与猪轮状病毒VP6蛋白相互作用的细胞蛋白研究

发布时间：2018-11-01 16:43

【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。
[Abstract]:Porcine rotavirus (PoRV) is the main pathogen causing acute gastroenteritis in piglets. At present, the prevalence rate of G9 strain in swine population is 3. 3% ~ 67. 3%. PoRV's simple infection or mixed infection with other pathogens (porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine circovirus, etc.) directly or indirectly affects pig population productivity, severely restricts the healthy development of pig industry in China, and causes huge economic loss. Therefore, it is very urgent and important to study the replication process of PoRV in organism and study the mechanism of interaction between virus protein and host protein. Rotavirus A pig/ China/ NMTL/ 2008/ G9P[23] strain (NMTL strain) isolated from the laboratory was used as the research object. The strain has strong pathogenicity to the pig, and the pig is the main storage host of rotavirus, and there is a potential danger to the human being. Therefore, it has important research value. PoRV VP6 protein is the most important structural protein of viral particles and is located in the middle layer of three-layer capsid structure of virus particles. It is related to the transcription activity of the double-layer virus particles (DLPs) released into the cytoplasm of the virus. According to the study, VP6 protein has a series of interactions with different cell proteins in virus-infected cells, playing a key role in the replication of viruses, but the cellular proteins involved in this process, as well as related functions, remain unclear. In this study, the full length of VP6 gene of PoRV NMTL strain was amplified by RT-PCR, and VP6 PCR products were cloned into prokaryotic expression vector pGEX-6P-1 and transformed to DH5 Jurkat cells to obtain p-GEX-PoRV-VP6 recombinant plasmid, and then transformed into E. coli expression bacteria BL21 (DE3) after sequencing. The fusion protein was induced by IPTG to obtain GST-VP6 recombinant protein. Three kinds of cellular proteins interacting with PoRV VP6 protein were identified by GST pull-down technique and Capture-TOF/ TOF mass spectrometry. The results showed that there were three kinds of cellular proteins interacting with PoRV VP6 protein, namely: actin-actin (beta-actin), tropomyosin type 1 (TPM1), and 40S ribosomal protein S16 (RPS16), and verified by co-precipitation (Co-IP) test. In addition, the interaction between the PoRV VP6 protein and the actin-actin was studied. The expression of VP6 protein in the cell lysate was detected by western blot. The high-throughput screening system was used to detect the infection rate of virus released to the culture solution. The time required for the PoRV round replication cycle was determined by the fluorescence quantitative q RT-PCR method, the virus content released to the culture broth at different times after the incubation. The different replication phases of PoRV were determined by combining the results of electron microscope observation and VP6 protein expression. The effects of actin cytoskeleton on the proliferation of PoRV were determined by using actin polymerization inhibitor Cyto D to treat infected cells at different replication stages of PoRV (post-toxin 0h/ 2h/ 4h/ 6h). The PoRV VP6 protein was observed under confocal microscope after double immunofluorescence staining. Whether actin was co-located in infected cells, and the transport pathway of PoRV VP6 protein in cells was observed by immunoelectron microscopy. PoRV DLPs were prepared by non-continuous density gradient centrifugation with cesium chloride and the ability of PoRV DLPs to enhance actin in vitro was determined by the actin-binding protein (ABPs) spin-down assay. It has been found by the above studies that actin-actin, tropomyosin type 1, 40S ribosomal protein S16 is a cellular protein interacting with VP6 protein in the process of PoRV infection. The three proteins are widely present in various histiocytes, and are highly conserved in the evolution of each species, virus is proliferated in the body, the cell protein which is easy to obtain can be utilized, the adaptability of the virus to the cells can be enhanced, and the tissue range of the virus infection is expanded, It is also possible to provide inter-seed propagation for the RV. A significant reduction in viral VP6 protein synthesis and viral particle release was observed with poRV-infected si RNA interfering with the expression of low-actin-actin-expressing cells. The expression level of PoRV VP6 in cell lysates was significantly lower than that in the control group after treatment with si ACTB for 72h, and the number of positive cells of PoRV-infected cells in the si ACTB treated group was significantly lower than that in untreated group after the treatment was infected with MA104 cells for 18h. Mock group, si FAM group and other control group. In the above studies, PoRV VP6 and? Actin interaction, which can effectively mediate viral infection, plays an important role in the transport of DLPs in cytoplasm, the transcription of the starting m RNA, the synthesis of viral proteins, the assembly and release of TLPs. Two immunofluorescent staining experiments showed that the PoRV VP6 protein with different fluorescent labels and the actin-actin were co-located in the cytoplasm of the virus infected cells. Immunoelectron microscopy showed that the VP6 protein with colloidal gold was located on actin microfilaments early in PoRV infection, after which VP6 protein was distributed in the ribosomes, mitochondria, ER and nucleus of infected cells. The results show that PoRV DLPs are dependent on actin network structure in infected cells and are transported to specific sites within the cells for replication. These findings reveal the transport pathways of PoRV VP6 proteins in infected cells, as well as cellular components involved in PoRV replication. The ABPs spin-down assay demonstrated in vitro that the PoRV VP6 protein binds to actin microfilaments, but DLPs do not enhance actin polymerization activity. Through the interaction between these two proteins, the PoRV DLPs abducted the actin cytoskeleton system of the cells so that it was in the cells. "Goods" the same is transported to a specific cell organelle to facilitate viral replication. However, in vivo PoRV infection, VP6 protein was used to recruit other host cell proteins (e.g., VP6 interacting protein TPM1 demonstrated in this study) to stimulate actin polymerization, as well as follow-up experimental verification. In the process of poRV infection, there is a wide variety of protein interactions between the virus and the host cell, which forms an inseparable protein network, and plays an important role in regulating the replication of the virus and starting the innate immune response of the host. The study of this paper will help to further reveal how PoRV VP6 protein and its interacting protein can be involved in mediating and regulating the virus replication process. It provides a reliable scientific basis and reference for further elucidating the pathogenesis of PoRV complicated pathogenesis, preventing and controlling the spread of diarrhea-related diseases.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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