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: 7598344
• 负责人: JOEL D. BAINES
• 金额: $ 1.15万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598344
• 关键词: 3-Dimensional; ATP Hydrolysis; Accounting; Actins; Biological Preservation; Blindness; Caliber; Candidate Disease Gene; Cell Nucleus; Cell physiology; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Cryoelectron Microscopy; Cytoplasm; Cytoskeletal Proteins; DNA Packaging; Electrons; Encephalitis; Extracellular Space; Extracellular Structure; Fiber; Freezing; Funding; Genes; Goals; Grant; Herpesviridae; Herpesvirus 1; Human; Immune; In Situ; Indium; Individual; Institution; Laboratories; Lamins; Manuscripts; Medical; Membrane; Morphologic artifacts; Nature; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Nucleocapsid; Numbers; Pathway interactions; Patients; Plastics; Preparation; Process; Production; Proteins; Protocols documentation; Reaction; Recruitment Activity; Reporting; Research; Research Personnel; Resources; Science; Shapes; Simplexvirus; Source; Staging; Structure; Techniques; Testing; Transport Vesicles; UL34 gene; United States National Institutes of Health; Universities; Viral; Viral Genes; Viral Proteins; Virion; Virus; Virus Assembly; Work; abstracting; electron tomography; experience; extracellular; insight; mutant; novel; particle; pathogen; pressure; reconstruction; sample fixation; terminase; three dimensional structure; tomography; viral DNA; viral envelope lipids

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. In the previous reporting period, eight new double-tilt tomographic reconstructions were made from the high-pressure frozen material, supplementing our previous work with conventionally-prepared material. We studied virus particles just inside the nuclear envelope, between the leaflets of the nuclear membrane, and just outside the membrane at various stages of envelopment, and compared these particles to mature extracellular particles.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它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 乔尔·D·贝恩斯康奈尔大学 NIH 2R01GM050740-09 单纯疱疹病毒终止酶，01/95-12/07 乔尔·D·贝恩斯康奈尔大学 NIH F32GM067519 HSV DNA 包装中的 ATP 水解，C.L.，2003-2005 年。 C.L.达菲康奈尔大学 抽象的 核衣壳的胞外疱疹病毒病毒粒子被称为外皮的无定形蛋白质层包围，外皮又由脂质病毒包膜组成，已知病毒粒子从内核膜出芽。获得初始的病毒体包膜，然后在核膜的小叶之间积聚，病毒体包膜最终与外核膜融合。小叶，将去包膜的核衣壳倾倒到细胞质中，最终被重新包膜并分泌到细胞外空间中，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，因为断层扫描可以对单个粒子进行原位分析，并且不会由于对称平均而产生偏差，这改变了包络过程如何发生的现有范式，并且表明疱疹病毒以意想不到的方式利用细胞。 疱疹病毒会导致人类许多重要的医疗状况，包括免疫受损和免疫功能正常的患者失明和脑炎。这项研究的最终目标是了解疱疹病毒包膜在核膜上的机制及其从核膜中排出的途径。所有病毒粒子的产生和退出都经历相同的途径，因此这些研究将为这一重要的病原体群体建立范例。这些步骤的保守性表明，干扰的化合物是可能的。该途径对所有疱疹病毒均有效。 使用断层扫描对疱疹病毒颗粒进行表征将为病毒体出口途径提供新的视角，并为病毒体和病毒蛋白的结构和功能提供新的见解。 该项目的首要目标是阐明野生型和突变体颗粒与内核膜包封机制的结构。尽管贝恩斯实验室已经确定了三种包封机制，但内核膜包封机制的性质尚不清楚。候选基因（基因 UL11、UL31 和 UL34）对于包膜反应同样重要（Baines 和 Roizman，1992；Reynolds 等人，2001）。野生型和缺乏编码这些蛋白质的基因的突变病毒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，存在桥接单纯疱疹病毒（HSV）病毒粒子包膜和核衣壳之间空间的新型纤维。这些纤维的推导质量。纤维的直径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。与肌动蛋白或核纤层蛋白等细胞骨架蛋白一致，尽管核肌动蛋白的存在存在争议，但其存在现已得到广泛认可，但其功能仍然不清楚（Bettinger 等，2004）。时尚，并且重要的是要验证这种不对称性不是固定过程的伪影，因此冷冻电子显微镜对于测试一个或多个逻辑假设至关重要。如果包膜蛋白的数量参与将纤维募集到病毒体中，则将对突变病毒进行分析，如果给定的蛋白质参与纤维的募集或形成，则位于内核的颗粒中此类纤维应该不存在或畸形。感染相关突变病毒的细胞膜。 一个单独但相关的问题是外皮蛋白是否在出口途径的这个阶段被掺入。目前的教条表明外皮蛋白仅在去包膜后掺入细胞质中。核周空间中单个颗粒的结构将表明是否在该阶段。至少一部分外皮蛋白通过与核衣壳或内核膜的相互作用被带入病毒体。 第二个目的是将细胞外病毒体的结构与新生病毒体的结构进行比较，这种比较可能表明在过去仅提到过的某些步骤发生了，例如，掺入了额外的蛋白质的假设。与核衣壳一起在细胞质中形成包膜是一个长期存在的问题，并预测细胞外病毒粒子的外皮中存在额外的质量（Gr¿newald 等人， 2003）而不是新生病毒粒子。 我们在 RVBC 的最初断层扫描工作是使用传统处理的细胞的塑料切片，我们看到了核膜内叶的特殊化，与内部病毒颗粒的二十面体形状相邻并一致。核膜是病毒转运囊泡的起源（手稿正在准备中）然而，传统的制备方法不足以可靠地保存病毒体及其周围环境的更精细的结构细节。在等待制定使用冷冻水合切片进行这项工作的方案时，我们将使用高压冷冻、冷冻替代材料继续我们的工作。压力冷冻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能够在原位获得单个病毒颗粒的 3D 超微结构信息的技术，为了使结果可信，需要冷冻水合制剂的天然保存。在 RVBC 开发的，将成为我们工作的宝贵财富。 参考 1. Baines, J.D. 和 Roizman, B. (1992)。单纯疱疹病毒 1 的 UL11 基因编码促进核衣壳包封和从细胞中排出的功能。 J. Virol 66, 5168-5174。 2. Bettinger, B.T.、Gilbert, D.M. 和 Amberg, D.C. (2004)，《自然细胞生物学》。 3.Gr¿ newald, K.、Desai, P.、Winkler, D.C.、Heymann, J.B.、Belnap, D.M.、Baumeister, W. 和 Steven, A.C.（2003 年）来自冷冻电子断层扫描的单纯疱疹病毒三维结构。 302、1396-1398。 4. Reynolds, A.E.、Ryckman, B.、Baines, J.D.、Zhou, Y.、Liang, L. 和 Roller, R.J. (2001) 单纯疱疹病毒 UL31 和 UL34 蛋白形成复合物，在核边缘，是包裹核衣壳所必需的，J. Virol 75， 8803-8817。 在上一个报告期内，我们用高压冷冻材料进行了八次新的双倾斜断层扫描重建，以补充我们之前用常规制备的材料进行的工作，我们研究了核膜内部、核膜小叶之间的病毒颗粒。 ，以及处于不同包封阶段的膜外，并将这些颗粒与成熟的细胞外颗粒进行比较。