3-D ULTRASTRUCTURAL STUDIES OF RETROVIRUS FACTORIES

逆转录病毒工厂的 3-D 超微结构研究

• 批准号: 7357292
• 负责人: John S Parker
• 金额: $ 0.56万
• 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/7357292
• 关键词: ULTRASTRUCTURAL; STUDIES; RETROVIRUS; FACTORIES

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. Reoviruses cause mild gastrointestinal and respiratory disease in children and have been used for many years to study the molecular genetics of viral replication and pathogenesis in murine models. Rotavirus is a leading cause of severe gastroenteritis in children. Recently, reoviruses have been shown to target and kill cancer cells and are being evaluated as potential human anti-neoplastic agents. Reoviruses are non-enveloped double-shelled virus particles that contain a segmented genome (10 segments) of double-stranded RNA. The double-layered virions are ~85 nm in diameter and the single-layered core particles are ~ 70 nm in diameter. Reoviruses replicate in the cytoplasm within structures called viral factories. Viral transcription begins when a viral core particle is deposited in the cytoplasm following the removal of the outer capsid layer during the virus entry and penetration processes. Each core functions as a molecular machine that contains all of the enzymatic machinery to replicate the genome, and transcribe and cap new viral mRNAs. Following initial transcription and production of new viral proteins, the viral cores are rapidly embedded within a dense matrix formed by the reovirus nonstructural ¿NS protein and a new viral factory begins to form around the core. By 18 h post-inoculation the factories are large, occupy a significant volume of the cytosol, and often surround the nucleus. I would estimate the thickness of the factories to be beween 0.1 ? 1 ?m. My interest is in trying to get more ultrastructural information about the virus factories. What we know at present is that the factories are large matrices that form in the cytosol of reovirus (and rotavirus) infected cells. We have identified the viral protein (¿NS) that forms this matrix and also a viral microtubule-associated protein (¿2) that connects the matrix to the microtubule cytoskeleton. By thin section EM, the factories are packed full of assembling and assembled viral particles, surrounded by a fine filamentous matrix, presumably ¿NS. This matrix is in turn linked to microtubules, which can be seen within the matrix and extending outside of it into the surrounding cytosol (please see attached PDF file). What I'd like to know more about is how these structures are organized in three dimensions and how they interact with the cellular cytoskeleton (microtubules, intermediate filaments, and actin filaments), membranes and other organelles. We believe that the ?NS protein may form filaments that are somewhat similar to intermediate filaments (10 nm diam.). Thin section electron microscopy studies of the factories that were done in the late 1960's early 70's described a dense filamentous matrix in which were embedded numerous assembling and assembled viral particles, and ?coated? microtubules. The filamentous nature of the matrix was described as ?Kinky? filaments and was distinguished from the more ?wavy? intermediate filaments. Some authors have suggested that the ?NS filaments and the intermediate filaments might form heteropolymers. Although we know which viral proteins localize to viral factories, we have no immunoEM data to confirm that ?NS forms the filamentous matrix or confirming that ?2 interacts with microtubules. By thin-section EM I have noted that mitochondria often appear to surround the factories. In addition, there appears to be membranes that collect near the margins of the factories, but not within them or directly contacting the factory matrix. Both immunoEM and 3-D EM data should help to clarify the localization and organization of these proteins and structures within the factories. This is turn will help us to understand how the factories work to promote viral replication and assembly. Some of the work I've been doing has been characterizing which viral proteins are recruited to the factories and how they are recruited. What we have so far is that all of the viral core proteins are recruited to viral factories or factory-like inclusions that form when you express the ?NS protein in cells. The nonstructural proteins, ?NS, is also recruited and concentrated within the factories. ?NS is a non-specific ssRNA-binding protein and we believe it functions to retain the capped viral mRNA within the factories for incorporation into new particles (the template for replication is each of the capped viral mRNAs, which are assorted and then packaged into new cores. I also have data that suggests that the molecular chaperones Hsp40 and Hsp70 are concentrated within the factories and that proteasomes are incorporated within the factories. We hypothesize that the factories are designed to sequester viral mRNA that will be packaged into new particles and to protect potential double-stranded regions of viral mRNA that may arise during assortment and packing from being exposed to PKR in the surrounding cytosol. There is now evidence from John Patton's lab at the NIH using rotaviruses supporting this idea; they have identified 2 pools of viral mRNA - one associated with the factories that is resistant to siRNA knock down and another that is free in the cytosol and presumably used as the template for new protein synthesis. The proteasome-connection to factories is interesting as the outer shell proteins of the virions are not always localized to factories and in vitro, the ¿NS protein inhibits recoating of the core particles with the outer shell proteins. One hypothesis, we have is that the ¿NS protein is dynamically being added to the factory and then removed by proteasomal degradation to expose newly assembled cores to outer capsid proteins free in the cytosol. The evidence supporting this hypothesis is that the ¿NS protein is ubiquitinated and has a half-life of approx 3-4 h. Our working model is that assortment of the 10 genomic RNAs and packaging of these into assembling core particles is coordinated within viral factories. The addition of the outer capsid proteins to the cores may then require removal of the ?NS matrix perhaps by proteasomal degradation. We hypothesize that this occurs in situ within the factory. This would imply that proteasomes also become embedded within the ?NS matrix. In support of this hypothesis, we can localize proteasomal subunits to the factories by IF microscopy. Another interesting aspect of the cell biology of these viruses we are studying is that one of the outer capsid proteins (?1) forms multiple, highly regular ring-like structures in the cytosol. These ring-like structures are sometimes seen within the factories by IF microscopy. These structures also form when the ?1 protein is expressed alone in cells. We do not know what membranes these ring-structures derive from although I have some partial colocalization data with ER and Golgi markers. We are currently investigating whether these structures are autophagosome-like. Again EM studies will help clarify this hypothesis. In summary, the reovirus factories are complex structures that form in the cytosol of reovirus infected cells. We have data on the viral proteins that are responsible for factory morphogenesis and the viral and cellular proteins that are recruited to and concentrated within the factories. The factory functions are not completely understood, but they are the sites of new viral particle assembly and they retain viral RNA. A more detailed ultrastructural understanding of how the factories associate with cellular structures such as microtubules, intermediate filaments, mitochondria, and membranes should help us to understand how these complex molecular machines are built within the cytosol of the cell in the face of cellular defenses.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。呼肠孤病毒会引起儿童轻度胃肠道和呼吸道疾病，多年来一直用于研究小鼠模型中病毒复制和发病机制的分子遗传学。轮状病毒是儿童严重胃肠炎的主要原因。最近，呼肠孤病毒已被证明可以靶向并杀死癌细胞，并被评估为潜在的人类抗肿瘤剂。呼肠孤病毒是无包膜双壳病毒颗粒，含有双链 RNA 分段基因组（10 个片段）。双层病毒颗粒直径约为 85 nm，单层核心颗粒直径约为 70 nm。呼肠孤病毒在称为病毒工厂的结构内的细胞质中复制。当病毒进入和渗透过程中去除外衣壳层后，病毒核心颗粒沉积在细胞质中，病毒转录就开始了。每个核心都充当分子机器，包含复制基因组、转录和封盖新病毒 mRNA 的所有酶机制。在最初转录和产生新病毒蛋白后，病毒核心迅速嵌入由呼肠孤病毒非结构ns蛋白形成的致密基质中，并且新的病毒工厂开始在核心周围形成。接种后 18 小时，工厂变得很大，占据了细胞质的很大一部分，并且通常围绕着细胞核。我估计工厂的厚度在 0.1 之间？ 1 米。我的兴趣是尝试获得更多有关病毒工厂的超微结构信息。目前我们所知道的是，这些工厂是在呼肠孤病毒（和轮状病毒）感染细胞的细胞质中形成的大型基质。我们已经鉴定出形成该基质的病毒蛋白（¿NS）以及将基质连接到微管细胞骨架的病毒微管相关蛋白（¿2）。通过薄切片电子显微镜，工厂里充满了组装和组装的病毒颗粒，周围环绕着细丝状基质，大概是“NS”。该基质又与微管相连，微管在基质内可见，并延伸到基质外，进入周围的细胞质（请参阅随附的 PDF 文件）。我想更多地了解的是这些结构如何在三个维度上组织以及它们如何与细胞骨架（微管、中间丝和肌动蛋白丝）、膜和其他细胞器相互作用。我们认为，?NS 蛋白可能形成与中间丝（直径 10 nm）有些相似的丝。 20世纪60年代末70年代初对工厂进行的薄切片电子显微镜研究描述了一种致密的丝状基质，其中嵌入了许多组装和组装的病毒颗粒，并被“包被”。微管。基质的丝状性质被描述为“Kinky”。细丝并与更多的“波浪形”区别开来。中间丝。一些作者提出，?NS 丝和中间丝可能形成杂聚物。尽管我们知道哪些病毒蛋白定位于病毒工厂，但我们没有免疫电镜数据来证实 ?NS 形成丝状基质或证实 ?2 与微管相互作用。通过薄片电镜，我注意到线粒体经常出现在工厂周围。此外，似乎有膜聚集在工厂边缘附近，但不在工厂内部或直接接触工厂基质。免疫电镜和 3-D 电镜数据都应有助于阐明工厂内这些蛋白质和结构的定位和组织。这反过来将帮助我们了解工厂如何促进病毒复制和组装。我一直在做的一些工作是描述哪些病毒蛋白被招募到工厂以及它们是如何被招募的。到目前为止，我们所掌握的是，所有病毒核心蛋白都被招募到病毒工厂或工厂样内含物中，这些内含物是在细胞中表达 ?NS 蛋白时形成的。非结构蛋白，?NS，也被招募并集中在工厂内。 ?NS 是一种非特异性 ssRNA 结合蛋白，我们相信它的功能是将带帽病毒 mRNA 保留在工厂内，以便掺入新颗粒中（复制的模板是每个带帽病毒 mRNA，将其分类，然后包装到新的核心中。我还有数据表明分子伴侣 Hsp40 和 Hsp70 集中在工厂内，并且蛋白酶体 并入工厂内。我们假设这些工厂的设计目的是隔离将被包装成新颗粒的病毒 mRNA，并保护在分类和包装过程中可能出现的病毒 mRNA 的潜在双链区域，以免暴露于周围细胞质中的 PKR。现在，美国国立卫生研究院 (NIH) 约翰·巴顿 (John Patton) 实验室使用轮状病毒的证据支持了这一想法。他们已经鉴定出 2 个病毒 mRNA 池——其中一个与 其中一个工厂对 siRNA 敲低有抵抗力，另一个工厂游离在细胞质中，可能用作新蛋白质合成的模板。蛋白酶体与工厂的连接很有趣，因为病毒体的外壳蛋白并不总是局限于工厂，并且在体外，NS蛋白抑制外壳蛋白对核心颗粒的重新涂覆。我们的一个假设是 ¿NS 蛋白质被动态地添加到工厂中，然后通过蛋白酶体降解去除，以使新组装的核心暴露于胞质溶胶中游离的外衣壳蛋白。支持这一假设的证据是 ¿NS 蛋白被泛素化，半衰期约为 3-4 小时。我们的工作模型是 10 种基因组 RNA 的分类以及将它们包装成组装核心颗粒的过程是在 病毒工厂。然后，将外部衣壳蛋白添加到核心可能需要通过蛋白酶体降解去除?NS基质。我们假设这发生在工厂内。这意味着蛋白酶体也嵌入到 ?NS 基质中。为了支持这一假设，我们可以通过 IF 显微镜将蛋白酶体亚基定位到工厂。另一个有趣的方面 我们正在研究的这些病毒的细胞生物学特征是，其中一种外衣壳蛋白（?1）在胞质溶胶中形成多个高度规则的环状结构。有时可以通过 IF 显微镜在工厂内看到这些环状结构。当α1蛋白在细胞中单独表达时，也会形成这些结构。尽管我有一些与内质网和高尔基体的部分共定位数据，但我们不知道这些环状结构源自什么膜 标记。我们目前正在研究这些结构是否是自噬体样的。电磁研究将再次有助于澄清这一假设。 总之，呼肠孤病毒工厂是在呼肠孤病毒感染细胞的细胞质中形成的复杂结构。我们拥有负责工厂形态发生的病毒蛋白以及被招募并集中在工厂内的病毒和细胞蛋白的数据。工厂功能没有完全理解， 但它们是新病毒颗粒组装的位点，并且保留了病毒 RNA。对工厂如何与微管、中间丝、线粒体和膜等细胞结构相关联的更详细的超微结构了解，应该有助于我们了解这些复杂的分子机器在面对细胞防御时如何在细胞的胞浆内构建。