【作者】 袁婧；

【导师】 魏萍；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2017， 博士

【摘要】 猪轮状病毒(PoRV)是引起幼龄仔猪发生急性胃肠炎的主要病原,一般通过接触病畜的排泄物后经粪口途径进行传播,呈地方性流行。目前在我国流行范围较广的是A群PoRV,在猪群中流行率为3.3%~67.3%,其中G9型毒株呈再度流行趋势。PoRV单纯感染或与其它病原(猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪圆环病毒等)混合感染,直接或间接地影响猪群生产力,严重制约了我国养猪业的健康发展,造成巨大经济损失。因此,深入了解PoRV在机体内的复制过程,研究病毒蛋白与宿主蛋白之间的相互作用机制,对于有效控制疫病流行、降低对养猪业的危害极为迫切和重要。本研究将实验室分离的Rotavirus A pig/China/NMTL/2008/G9P[23]毒株(简称NMTL株)作为研究对象,该毒株对猪有较强致病力,猪也是轮状病毒主要的储存宿主,存在动物病原传染给人的潜在危险,因此具有重要的研究价值。PoRV VP6蛋白是病毒粒子最主要的结构蛋白,位于病毒粒子三层衣壳结构的中间层。它与病毒脱壳释放至胞浆中的双层病毒粒子(DLPs)的转录活性有关。根据研究报道,VP6蛋白在病毒感染细胞中与不同细胞蛋白发生一系列相互作用,在病毒的复制过程中起关键作用,但对参与这个过程的细胞蛋白以及有关功能仍不十分清楚。在本研究中,采用RT-PCR方法扩增出PoRV NMTL株VP6基因c DNA全长,将VP6 PCR产物克隆至原核表达载体p GEX-6P-1中,并转化至DH5α感受态细胞,获得p GEX-PoRV-VP6重组质粒,测序正确后转化至大肠杆菌表达菌BL21(DE3),IPTG诱导融合蛋白表达,获得GST-VP6重组蛋白。利用GST pull-down技术和MALDI-TOF/TOF质谱分析鉴定,发现了3种与PoRV VP6蛋白存在相互作用的细胞蛋白,分别是:β-肌动蛋白(beta-actin)、原肌球蛋白1型(TPM1)以及40S核糖体蛋白S16(RPS16),并通过免疫共沉淀(Co-IP)试验加以验证。此外,还对PoRV VP6蛋白与β-肌动蛋白相互作用进行了后续研究:用si RNA干扰敲低细胞β-肌动蛋白表达量后,感染PoRV,用western blot方法检测细胞裂解物中VP6蛋白表达量变化,用高通量内涵筛选系统检测释放至培养液上清中病毒的感染率。通过荧光定量q RT-PCR方法,检测接毒后不同时间释放至培养液上清中的病毒含量,确定PoRV一轮复制周期所需时间。结合电镜观察和VP6蛋白表达量检测结果,确定PoRV的不同复制阶段。分别在PoRV不同复制阶段(接毒后0h/2h/4h/6h),用肌动蛋白聚合抑制剂Cyto D处理感染细胞,用荧光定量q RT-PCR方法检测接毒后8h释放至培养液上清中的病毒含量差异,确定抑制肌动蛋白聚合作用对PoRV增殖释放的影响。双免疫荧光染色后在共聚焦显微镜下观察PoRV VP6蛋白与?-肌动蛋白在感染细胞中是否共定位,用免疫电镜观察PoRV VP6蛋白在细胞内的运送途径。用氯化铯不连续密度梯度离心方法制备PoRV DLPs,通过肌动蛋白结合蛋白(ABPs)spin-down试验确定PoRV DLPs能否在体外增强肌动蛋白的聚合作用。通过以上研究发现,β-肌动蛋白、原肌球蛋白1型、40S核糖体蛋白S16,是PoRV感染过程中与VP6蛋白相互作用的细胞蛋白。这3种蛋白广泛存在于各种组织细胞中,在各物种进化过程中高度保守,病毒在体内增殖,利用容易获得的细胞蛋白,有助于增强病毒对细胞的适应性,扩大病毒感染的组织范围,也为RV的跨种间传播提供了可能。用PoRV感染si RNA干扰敲低β-肌动蛋白表达量的细胞,病毒VP6蛋白合成和病毒粒子释放显著降低。MA104细胞经si ACTB转染72h后,感染PoRV NMTL株48h,细胞裂解物中PoRV VP6蛋白表达量与对照组相比显著降低;将上述处理后收集到的上清样品感染MA104细胞18h后,si ACTB处理组PoRV感染阳性细胞数量,明显少于未处理组、Mock组、si FAM组等对照组。以上研究证实,PoRV VP6与?-肌动蛋白相互作用,能够有效介导病毒感染,在胞浆中运输DLPs、起始m RNA转录翻译合成病毒蛋白、组装和释放TLPs过程中起重要作用。双免疫荧光染色实验发现,带有不同荧光标记的PoRV VP6蛋白和β-肌动蛋白,在病毒感染细胞的胞浆中共定位。免疫电镜观察发现,在PoRV感染早期,带有胶体金标记的VP6蛋白位于肌动蛋白微丝上,此后VP6蛋白分布于感染细胞核糖体、线粒体、内质网、细胞核中。研究结果表明,PoRV DLPs在感染细胞中依赖肌动蛋白网络结构,运送至细胞内的特定位置进行复制。这些发现揭示了PoRV VP6蛋白在感染细胞内的运输途径,以及参与PoRV复制的细胞成分。ABPs spin-down试验体外证实,PoRV VP6蛋白结合至肌动蛋白微丝上,但DLPs并不能增强肌动蛋白的聚合活性。通过这2种蛋白之间的相互作用,PoRV DLPs劫持了细胞的肌动蛋白骨架系统,使它在细胞中像“货物”一样被运输至特定的细胞器中,以便于病毒复制。但在体内PoRV感染过程中,VP6蛋白是否招募其它宿主细胞蛋白(如:本研究已证实的VP6互作蛋白TPM1)协同刺激肌动蛋白聚合,还需要后续实验验证。在PoRV感染细胞过程中,病毒与宿主细胞之间存在着多种多样的蛋白相互作用,形成密不可分的蛋白网络,对于调控病毒复制、启动宿主天然免疫反应,都发挥着重要的功能。本论文的研究发现,将有助于进一步揭示PoRV VP6蛋白及其互作蛋白如何参与介导和调控病毒复制过程,对于深入阐明PoRV复杂的致病机制,预防和控制腹泻相关疾病传播,提供可靠的科学依据和研究参考。更多还原

【Abstract】 Porcine rotaviruses(PoRV)are the major cause of acute viral gastroenteritis in piglets.PoRV infection is transmissive by fecal-oral route,and these diseases are endemic.PoRV group A(RVA)spread widely in China now,and the prevalence rates in pigs vary from 3.3% to 67.3%.G9 genotypes of PoRV strains re-emerge in recent years.PoRV infection alone or mixed infection with other pathogens,such as porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV)and porcine circovirus(PCV),impact productivity in swine herds.This becomes an important factor to hinder pig industry development,and it causes huge economic losses.Therefore,it is extremely urgent and important that we start to have an insight into PoRV replication process in vivo,and study the interaction mechanisms of viral and cellular proteins,for controlling these diseases prevalence effectively,and reduce the hazard.Rotavirus A pig/China/NMTL/2008/G9P[23](abbreviated as NMTL strain)was isolated in our lab.We selected PoRV NMTL strain as object of this study,not only because it had high pathogenicity for piglets,but also there are potential risks for G9P[23] genotype strains of RVA transmission from porcine to human,which pose a great threaten to the public health,and should be given full consideration.PoRV VP6 is the major structural protein,and locates in the intermediate layer of virion.It involves in the transcriptive activity of double-layered particles(DLPs),which were released into the cytoplasm after virus uncoating.Recent researches suggest that VP6 may associate with plenty of cellular factors,and these interactions play essential roles during RV infection,but the interaction partners of VP6 and their fuctions are still unclear.In this study,we amplified VP6 gene of the PoRV NMTL strain by reverse transcriptionpolymerase chain reaction(RT-PCR),and the VP6 PCR products were ligated into the glutathione S-transferase(GST)fusion vector p GEX-6P-1.After that,it was transformed into DH5α competent cells.The resulting recombinant plasmid p GEX-PoRV-VP6 was verified by DNA sequencing.GST and the GST-VP6 fusion protein were expressed in the Escherichia coli BL21(DE3)strain by induction with 1 m M isopropyl-β-D-thiogalactopyranoside.By GST pull-down assay and matrix-assisted laser desorption/ionization dual time of flight(MALDI-TOF/TOF)mass spectrometry(MS),we discovered three interactive proteins of PoRV VP6 in MA104 cells,and they are beta-actin,TPM1 and RPS16.These interactions were further confirmed by Co-immunoprecipitation(Co-IP)assay.The interaction with beta-actin was further studied.Cellular beta-actin protein expression was knocked down by si RNA interference,and then MA104 cells were infected with PoRV.PoRV VP6 protein expression in the cell lysates were detected bywestern blotting analysis,and the culture supernatants of differently treated groups were collected to perform infectivity assays.Virus particles releasing from MA104 cells to media were measured at different time points post PoRV infection(2h/4h/6h/8h/10h/12h)using q RT-PCR analysis,to determine the time necessary for a single lifecycle of PoRV infection.On the basis of electron microscopy observation and expression kinetics of PoRV VP6,define PoRV replication stages.Cyto D were added to MA104 cells at different time points after virus inoculation(0h/2h/4h/6h),and releasing viruses were detected at 8hpi by q RT-PCR,to make sure whether actin polymerization have an effect on PoRV propagation and virus release.Co-localization of beta-actin and PoRV VP6 were detected in PoRV infected cells by double immunofluorescence,and transport pathways of PoRV VP6 were observed by immuno-electron microscope.PoRV DLPs were purified by cesium chloride(Cs Cl)density-gradient centrifugation,in order to determine the roles PoRV DLPs played in actin polymerization by actin-binding proteins(ABPs)spin-down assay.Our study discovered beta-actin,tropomyosin1(TPM1)and 40 S ribosomal protein S16(RPS16)are interactive proteins of VP6 in PoRV infection.These three proteins mentioned above exist extensively in various histocytes,and they are highly conservative during species evolution.PoRV utilizing these common cellular proteins is beneficial to virus adaptation and infectivity,furthermore make it possible for RV interspecies transmission.PoRV VP6 synthesis and virions release were significantly reduced when beta-actin was knocked down by si RNA in PoRV infected cells.MA104 cells were transfected with si ACTB for72 h,and then were infected with the PoRV NMTL strain for another 48 h.Cell lysates were collected for western blot analysis,and PoRV VP6 protein expression was significantly reduced compared with that of the control groups.The culture supernatants were collected to infect MA104 cells.After 18 hours post infection,infectivity rates of different groups were examined with the Operetta TM High Content Screening System.We found that there were less PoRV-infected cells in the si ACTB treatment group than the others(Untreated,Mock and si FAM groups).These results suggest that the interaction between PoRV VP6 and beta-actin might efficiently mediate a productive virus infection,and plays essential roles in DLPs delivery,m RNA transcription initiation to synthesize viral proteins,and TLPs maturation and release.By double immunofluorescence assay,we found that PoRV VP6 extensively co-localized with beta-actin throughout the cell cytoplasm in the infected cells.By immunoelectron microscopy observation,we found VP6 localized on the actin microfilaments at the early stage of PoRV infection,and then they distributed in the ribosome,mitochondria,endoplasmic reticulum(ER)and nucleus.These findings suggest that depending on the actin network,DLPs can be delivered to specified sites for replication in PoRV infected cells.It revealed the pathways of VP6 travelling in the infected cells and cellular components that involved in PoRV replication.ABPs spin-down assays confirmed that PoRV VP6 bound to actin filaments,but PoRV DLPs didn’t have actin polymerization enhancement activity in vitro.Rely on this interaction,PoRV DLPs hijack cellular actin network,and DLPs can be delivered to specified organelles in thecytoplasm like cargoes for replication.But during PoRV infection in vivo,whether PoRV VP6 recruit other cellular proteins to co-stimulate actin polymerization,such as TPM1,it needs further verified.During the PoRV infection,there are various interactions between virus and host cells,which establish a complex network with proteins.They play critical roles in virus replication and innate immune response.These findings in this study reveal the ways that PoRV VP6 and its interactive proteins involving in virus replication process.It will provide a scientific reference for us to understand PoRV pathogenic mechanisms,control and prevent diarrheal diseases.更多还原