猪繁殖与呼吸系统综合征病毒非结构蛋白1β结构与功能
摘要
猪繁殖与呼吸系统综合征病毒属于套病毒目，动脉炎病毒科，动脉炎病毒属，是导致猪繁殖与呼吸系统综合征的病原体。该病主要引起怀孕母猪流产、早产、死胎、木乃伊胎增多等症状，也有人根据其临床特征将其称为“猪蓝耳病”。自1987年首次爆发以来，在短短的几年时间内，该病已蔓延至世界各个养猪业发达的国家和地区，造成了巨大的经济损失。该病自1995年底在我国爆发，迅速传播至各地，已成为困扰养猪业的主要问题之一。由于其基因组的高度变异性，使得不同毒株存在较大差异，为有效疫苗的开发增加了难度。
非结构蛋白nsp1β是猪繁殖与呼吸系统综合征病毒基因组编码的第二个非结构蛋白，通过C端类木瓜蛋白酶半胱氨酸蛋白酶结构域PCPβ进行自剪切，进而从复制酶多蛋白上释放出来。研究者已经发现了nsp1β的蛋白酶活性，且有报道称nsp1β在病毒的复制过程和发病机理中发挥重要作用，但对nsp1β在上述过程中的作用机制并没有明确的阐释。
本论文解析了猪繁殖与呼吸系统综合征病毒nsp1β的三维晶体结构，通过对PCPβ结构域与C端延伸区域之间相互作用的分析，一定程度上揭示了PCPβ与底物之间的作用机制，并确认了nsp1β与下游蛋白nsp2之间的切割位点，发现并解释了成熟的nsp1β的蛋白酶活性被抑制的现象。同时，通过对溶液中nsp1β二聚体形式的确认，结合二聚体晶体结构，提出了可能的顺式自剪切机制。另外，通过三维结构比对，我们预测了nsp1β的N端结构域的核酸酶活性，并通过对λ-DNA的体外降解实验，验证了这一预测。我们进一步对nsp1β的核酸酶活性进行了研究，发现了其金属离子依赖性，即nsp1β在镁离子或锰离子存在的条件下行使核酸酶活性。我们还分别尝试了以猪肝脏总RNA提取物、人工合成的单链DNA、双链RNA和单链RNA为底物的情况下nsp1β的核酸酶活性，发现了其底物的选择性，即nsp1β能够切割双链DNA和单链RNA，而对一些人工合成的双链RNA和单链DNA不具有切割活性。通过nsp1β的亚细胞定位实验，进一步暗示了nsp1β对猪繁殖与呼吸系统综合征病毒宿主细胞的可能影响。综上所述，上述实验结果提示了nsp1β的生物学功能，并为抗病毒药物的开发提供了一个多位点靶标。
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of theArteriviridae family of Nidovirales, is the etiological agent responsible for a disease inpigs called porcine reproductive and respiratory syndrome (PRRS). PRRS ischaracterized by late-term abortion, early farrowing, stillbirth, and the birth of weakpiglets, which is initially referred to as “blue-ear pig disease” by its clinical symptoms.Since its first report in1987, it has spread to the majority of swine-producing countriesaround the world and has caused enormous economic losses in the swine industry. APRRS epidemic occurred in China in late1995, quickly transmitted to most provincesand is considered as one of the major problems in the swine industry. Due to the highmutation rate of its genome, various strains possess great differences, which curbs thedevelopment of vaccines, and more effective medications are needed.
As the second nonstructural protein coded in the genome of PRRSV, nsp1βreleases itself from the replicase polyprotein through self-cleavage by itsC-terminal papain-like cysteine protease (PCP) domain, which is named PCPβ.It is known that nsp1β is able to function as protease, and is of vital importanceto the replication of PRRSV and the pathogenesis of PRRS, yet the mechanismof such processes is lack of sufficient illustration.
We report here the three-dimensional structure of PRRSV nsp1β, revealing thecommon known PCPβ domain and some other regions. N-terminal sequencing ofauto-cleaved nsp2fragment from an nsp1β-nsp2N fusion protein shows the exactcleavage site between nsp1β and nsp2. By analyzing the interaction between thePCPβ domain and the C-terminal extension (CTE) as a putative substrate, wecan explain the mechanism in the interaction between PCPβ and its substrate.We also discovered and explained the phenomenon that mature nsp1β lost itsproteolytic activity. The results of gel-filtration and cross-linking assay showsthat nsp1β should exist as a homodimer in solution, which suggests a cisself-processing mode of nsp1β by the crystal structure of the homodimer.Besides, we expect a possible nuclease activity of N-terminal domain (NTD) bycomparing protein structures in3D, and demonstrate our anticipation by in vitro nuclease assay with λ-DNA. Further examination on the nuclease activity ofnsp1β reveals its metal dependence on Mn2+and Mg2+and substrate selectivityinclined to dsDNA and ssRNA. The combination of the fact that nsp1βpossesses the nuclease activity on total RNA extracted from porcine liver andthe result of the sub-cellular localization assay of nsp1β hints some possibleinfluence on the host cell of PRRSV. In sum, these results shed light on thebiological function of nsp1β and offer a multi-target template for future drugdiscovery.
非结构蛋白nsp1β是猪繁殖与呼吸系统综合征病毒基因组编码的第二个非结构蛋白，通过C端类木瓜蛋白酶半胱氨酸蛋白酶结构域PCPβ进行自剪切，进而从复制酶多蛋白上释放出来。研究者已经发现了nsp1β的蛋白酶活性，且有报道称nsp1β在病毒的复制过程和发病机理中发挥重要作用，但对nsp1β在上述过程中的作用机制并没有明确的阐释。
本论文解析了猪繁殖与呼吸系统综合征病毒nsp1β的三维晶体结构，通过对PCPβ结构域与C端延伸区域之间相互作用的分析，一定程度上揭示了PCPβ与底物之间的作用机制，并确认了nsp1β与下游蛋白nsp2之间的切割位点，发现并解释了成熟的nsp1β的蛋白酶活性被抑制的现象。同时，通过对溶液中nsp1β二聚体形式的确认，结合二聚体晶体结构，提出了可能的顺式自剪切机制。另外，通过三维结构比对，我们预测了nsp1β的N端结构域的核酸酶活性，并通过对λ-DNA的体外降解实验，验证了这一预测。我们进一步对nsp1β的核酸酶活性进行了研究，发现了其金属离子依赖性，即nsp1β在镁离子或锰离子存在的条件下行使核酸酶活性。我们还分别尝试了以猪肝脏总RNA提取物、人工合成的单链DNA、双链RNA和单链RNA为底物的情况下nsp1β的核酸酶活性，发现了其底物的选择性，即nsp1β能够切割双链DNA和单链RNA，而对一些人工合成的双链RNA和单链DNA不具有切割活性。通过nsp1β的亚细胞定位实验，进一步暗示了nsp1β对猪繁殖与呼吸系统综合征病毒宿主细胞的可能影响。综上所述，上述实验结果提示了nsp1β的生物学功能，并为抗病毒药物的开发提供了一个多位点靶标。
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of theArteriviridae family of Nidovirales, is the etiological agent responsible for a disease inpigs called porcine reproductive and respiratory syndrome (PRRS). PRRS ischaracterized by late-term abortion, early farrowing, stillbirth, and the birth of weakpiglets, which is initially referred to as “blue-ear pig disease” by its clinical symptoms.Since its first report in1987, it has spread to the majority of swine-producing countriesaround the world and has caused enormous economic losses in the swine industry. APRRS epidemic occurred in China in late1995, quickly transmitted to most provincesand is considered as one of the major problems in the swine industry. Due to the highmutation rate of its genome, various strains possess great differences, which curbs thedevelopment of vaccines, and more effective medications are needed.
As the second nonstructural protein coded in the genome of PRRSV, nsp1βreleases itself from the replicase polyprotein through self-cleavage by itsC-terminal papain-like cysteine protease (PCP) domain, which is named PCPβ.It is known that nsp1β is able to function as protease, and is of vital importanceto the replication of PRRSV and the pathogenesis of PRRS, yet the mechanismof such processes is lack of sufficient illustration.
We report here the three-dimensional structure of PRRSV nsp1β, revealing thecommon known PCPβ domain and some other regions. N-terminal sequencing ofauto-cleaved nsp2fragment from an nsp1β-nsp2N fusion protein shows the exactcleavage site between nsp1β and nsp2. By analyzing the interaction between thePCPβ domain and the C-terminal extension (CTE) as a putative substrate, wecan explain the mechanism in the interaction between PCPβ and its substrate.We also discovered and explained the phenomenon that mature nsp1β lost itsproteolytic activity. The results of gel-filtration and cross-linking assay showsthat nsp1β should exist as a homodimer in solution, which suggests a cisself-processing mode of nsp1β by the crystal structure of the homodimer.Besides, we expect a possible nuclease activity of N-terminal domain (NTD) bycomparing protein structures in3D, and demonstrate our anticipation by in vitro nuclease assay with λ-DNA. Further examination on the nuclease activity ofnsp1β reveals its metal dependence on Mn2+and Mg2+and substrate selectivityinclined to dsDNA and ssRNA. The combination of the fact that nsp1βpossesses the nuclease activity on total RNA extracted from porcine liver andthe result of the sub-cellular localization assay of nsp1β hints some possibleinfluence on the host cell of PRRSV. In sum, these results shed light on thebiological function of nsp1β and offer a multi-target template for future drugdiscovery.
引文
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[2] Wensvoort G, Terpstra C, Pol J M, et al. Mystery swine disease in the Netherlands:theisolation of Lelystad virus. Vet Q,1991,13(3):121-130.
[3] Benfield D, Collins J, Dee S, et al. Porcine reproductive and respiratory syndrome..Diseases of the swine.8th ed. Ames, Iowa: Iowa State University Press,1999:201-232.
[4] Paton D J, Brown I H, Edwards S, et al.'Blue ear' disease of pigs. Vet Rec,1991,128(26):617-620
[5] Ahl R, Pensaert M, Rovertson I B, et al. Porcine reproductive and respiratory syndrome(PRRS or blue-eared disease). Vet Rec,1992,130:87-89.
[6] Baron T, Albina E, Leforban Y, et al. Report on the first outbreak of porcine reporductiveadn respiratory syndrome (PRRS) in France. Ann Rech Vet,1992,23:161-166.
[7] Neumann E J, Kliebenstein J B, Johnson C D, et al. Assessment of the economic impactof porcine reproductive and respiratory syndrome on swine production in the UnitedStates. Journal of the American Veterinary Medical Association,2005,227(3):385-392.
[8]郭宝清,陈章水,刘兴文,等.从疑似PRRS流产胎儿分离PRRSV的研究.中国畜禽传染病,1996,18(2):21-23.
[9]杨汉春,管山红,尹晓敏,等.猪繁殖与呼吸综合征病毒的分离与初步鉴定.中国兽医杂志,1997,23(10):9-10.
[10] Mardassi H, Mounir S, Dea S. Molecular analysis of the ORFs3to7of porcinereproductive and respiratory syndrome virus, Quebec reference strain. Arch Virol,1995,140(8):1405-1418.
[11] Suarez P, Zardoya R, Martin M J, et al. Phylogenetic relationships of European strains ofporcine reproductive and respiratory syndrome virus (PRRSV) inderred from DNAsequences of putative ORF-5and ORF-7genes. Virus Res,1996,42(12):159-165.
[12] Thiel H J, Meyers G, Stark R, et al. Molecular characterization of positive-strand RNAviruses: pestiviruses and the porcine reproductive and respiratory syndrome virus(PRRSV). Arch Virol Suppl,1993,7:41-52.
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