ELECTRON TOMOGRAPHIC STUDY OF HERPES SIMPLEX VIRUS 1 ENVELOPMENT AND EGRESS

单纯疱疹病毒 1 包络和流出的电子断层扫描研究

• 批准号: 7357272
• 负责人: JOEL D. BAINES
• 金额: $ 2.25万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357272
• 关键词: ELECTRON; TOMOGRAPHIC; STUDY; HERPES; SIMPLEX

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SUPPORT: NIH 5R01AI052341-02 ¿Nucleocapsid envelopment Herpes simplex virus-1¿, 03/03-02/08 Joel D. Baines Cornell University NIH 2R01GM050740-09 ¿Herpes Simplex Virus Terminases¿, 01/95-12/07 Joel D. Baines Cornell University NIH F32GM067519 ¿ATP hydrolysis in HSV DNA packaging¿ to C.L. Duffy, 2003-2005. C.L. Duffy Cornell University ABSTRACT Extracellular herpesvirus virions consist of the nucleocapsid surrounded by an amorphous proteinaceous layer called the tegument that is in turn surrounded by a lipid viral envelope. The latter is derived from host membranes from which virions bud. The particles are known to bud from the inner nuclear membrane where they acquire an initial virion envelope. Enveloped virions then accumulate between the leaflets of the nuclear membrane, and the virion envelope eventually fuses with the outer nuclear membrane leaflet, dumping the de-enveloped nucleocapsid into the cytoplasm, where it eventually becomes re-enveloped and is secreted into the extracellular space. The involvement of possible host cytoskeletal elements in the production of virions can only be studied by the use of electron tomography because tomography allows analysis of individual particles in-situ and without bias due to symmetry-averaging. Preliminary observations have changed the existing paradigm of how the envelopment process occurs, and suggest that herpesviruses exploit the cell in ways that were not predicted. Herpesviruses cause a number of important medical conditions in humans including blindness and encephalitis in both immune-compromised and immune competent patients. The ultimate goal of this study is to understand the mechanisms by which herpesviruses become enveloped at the nuclear membrane and their pathway of egress from the perinuclear space. All undergo the same pathway of virion production and exit, thus the studies will establish the paradigm for this important group of pathogens. The conservation of these steps suggest that it is conceivable that compounds that interfere with this pathway would be effective against all herpesviruses. The characterization of herpesvirus particles using tomography will provide a novel view of the virion egress pathway, and new insight for structure and function of virions and viral proteins. The first aim in this project is to elucidate the structure of wild type and mutant particles as they engage the envelopment machinery at the inner nuclear membrane. The nature of the envelopment machinery at the inner nuclear membrane is unknown, although the Baines laboratory has identified three candidates (genes UL11, UL31 and UL34) as important for the envelopment reaction (Baines and Roizman, 1992; Reynolds et al., 2001). Cells infected with wild type and available mutant viruses lacking genes encoding these proteins will be examined by cryoelectron tomography. Preliminary analyses of wild type nascent virions (those located in the perinuclear space), using fixed material, indicate the presence of novel fibers that bridge the space between the herpes simplex virus (HSV) virion envelope and the nucleocapsid. The deduced mass of these fibers is higher than can be accounted for by any viral gene, suggesting the novel possibility that host proteins are used as a structural component during virus assembly. This would be unprecedented. The diameter of the fibers is consistent with cytoskeletal proteins such as actin or lamins. Although the existence of nuclear actin was controversial, its presence is now widely acknowledged, but remains functionally obscure (Bettinger et al., 2004). The fibers are connected to the nucleocapsid in an asymmetrical fashion, and it will be important to verify that this asymmetry is not an artifact of the fixation process, thus cryoelectron microscopy is essential. To test the logical hypotheses that one or more of the envelopment proteins are involved in recruiting the fibers into virions, analysis of the mutant viruses will be performed. If a given protein is involved in recruitment or formation of the fibers, such fibers should be absent or misshapen in particles located at the inner nuclear membrane of cells infected with the relevant mutant virus. A separate, but related issue is whether tegument proteins are incorporated at this stage of the egress pathway. Current dogma suggests that tegument proteins are incorporated only in the cytoplasm after de-envelopment. The structure of individual particles in the perinuclear space will indicate whether at least a subset of tegument proteins are brought into the virion through interaction with the nucleocapsid or the inner nuclear membrane. The second aim is to compare the structure of extracellular virions with that of nascent virions. The comparison might indicate that certain steps occur in the egress pathway occur that have only been alluded to in the past. For example, the hypothesis that additional proteins are incorporated along with nucleocapsids as they become enveloped in the cytoplasm is a longstanding one, and predicts the presence of additional mass in the tegument of extracellular virions (Gr¿newald et al., 2003) as opposed to nascent virions. Our initial tomography work at the RVBC was with plastic sections of conventionally-processed cells. We saw a specialization of the inner leaflet of the nuclear envelope adjacent to, and conforming to, the icosohedral shape of an internal virus particle. We also saw evidence that the nuclear envelope is the origin of viral ¿transport vesicles¿ (manuscript in preparation). However, conventional preparation was inadequate to reliably preserve the finer structural details of the virions and their immediate surroundings. Ultimately, we feel that the ¿native¿ preparation afforded by frozen-hydrated sections will be required. While awaiting establishment of protocols to do this work with frozen-hydrated sections, we are continuing our work by using high-pressure frozen, freeze-substituted material. Our experience with high-pressure freezing will help us establish optimal freezing protocols with our material prior to starting the frozen-hydrated section work. Electron tomography is the only technique that can yield 3-D ultrastructural information on individual virus particles in-situ. In order to be confident of the results, the native preservation of frozen-hydrated preparation will be required. The technique of frozen-hydrated sections, developed at the RVBC, will be a great asset for our work. References 1. Baines,J.D. and Roizman,B. (1992). The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells. J. Virol. 66, 5168-5174. 2. Bettinger,B.T., Gilbert,D.M., and Amberg,D.C. (2004). Actin up in the nucleus. Nat. Rev. Mol. Cell Biol. 5, 410-415. 3. Gr¿newald,K., Desai,P., Winkler,D.C., Heymann,J.B., Belnap,D.M., Baumeister,W., and Steven,A.C. (2003). Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302, 1396-1398. 4. Reynolds,A.E., Ryckman,B., Baines,J.D., Zhou,Y., Liang,L., and Roller,R.J. (2001). UL31 and UL34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J. Virol. 75, 8803-8817.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。支持：NIH 5R01AI052341-02“核衣壳包膜单纯疱疹病毒-1”，03/03-02/08 Joel D. Baines康奈尔大学NIH 2R01GM050740-09“单纯疱疹病毒终止”，01/95-12/07 Joel D. Baines康奈尔大学NIH F32GM067519“HSV DNA包装中的ATP水解”到C.L. Duffy, 2003-2005。细胞外疱疹病毒病毒粒子由核衣壳组成，核衣壳被一种称为被膜的无定形蛋白质层包围，而被膜又被一种脂质病毒包膜包围。后者来源于病毒粒子发芽的宿主膜。已知这些颗粒从核膜内萌发，在那里它们获得初始病毒粒子包膜。被包膜的病毒粒子随后在核膜小叶之间积累，病毒粒子的包膜最终与外核膜小叶融合，将被包膜的核衣壳倾倒到细胞质中，在那里它最终被重新包膜并分泌到细胞外空间。可能的宿主细胞骨架元件参与病毒粒子的产生只能通过使用电子断层扫描来研究，因为断层扫描允许对单个粒子进行原位分析，并且没有由于对称平均而产生的偏差。初步观察改变了包膜过程如何发生的现有模式，并表明疱疹病毒以未预料到的方式利用细胞。疱疹病毒在人类中引起许多重要的疾病，包括免疫功能低下和免疫正常的患者的失明和脑炎。本研究的最终目的是了解疱疹病毒在核膜上被包裹的机制及其从核周空间流出的途径。它们都经历了相同的病毒粒子产生和退出的途径，因此这些研究将为这一重要病原体群体建立范式。这些步骤的保存表明，可以想象，干扰这一途径的化合物对所有疱疹病毒都有效。利用断层扫描技术表征疱疹病毒颗粒将为病毒粒子的输出途径提供新的视角，并为病毒粒子和病毒蛋白的结构和功能提供新的见解。本项目的第一个目的是阐明野生型和突变型粒子在参与核膜包膜机制时的结构。尽管Baines实验室已经确定了三个候选基因（UL11、UL31和UL34）对包膜反应很重要，但核膜包膜机制的性质尚不清楚（Baines和Roizman, 1992； Reynolds等人，2001）。被缺乏编码这些蛋白质的基因的野生型和可用的突变病毒感染的细胞将通过冷冻电子断层扫描进行检查。使用固定材料对野生型新生病毒粒子（位于核周空间的病毒粒子）进行的初步分析表明，存在连接单纯疱疹病毒（HSV）病毒粒子包膜和核衣壳之间空间的新型纤维。推断出的这些纤维的质量比任何病毒基因所能解释的都要高，这表明宿主蛋白在病毒组装过程中被用作结构成分的新可能性。这将是前所未有的。纤维的直径与细胞骨架蛋白如肌动蛋白或层粘连蛋白一致。尽管核肌动蛋白的存在存在争议，但它的存在现在已被广泛承认，但其功能仍不清楚（Bettinger et al., 2004）。纤维以一种不对称的方式连接到核衣壳上，重要的是要验证这种不对称不是固定过程的产物，因此冷冻电子显微镜是必不可少的。为了验证一种或多种包膜蛋白参与将纤维招募到病毒粒子中的逻辑假设，将对突变病毒进行分析。如果一种特定的蛋白质参与了纤维的募集或形成，那么这种纤维在被相关突变病毒感染的细胞内膜上的颗粒中应该是缺失的或畸形的。另一个独立但相关的问题是被膜蛋白是否在这个输出通路的阶段被掺入。目前的理论认为，被膜蛋白只有在脱包膜后才被纳入细胞质中。核周空间中单个颗粒的结构将表明是否至少有一部分被膜蛋白通过与核衣壳或核膜的相互作用被带入病毒粒子。第二个目的是比较细胞外病毒粒子和新生病毒粒子的结构。这种比较可能表明，在出口途径中发生的某些步骤在过去只被暗示过。例如，当核衣壳被包裹在细胞质中时，额外的蛋白质与核衣壳一起被合并的假设是一个长期存在的假设，并预测了与新生病毒粒子相反，细胞外病毒粒子的被膜中存在额外的质量（Gr¿newald et al., 2003）。我们最初在RVBC的断层扫描工作是用常规处理的细胞的塑料切片。我们看到核膜内小叶的特化与内部病毒颗粒的二十面体形状相邻，并与之一致。我们还看到了核膜是病毒运输囊泡起源的证据（手稿准备中）。然而，传统的制备方法不足以可靠地保存病毒粒子及其周围环境的精细结构细节。最终，我们认为冷冻水合部分提供的“天然”准备是必需的。在等待建立用冷冻水合部分进行这项工作的协议的同时，我们正在继续使用高压冷冻、冷冻替代材料进行这项工作。我们在高压冷冻方面的经验将帮助我们在开始冷冻水合部分工作之前建立最佳的材料冷冻方案。电子断层扫描是唯一一种可以在现场获得单个病毒颗粒的三维超微结构信息的技术。为了对结果有信心，需要对冷冻水合制剂进行天然保存。RVBC开发的冷冻水合切片技术将是我们工作的重要资产。引用1。贝恩斯,法学博士Roizman, B。(1992)。单纯疱疹病毒1的UL11基因编码一种促进核衣壳包膜和从细胞中退出的功能。[j] .中国生物医学工程学报，2016,33(5):568 - 574。2. 赌博,B.T.吉尔伯特,D.M.以及华盛顿特区的安伯格(2004)。细胞核中的肌动蛋白。《细胞生物学》，5(4),410-415。3. Gr newald, K。德赛,P。,温克勒特区。海曼,J.B.、Belnap D.M.鲍迈斯特,W。Steven，A.C.(2003)。单纯疱疹病毒的低温电子断层扫描三维结构。科学学报302,1396-1398。4. Reynolds A.E., Ryckman B。贝恩斯,J.D.周,Y。梁,L。R.J.罗勒(2001)。1型单纯疱疹病毒的UL31和UL34蛋白形成一个复合物，在核边缘积聚，是核衣壳包膜所必需的。中国生物医学工程学报，32(2):444 - 444。