] Beneke S, Burkle A. Poly(ADP-ribosyl)ation in mammalian ageing. Nucleic Acids Res, 2007, 35(22): 7456-7465
[18] Piskunova T S., Yurova M N., Ovsyannikov A I., et al. Deficiency in Poly(ADP-ribose) Polymerase-1 (PARP-1) Accelerates Aging and Spontaneous Carcinogenesis in Mice. Curr Gerontol Geriatr Res, 2008, 2008: 1-10
[19] Ratnam K, Low J. Current Development of Clinical Inhibitors of Poly(ADP-Ribose) Polymerase in Oncology. Clin Cancer Res, 2007, 13(5):1383-1388
[20] Cepeda V, Fuertes M A., Castilla J, et al. Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors in Cancer Chemotherapy. Recent Patents on Anticancer Drug Discov, 2006, 1(1):39-53
[21] Albert J M., Cao C, Kim K W, et al. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Cell Death and Improves Tumor Growth Delay in Irradiated Lung Cancer Models. Clin Cancer Res, 2007, 13(10): 3033-3042
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[1] Petrucco S. Sensing DNA damage by PARP-like fingers [J]. Nucleic Acids Res, 2003, 31(23): 6689-6699
[2] Woodhouse B C, Dianov G L. Poly ADP-ribose polymerase-1: An international molecule of mystery [J]. DNA Repair (Amst), 2008, 7(7):1077-1086
[3] Oliver A W, Ame J C, Roe S M, et al. Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2 [J]. Nucleic Acids Res, 2004, 32(2):456-464
[4] De Rycker M, Venkatesan R N, Wei C, et al. Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization [J]. Biochem J, 2003, 372(pt 1):87-96
[5] Ame C J, Spenlehauer C, De Murcia G. The PARP superfamily [J]. BioEssays, 2004, 26(8):882-893
[6] Aravind L. The WWE domain: a common interaction module in protein ubiquitination and ADP ribosylation [J]. Trends Biochem Sci, 2001, 26(5):273-275
[7] Mere-Fico M L., Meyer R G., Coyle D L., et al. Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments [J]. Exp.Cell Res, 2004, 297(2):521-532
[8] Fisher A E. O., Hochegger H, Takeda S, et al. Poly(ADP-Ribose) Polymerase 1 AcceleratesSingle-Strand Break Repair in Concert with Poly(ADP-Ribose) Glycohydrolase [J]. Mol Cell Biol, 2007, 27(15) 5597-5605
[9] Andrabi S A, Kim N S, Yu S W, et al. Poly(ADP-ribose) (PAR) polymer is a death signal [J]. Proc Natl Acad Sci U S A, 2006, 103(48):18308-18313
[10] Mortusewicz O, Ame J C, Schreiber V, et al. Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells [J]. Nucleic Acids Res, 2007, 35(22): 7665-7675
[11] Horton J K, Watson M, Stefanick D F, et al. XRCC1 and DNA polymeraseβin cellular protection against cytotoxic DNA single-strand breaks [J]. Cell Res, 2008, 18(1):48-63
[12] Piskunova T S, Iurova M N, Zabezhinski? M A, et al. Poly(ADP-ribosa)polymerase-the relationships with life span and carcinogenesis [J]. Adv Gerontol, 2007, 20(2):82-90
[13] Guastafierro T, Cecchinelli B, Zampieri M, et al. CTCF activates PARP-1 affecting DNA methylation machinery [J]. J Biol Chem, 2008, 283(32):21873-21880
[14] Nusinow D A, Munoz I H, Fazzio T G, et al. Poly(ADP-ribose) Polymerase 1 Is Inhibited by a Histone H2A Variant, MacroH2A, and Contributes to Silencing of the Inactive X Chromosome [J]. J Biol Chem, 2007, 282(17):12851-12859
[15] Menissier de Murcia J, Ricoul M, Tartier L, et al. Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse [J]. EMBO J., 2003, 22(9):2255-2263
[16] Wacker D A, Ruhl D D, Balagamwala E H, et al. The DNA binding and catalytic domains of Poly(ADP-Ribose) Polymerase 1 cooperate in the regulation of chromatin structure and transcription [J]. Mol Cell Biol, 2007, 27(21):7475-7485
[17] Chou H Y E, Chou H T, Lee S C. CDK-dependent Activation of Poly(ADP-ribose) Polymerase Member 10 (PARP10) [J]. J Biol Chem, 2006, 281(22): 15201-15207
[18] Von Kobbe C, Harrigan J A, May A, et al. Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1complex in the poly(ADP-ribosyl)ation pathway after DNA damage [J]. Mol. Cell. Biol, 2003, 23(23): 8601-8613
[19] Saenz L, Lozano J J, Valdor R, et al. Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation [J]. BMC Genomics, 2008, 9(171)
[20] Aldinucci A, Gerlini G, Fossati S, et al. A Key Role for Poly(ADP-Ribose) Polymerase-1 Activity during Human Dendritic Cell Maturation [J]. J Immunol, 2007, 179(1): 305-312
[21] Bai P, Houten S M, Huber A, et al. Poly(ADP-ribose) polymerase-2 [corrected] Controls Adipocyte Differentiation and Adipose Tissue Function through the Regulation of the Activity of the Retinoid X Receptor/PPARγHeterodimer [J]. J Biol Chem, 2007, 282(52): 37738-37746
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