ATP5J促进猪繁殖与呼吸综合征病毒复制研究
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摘要
为了研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)复制与细胞调控酶ATP5J的关系,本研究首先对猪源ATP5J基因进行了抗原表位分析,选取并合成了3段抗原小肽,制备ATP5J的多克隆的抗体。用制备的多抗检测了ATP5J质粒在293T细胞中过表达的情况。然后,用PRRSV感染猪肺泡巨噬细胞(PAMs),利用qPCR和Western blot检测PRRSV感染对PAM细胞中ATP5J表达的影响。最后,我们通过小RNA干扰以及过表达ATP5J,使用qPCR和Western blot技术,在PAM和Marc-145细胞上检测ATP5J变化对PRRSV复制增殖的影响。结果显示:PRRSV感染能够导致宿主细胞ATP5J蛋白表达下调;ATP5J下调时,PRRSV复制减弱;ATP5J过表达时,PRRSV复制增强,说明ATP5J能够促进PRRSV复制增殖。进一步分析证明:ATP5J促进PRRSV复制功能没有剂量依赖性。综上,PRRSV感染能够导致宿主细胞ATP5J蛋白表达下调,而ATP5J具有促进PRRSV复制的作用。该研究为阐明PRRSV与宿主相互作用提供了新思路,为解析PRRSV免疫抑制和免疫逃逸机制奠定了基础。
In order to study the relationship between porcine reproductive and respiratory syndrome virus( PRRSV) replication and cell regulatory enzyme ATP5J,the ATP5J amino acid sequence of Sus scrofa species was analyzed and three small peptides were synthesized as antigens for generation of rabbit polyclonal antibodies against ATP5J. The polyclonal antibodies were prepared to detect the overexpression of ATP5J in 293 T cells. PRRSV was used to infect pig alveolar macrophages( PAMs). Then,the expression of ATP5J in PRRSV-infected PAM cells was measured by real-time quantitative PCR( qPCR) and Western blot. Finally,qPCR and Western blot were performed to measure the effect of ATP5J on PRRSV replication in PAM and Marc-145 cells via either ATP5J silencing or overexpression. The results indicated that ATP5J m RNA and the protein levels were downregulated after infection with PRRSV. When ATP5J was downregulated,PRRSV replication was suppressed. In contrast,when ATP5J was overexpressed,PRRSV replication was enhanced. These findings suggested that ATP5J promoted PRRSV replication. Further analysis showed that ATP5J promoted PRRSV replication in a dose-independent manner. In conclusion,PRRSV infection leads to downregulation of ATP5J expression in host cells,but ATP5J promotes PRRSV replication. The present study provides new insights into the interaction between PRRSV and host cells and lays a foundation for exploring mechanisms of immunosuppression and immune escape of PRRSV.
In order to study the relationship between porcine reproductive and respiratory syndrome virus( PRRSV) replication and cell regulatory enzyme ATP5J,the ATP5J amino acid sequence of Sus scrofa species was analyzed and three small peptides were synthesized as antigens for generation of rabbit polyclonal antibodies against ATP5J. The polyclonal antibodies were prepared to detect the overexpression of ATP5J in 293 T cells. PRRSV was used to infect pig alveolar macrophages( PAMs). Then,the expression of ATP5J in PRRSV-infected PAM cells was measured by real-time quantitative PCR( qPCR) and Western blot. Finally,qPCR and Western blot were performed to measure the effect of ATP5J on PRRSV replication in PAM and Marc-145 cells via either ATP5J silencing or overexpression. The results indicated that ATP5J m RNA and the protein levels were downregulated after infection with PRRSV. When ATP5J was downregulated,PRRSV replication was suppressed. In contrast,when ATP5J was overexpressed,PRRSV replication was enhanced. These findings suggested that ATP5J promoted PRRSV replication. Further analysis showed that ATP5J promoted PRRSV replication in a dose-independent manner. In conclusion,PRRSV infection leads to downregulation of ATP5J expression in host cells,but ATP5J promotes PRRSV replication. The present study provides new insights into the interaction between PRRSV and host cells and lays a foundation for exploring mechanisms of immunosuppression and immune escape of PRRSV.
引文
[1]Wensvoort G,Terpstra C,Pol J M,et al.Mystery swine disease in The Netherlands:the isolation of Lelystad virus[J].Vet Quart,1991,13(3):121-130.
[2]Terpstra C,Wensvoort G,Pol J M.Experimental reproduction of porcine epidemic abortion and respiratory syndrome(mystery swine disease)by infection with Lelystad virus:Koch's postulates fulfilled[J].Vet Quart,1991,13(3):131-136.
[3]Collins J E,Benfield D A,Christianson W T,et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduction of the disease in gnotobiotic pigs[J].J Vet Diagn Invest,1992,4(2):117-126.
[4]Joshi S,Pringle M J.ATP synthase complex from bovine heart mitochondria.Passive H+conduction through mitochondrial coupling factor 6-depleted F0 complexes[J].J Biol Chem,1989,264(26):15548-15551.
[5]Collinson I R,van Raaij M J,Runswick M J,et al.ATP synthase from bovine heart mitochondria:in vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence[J].J Mol Biol,1994,242(4):408-421.
[6]Dorward A,.Sweet S,Moorehead R,et al.Mitochondrial contributions to cancer cell physiology:redox balance,cell cycle,and drug resistance[J].J Bioenerg biomembr,1997,29(4):385-392.
[7]Ko Y H,Smith B L,Wang Y C,et al.Advanced cancers:eradication in all cases using 3-bromopyruvate therapy to deplete ATP[J].Biochem Bioph Res Co,2004,324(1):269-275.
[8]王蓉.猪繁殖与呼吸综合征病毒对Ⅰ型干扰素下游信号通路影响的研究[D].杨凌:西北农林科技大学,2013.
[9]Overend C,Mitchell R,He D,et al.Recombinant swine beta interferon protects swine alveolar macrophages and Marc145 cells from infection with porcine reproductive and respiratory syndrome virus[J].J Gen Virol,2007,88:925-931.
[10]Shi X,Zhang X,Wang L,et al.Rcombinant beta interferon could clear the Low-dose infected porcine reproductive and respiratory syndrome virus(PRRSV)in MARC-145 cells[J].Acta Virol,2016,60:290-297.
[11]Fang J,Wang H,Bai J,et al.Monkey viperin restricts porcine reproductive and respiratory syndrome virus replication[J].PLo S ONE,2016,11(5):e0156513-e0156514.
[12]Ji L,Zhou X,Liang W,et al.Porcine interferon stimulated gene12a restricts porcine reproductive and respiratory syndrome virus replication in MARC-145 cells[J].Int J MOI Sci,2017,18(8):1613-1615.
[13]Wang H,Bai J,Fan B,et al.The interferon-induced MX2 inhibits porcine reproductive and respiratory syndrome virus replication[J].J Interferon Cytokine Res,2016,36(2):129-39.
[14]Chen J,Shi X,Zhang X,et al.Micro RNA 373 facilitates the replication of porcine reproductive and respiratory syndrome virus by its negative regulation of typeⅠinterferon induction[J].J virol,2017,18:91-93.
[15]Li L,Gao F,Jiang Y,et al.Cellular mi R-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo[J].Sci Rep,2015,19(5):17010-17011.
[16]Zhang Q,Huang C,Yang Q,et al.Micro RNA-30c Modulates typeⅠIFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1[J].J Immunol,2016,196(5):2272-82.
[17]Li L,Wei Z,Zhou Y,et al.Host mi R-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons[J].Virus Res,2015,195:86-94.
[18]Li L,Chen R,Zhao J,et al.LSM14A inhibits porcine reproductive and respiratory syndrome virus(PRRSV)replication by activating IFN-βsignaling pathway in Marc-145[J].Mol Cell Biochem,2015,399(1-2):247-256.
[2]Terpstra C,Wensvoort G,Pol J M.Experimental reproduction of porcine epidemic abortion and respiratory syndrome(mystery swine disease)by infection with Lelystad virus:Koch's postulates fulfilled[J].Vet Quart,1991,13(3):131-136.
[3]Collins J E,Benfield D A,Christianson W T,et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduction of the disease in gnotobiotic pigs[J].J Vet Diagn Invest,1992,4(2):117-126.
[4]Joshi S,Pringle M J.ATP synthase complex from bovine heart mitochondria.Passive H+conduction through mitochondrial coupling factor 6-depleted F0 complexes[J].J Biol Chem,1989,264(26):15548-15551.
[5]Collinson I R,van Raaij M J,Runswick M J,et al.ATP synthase from bovine heart mitochondria:in vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence[J].J Mol Biol,1994,242(4):408-421.
[6]Dorward A,.Sweet S,Moorehead R,et al.Mitochondrial contributions to cancer cell physiology:redox balance,cell cycle,and drug resistance[J].J Bioenerg biomembr,1997,29(4):385-392.
[7]Ko Y H,Smith B L,Wang Y C,et al.Advanced cancers:eradication in all cases using 3-bromopyruvate therapy to deplete ATP[J].Biochem Bioph Res Co,2004,324(1):269-275.
[8]王蓉.猪繁殖与呼吸综合征病毒对Ⅰ型干扰素下游信号通路影响的研究[D].杨凌:西北农林科技大学,2013.
[9]Overend C,Mitchell R,He D,et al.Recombinant swine beta interferon protects swine alveolar macrophages and Marc145 cells from infection with porcine reproductive and respiratory syndrome virus[J].J Gen Virol,2007,88:925-931.
[10]Shi X,Zhang X,Wang L,et al.Rcombinant beta interferon could clear the Low-dose infected porcine reproductive and respiratory syndrome virus(PRRSV)in MARC-145 cells[J].Acta Virol,2016,60:290-297.
[11]Fang J,Wang H,Bai J,et al.Monkey viperin restricts porcine reproductive and respiratory syndrome virus replication[J].PLo S ONE,2016,11(5):e0156513-e0156514.
[12]Ji L,Zhou X,Liang W,et al.Porcine interferon stimulated gene12a restricts porcine reproductive and respiratory syndrome virus replication in MARC-145 cells[J].Int J MOI Sci,2017,18(8):1613-1615.
[13]Wang H,Bai J,Fan B,et al.The interferon-induced MX2 inhibits porcine reproductive and respiratory syndrome virus replication[J].J Interferon Cytokine Res,2016,36(2):129-39.
[14]Chen J,Shi X,Zhang X,et al.Micro RNA 373 facilitates the replication of porcine reproductive and respiratory syndrome virus by its negative regulation of typeⅠinterferon induction[J].J virol,2017,18:91-93.
[15]Li L,Gao F,Jiang Y,et al.Cellular mi R-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo[J].Sci Rep,2015,19(5):17010-17011.
[16]Zhang Q,Huang C,Yang Q,et al.Micro RNA-30c Modulates typeⅠIFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1[J].J Immunol,2016,196(5):2272-82.
[17]Li L,Wei Z,Zhou Y,et al.Host mi R-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons[J].Virus Res,2015,195:86-94.
[18]Li L,Chen R,Zhao J,et al.LSM14A inhibits porcine reproductive and respiratory syndrome virus(PRRSV)replication by activating IFN-βsignaling pathway in Marc-145[J].Mol Cell Biochem,2015,399(1-2):247-256.