科研快讯

<正>PLoS Pathog:埃博拉病毒蛋白导致大量炎症和血管渗漏发表在PLOS Pathogens杂志上的一则报告:覆盖在病毒表面的,并在感染过程中从受感染细胞脱落的埃博拉病毒GP蛋白,可以引发免疫反应失调,影响血管的通透性。埃博拉病毒有7个基因。其中一个叫GP,其编码两个相关的蛋白质:一个较短的分泌蛋白,一个为更长的贯穿病毒膜、伸出病毒表面的蛋白。在病毒感染中,一些表面的GP被一个人源酶切断,并随后从被感染的细胞脱落。大量脱落的GP和分泌的GP存在于受感染人类和动物的血中。没有利用完整的埃博拉病毒开展工作,Viktor Volchkov和同事在组织培养中制造出脱落的GP和分泌的GP,利用这些蛋白质在人细胞中测试其作用。他们发现,脱落的GP而不是分泌的GP能够结合巨噬细胞和树突状细胞,这两类细胞是埃博拉病毒感染的靶细胞。     

埃博拉病毒GP和VP40蛋白在哺乳动物细胞中的共表达及病毒样颗粒装配

目的：制备基因重组埃博拉病毒样颗粒,为疫苗研究及埃博拉病毒特异抗原、抗体检测提供基础。方法：根据埃博拉病毒扎伊尔株的GP和VP40蛋白氨基酸序列,以哺乳动物细胞基因表达密码子偏好性进行基因优化设计;化学合成GP和VP40基因片段并分别构建于表达质粒pcDNA3.1或同时构建到具有双表达单元的质粒pBudCE4.1;重组质粒经lipofectamine2000转染293FT细胞;以Western blot检测重组蛋白GP和VP40的表达;通过电镜观察病毒样颗粒。结果：构建的重组质粒经酶切鉴定及测序分析证实构建成功;Western blot结果显示,共转染分别表达GP和VP40的两个质粒或转染共表达两个蛋白的质粒都发现GP特异反应条带产生,且大小与预期相符,此外,转染共表达质粒产生的GP蛋白表达明显强于两个质粒共转染,并同时可检测到VP40的表达;电镜观察到典型的丝状的埃博拉病毒样颗粒。结论：在293FT细胞中基因优化的埃博拉病毒GP和VP40可有效表达并装配为病毒样颗粒,为进一步研究奠定了基础。     

HSV-1间层蛋白VP22转录调控功能的分析

HSV-1间层蛋白是一类重要的功能蛋白,在病毒复制的多个环节中具有重要意义．为了解主要间层蛋白VP22在病毒复制过程中的生物学特性,本实验利用氯霉素乙酰转移酶系统,分析了VP22对病毒启动子的转录调控功能,结果表明VP22对HSV-1觯,TK和gC基因启动子明显具有剂量效应关系的转录抑制作用;VP22也能抑制病毒不同转录调控因子（VP16和ICP0）对启动子的转录激活作用,尤其明显抑制ICP0对TK和gC基因启动子的转录激活作用;VP22能明显抑制组蛋白乙酰转移酶PCAF对ICP0转录激活的促进作用,此抑制作用较VP22抑制ICP0的转录激活作用更为显著．VP22对其他病毒启动子的转录抑制效应,在内对照实验β-gal活性分析中也得到了证明．     

埃博拉病毒(EBOV)蛋白的最新研究进展

埃博拉病毒(EBOV)是一种高致死性的病毒,在其RNA编码的7种蛋白中,糖蛋白GP、基质蛋白VP40以及膜蛋白VP24在病毒粒子的装配、出芽以及致病过程中起着关键的作用。详细介绍了这三种蛋白的结构、功能以及作用机制的最新研究进展,并对EBOV蛋白的研究前景作出了展望。     

口蹄疫病毒VP1基因在番茄中的表达及转基因番茄对豚鼠诱导免疫保护

构建克隆有O型口蹄疫病毒China99株VP1基因的植物双元表达载体pBin438/VP1。通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得60株抗性植株。对抗性植株分别做PCR、RT-PCR检测目的基因的整合与转录,ELISA筛选约40%的卡那抗性植株阳性,分别提取两株ELISA和Western blot检测阳性的转基因番茄叶片蛋白与弗氏佐剂乳化,于0、15、30d经肌肉途径免疫豚鼠,第三次免疫后28d用100ID50的同源强毒攻击,根据豚鼠抗体水平的消长动态和免疫豚鼠抗强毒攻击的保护率进行转基因植物疫苗免疫原性的评估。结果表明,双元表达载体pBin438/VP1构建正确,PCR、RT-PCR结果证实口蹄疫病毒VP1基因已整合到番茄基因组并在转录水平表达,ELISA和Western blot检测重组蛋白能够与FMDV阳性血清反应。转基因番茄第三次免疫豚鼠后21d血清效价最高可达1∶64,攻毒后两组免疫豚鼠保护率分别达80%和40%,证明转基因番茄表达的VP1蛋白具有良好的免疫原性。     

埃博拉病毒疫苗rVSV-ZEBOV的研究进展

埃博拉病毒被列为A类病原体,感染后可引起埃博拉出血热,具有高传染率和高致死率。研发安全有效的抗病毒疫苗迫在眉睫。目前正在研发的埃博拉病毒疫苗包括病毒载体疫苗、蛋白疫苗、DNA疫苗等,其中最有希望的是重组水疱性口炎病毒载体疫苗rVSV-ZEBOV。该疫苗在预防和治疗埃博拉出血热方面具有较高的安全性和有效性,有望在2018年上市。为了深入了解rVSV-ZEBOV疫苗,现主要从制备方法、药理学研究和作用机制等方面对该疫苗进行介绍。     

疱疹病毒VP16研究进展

疱疹病毒VP16蛋白是疱疹病毒重要的皮层蛋白,参与病毒立即早期基因转录的激活、病毒在宿主细胞内的装配与释放等过程,与许多病毒蛋白和宿主蛋白都存在蛋白相互作用,且部分疱疹病毒VP16具有去泛素活性以及帮助病毒抵御宿主免疫的功能。本文将以疱疹病毒VP16蛋白的结构特点为基础来阐述VP16的功能及其复杂的相互作用,为深入研究疱疹病毒的成熟过程以及VP16涉及的相互作用提供参考。     

型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

HSV-1立即早期蛋白ICP22与VP16共同参与α基因转录调控的初步分析

Ⅰ型单纯疱疹病毒（herpes simplex virus1,HSV-1）在宿主体内形成两种感染模式,不同感染模式的建立与病毒α基因的表达相关.作为病毒α基因表达产物之一的ICP22在病毒复制中发挥了多重作用,但其确切功能尚不清楚.实验利用氯霉素乙酰转移酶（chloram-phenicol acetyl transferase,CAT）报告系统发现ICP22非特异地抑制多种病毒或细胞启动子的转录启动作用,而且该抑制作用不受特定的病毒或细胞启动子上游调控元件影响.进一步的实验发现,HSV病毒蛋白VP16通过结合α4基因启动子上游特定元件解除ICP22对α4基因的转录抑制.这些结果提示,ICP22和VP16可能共同参与α基因的转录调控,从而建立HSV-1裂解性增殖或潜伏性感染.     

Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP

Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.     

Contribution of ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i). VP40 L domains are not required for GP-mediated enhancement of budding; (ii). the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii). NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv). the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.     

Interaction between Ebola Virus Glycoprotein and Host Toll-Like Receptor 4 Leads to Induction of Proinflammatory Cytokines and SOCS1

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection.Ebola virus and Marburg virus comprise the Filoviridae family and represent important human pathogens and potential agents of bioterrorism. Currently there are no approved vaccines or specific treatments available to prevent or treat filovirus infections. The filoviruses are the cause of severe hemorrhagic disease in humans (7). Ebola virus initially targets monocytes/macrophages and dendritic cells (DCs), which can lead to the release of proinflammatory cytokines and chemokines (3, 7). A better understanding of the physical and functional interactions between Ebola virus proteins and cellular factors regulating the host innate immune response may reveal novel insights into the pathogenesis of Ebola virus and offer new strategies to inhibit Ebola virus replication.The VP40 matrix protein of Ebola virus is a key structural protein critical for budding virus-like particles (VLPs) and virion egress. Interactions between late budding domains of VP40 and specific host proteins facilitate efficient release of VLPs and infectious virus. Viral proteins other than VP40 also contribute to efficient budding of VLPs. Ebola virus glycoprotein (GP), when coexpressed with VP40, is incorporated into budding VLPs and enhances VLP egress (15), possibly by antagonizing the function of host proteins (12).Several studies have reported the induction of an innate immune response following infection or stimulation of macrophages/monocytes and DCs with Ebola virus or VLPs, respectively (2, 31). For example, incubation of Ebola virus VP40+GP VLPs with DCs led to the induction of interleukin-6 (IL-6), IL-8, NF-κB and ERK1/2 (18, 31). The triggering mechanism by which Ebola virus VLPs stimulate cytokine production is unknown. Here, we present evidence that Ebola virus VLPs stimulate induction of proinflammatory cytokines as well as SOCS1 (a ubiquitin ligase and negative feedback regulator of cytokine production) by interacting with host Toll-like receptor 4 (TLR4). Importantly, Ebola virus VP40+GP VLPs, but not VP40 VLPs, induced cytokine and SOCS1 expression in a TLR4/MD2 dependent manner both in a human monocytic cell line (THP-1 cells) and in 293T cells expressing a functional TLR4/MD2 receptor. These results indicate that the stimulation of TLR4 by Ebola virus envelope GP results in an innate host response, induction of SOCS1 protein, and potential enhancement of virus egress.     

Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing

The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP) of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1). However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin) Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER), Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP), it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.     

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.