猪瘟病毒NS5A蛋白在病毒致病中作用的研究
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摘要
猪瘟(Clssical swine fever,CFS)是猪的一种急性烈性的传染病,其传播迅速,造成的死亡率高,因而被世界动物卫生组织(OIE)列为必须上报的传染病之一,我国也将猪瘟列为一类动物疫病。目前,针对CSFV NS5A蛋白在病毒致病中作用的研究文献较少。本文以CSFV NS5A蛋白为研究对象,通过其在猪脐静脉血管内皮细胞(SUVEC)中表达,开展了NS5A蛋白的亚细胞定位、NS5A蛋白对宿主细胞氧化应激的影响、NS5A蛋白与细胞相互作用的蛋白及其对宿主细胞的与炎症相关因子的调控等方面的研究,获得了以下研究结果:
     (1)以CSFV石门株感染的SUVEC的总mRNA为模板进行RT-PCR,成功的扩增了CSFV NS5A基因的DNA片段。经酶切鉴定和测序鉴定表明,所扩增片段即为CSFVNS5A基因,其全长为1491bp,共编码497个氨基酸。CSFV NS5A蛋白的分子质量约为60ku。生物信息学分析表明,CSFV NS5A蛋白没有信号肽序列,但是其在226~247位氨基酸之间可能存在膜结合区域。分析发现CSFV NS5A蛋白的氨基酸序列中含有一段低复杂性区域VVVDTTDVTVTVV,提示CSFV NS5A蛋白可能通过被该序列分为两个不同的蛋白功能区。此外,对CSFV NS5A蛋白的磷酸化分析表明该蛋白是一个磷酸化水平很高的蛋白,存在S15、S81、S92、T274、T401等多个重要的磷酸化位点。
     (2)根据pEGFP-C1和pEGFP-N1的图谱信息,将CSFV NS5A基因成功克隆至真核表达载体pEGFP-C1和pEGFP-N1中,并将阳性质粒pEGFP-NS5A、pNS5A-EGFP和对照质粒pEGFP-C1转染SUVEC,通过含有1500μg/mLG418的抗性筛选,经过荧光观察、RT-PCR鉴定和Western blot鉴定,建立了稳定表达CSFV NS5A蛋白的细胞株。并通过激光共聚焦显微镜扫描观察,发现CSFV NS5A蛋白定位于细胞内的内质网上面。
     (3)在建立了稳定表达CSFV NS5A蛋白的细胞株的基础上,通过荧光观察、流式细胞术检测和活性氧的原位比色的试剂盒检测了表达CSFV NS5A蛋白的SUVEC的ROS产生情况。结果表明,表达NS5AGFP和GFPNS5A的SUVEC产生大量的ROS,而只表达标签蛋白GFP的细胞ROS没有升高。Real-time RT-PCR检测结果表明,CSFV NS5A蛋白表达后,抗氧化应激蛋白PRDX-6mRNA水平升高9~10倍,Trx蛋白升高5~6倍,而HO-1的mRNA水平则下降4~5倍,可见CSFV NS5A蛋白的表达可以调节抗氧化应激蛋白的mRNA水平的变化;与氧化应激关系密切的抗炎蛋白COX-2和抑炎蛋白PPAR-γ的检测结果表明,CSFV NS5A蛋白可以促进COX-2的表达和抑制PPAR-γ的表达,说明CSFVNS5A蛋白对猪血管内皮细胞有明显的促炎作用。
     (4)通过缺失表达的方法明确了CSFV NS5A蛋白的5个功能区在SUVEC内的分布,其中N端的NS5A/1-84的功能区和NS5A蛋白在内质网上的特异性分布有关。CSFVNS5A蛋白N端的NS5A/1-804功能区在NS5A蛋白引起的氧化应激中发挥主要作用。
     (5)采用酵母双杂交的方法明确了CSFV NS5A蛋白在血管内皮细胞中的相互作用的胞内蛋白。结果表明,CSFV NS5A蛋白可以和宿主细胞28个胞内蛋白相互作用,其中包括ATP依赖的RNA解旋酶(DDX5)、锌指蛋白、热休克蛋白70、G蛋白、神经刺激因子1(NAV1)、RNA聚合酶1亚基39(hRPA39)等共19个涉及细胞的细胞周期、RNA转录、蛋白质的正确折叠、血管生成和细胞通透性等方面功能的蛋白。
     (6)CSFV在感染SUVEC后,引起ROS水平的显著升高和抗氧化应激蛋白的水平明显变化,该现象说明CSFV自身可以引起SUVEC发生氧化应激。并且抗氧化剂NAC、GSH、BHA和姜黄素加入CSFV感染的SUVEC培养基后,CSFV基因的复制下降。
     综上所述,本研究首次通过试验发现CSFV NS5A蛋白定位于细胞的内质网上,并且可以引起细胞发生氧化应激,并进一步明确了CSFV NS5A蛋白与氧化应激相关的功能区。通过体外试验揭示了该蛋白可以和宿主细胞28个胞内蛋白存在着相互作用,首次发现CSFV NS5A蛋白的表达有助于炎症因子相关蛋白COX-2和PPAR-γ的表达量发生变化,为明确CSFV NS5A蛋白的功能及其在病毒的致病过程中的作用提供了新的科学资料。
Classical swine fever (CSF) is a fatal disease of pigs worldwide and classified as anotifiable (previously list A) disease by the World Organization for Animal Health (OIE) dueto its potential for rapid spread across national borders and the considerable socio-economicimpact on the pig industry. The effects of CSFV nonstructural protein5A (NS5A) protein onhost cells are still unknown by now. To elucidate the function of CSFV NS5A, the CSFVNS5A protein was expressed in the swine umbilical vein endothelial cells (SUVEC) cells inthis present study. Then subcellular localization of CSFV NS5A protein, the effect of CSFVNS5A protein on the oxidative stress in SUVEC, the regulation of CSFV NS5A protein on theinflammatory response and the intracellular proteins interacting with CSFV NS5A wereextensively investigated. The results were shown as fellow:
     (1) The CSFV NS5A gene was successfully amplified from the total RNA of CSFVinfected cells by reverse transcription polymerase chain reaction (RT-PCR). The analysis ofthe obtained sequences showed that, CSFV NS5A gene was1491bp encoding497aminoacids (aa). Bioinformatics analysis showed that there were no signal sequences and CSFVNS5A probably attached to the membranes by the sequences between the226th aa and247thaa. The analysis also showed that a low complexity region (LCR) VVVDTTDVTVTVV wasfound, it indicated that CSFV NS5A protein probably has two domains. Besides, CSFVNS5A protein was a phosphorylated protein and the position of Ser15, Ser81, S9er2, Thr27and Thr401showed great possibility of phosphorylation.
     (2) CSFV NS5A gene was cloned into the pEGFP-C1and pEGFP-N1expression vectoraccording the vector information. the plasmids pEGFP-NS5A, pNS5A-EGFP and pEGFP-C1vector were transfected into SUVEC, then the resulting stably transfected cell linesexpressing fusion proteins GFPNS5A, NS5AGFP and GFP were established by adding theselective media containing1500μg/ml G418to each well. RT-PCR and western blot analysisshowed that CSFV NS5A gene is successfully expressed in SUVEC and the molecular weightof CSFV NS5A is approximately60ku. Co-localization suggested that CSFV NS5A proteinlocalizes in the endoplasmic reticulum (ER) by confocal laser scanning microscope detection.
     (3) On the basic of the establishing of SUVEC cells expressing CSFV NS5A, the role ofCSFV NS5A on oxidative stress was explored. CSFV NS5A induces oxidative stressassociated with enhanced reactive oxygen species (ROS) production. The expression of CSFVNS5A protein exerts different effects on the three major antioxidants. Particularly, it exhibits asignificant increase in transcriptional activities of antioxidant proteins thioredoxin (Trx) andperoxiredoxin-6(PRDX-6), but accompanied by a concomitant decrease of antioxidantprotein heme oxygenase-1(HO-1). Further studies showed that cyclooxygenase-2(COX-2), apro-inflammatory protein related to oxidative stress, is up-regulated while anti-inflammatoryprotein peroxisome proliferator-activated receptor-γ (PPAR-γ), an important mediator invascular functional regulation, is down-regulated in CSFV NS5A expressing cells. This studysuggested that CSFV NS5A plays important roles in the induction of oxidative stress andinflammatory response in vascular endothelial cells.
     (4) Five cDNAs of the NS5A gene were amplified by the PCR deletion method andcloned into a eukaryotic expression vector, which was transfected into SUVEC. Subcellularlocalization of the NS5A protein was characterized by confocal microscopy, and westernblots were carried out to analyze protein expression. It showed that the peptide NS5A/1-84was in charge of the subcellular localization of CSFV NS5A. and the domain NS5A/1-804were the peptide which play a key role of oxidative stress induced in SUVEC.
     (5) The intracellular proteins of SUVEC interacted with CSFV NS5A protein wereascertained by the yeast two-hybrid technique. The result showed that CSFV NS5A couldinteract with28proteins in SUVEC,19of them are known proteins, includingATP-dependent RNA helicase (DDX5), PHD finger protein5A, heat shock70, RNApolymerase I subunit39(hRPA39), G protein, neuron navigator1(NAV1) and so on, theseintracellular proteins may play roles in the cell cycle, RNA transcription, protein folding,angiopoiesis and cellular permeability.
     (6) CSFV itself induces oxidative stress in SUVEC after the infection of CSFV onSUVEC. The level of ROS was significantly enhanced and the antioxidant proteins wereregulated by CSFV as the time went on after the infection. And it is found that the addition ofthe antioxidant NAC, GSH, BHA, and curcumin in the media of CSFV inhibit the replicationof CSFV.
     In conclusion, we firstly found that the CSFV NS5A protein was located to ERmembrance and the level of ROS and antioxidants were significantly elevated in SUVECafter being transfected with NS5A expressing plasmids. The domain which plays a key rolein oxidative stress was indentified and28intracellular proteins interacted with CSFV NS5Awere explored. This is the first time to reveal the interaction of CSFV NS5A and its host cell, which might provide new evidences to the effect of NS5A protein in the pathogenesis andmechanism of CSFV.
引文
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