3-D ULTRASTRUCTURAL STUDIES OF RETROVIRUS FACTORIES

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。呼肠孤病毒在儿童中引起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。轮状病毒是引起儿童严重肠胃炎的主要原因。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药物。呼肠孤病毒是无包膜的双壳病毒颗粒，含有双链RNA的片段基因组（10段）。双层病毒粒子直径约为~85 nm，单层核粒子直径约为~ 70 nm。呼肠孤病毒在细胞质中复制，这种结构被称为病毒工厂。在病毒进入和渗透过程中，当病毒核心颗粒在去除外衣壳层后沉积在细胞质中时，病毒转录开始。每个核心都是一个分子机器，包含所有的酶机制来复制基因组，转录和盖帽新的病毒mrna。在初始转录和产生新的病毒蛋白之后，病毒核心迅速嵌入呼肠孤病毒非结构n - NS蛋白形成的致密基质中，新的病毒工厂开始在核心周围形成。接种后18小时，这些工厂很大，占据了相当大的细胞质体积，并且经常围绕在细胞核周围。我估计工厂的厚度在0.1 ？1 ? m。我的兴趣是试图获得更多关于病毒工厂的超微结构信息。我们目前所知道的是，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。我们已经确定了形成这种基质的病毒蛋白（¿NS），以及将基质与微管细胞骨架连接起来的病毒微管相关蛋白（¿2）。在薄片电镜下，工厂里充满了组装和组装的病毒颗粒，周围是细丝状基质，大概是¿NS。这个基质依次与微管相连，微管可以在基质内看到，并从基质外延伸到周围的细胞质中（请参阅附件的PDF文件）。我更想知道的是这些结构是如何在三维空间中组织的，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。我们相信？NS蛋白可形成类似于中间丝（直径10纳米）的丝。60年代末70年代初对工厂进行的薄片电子显微镜研究描述了一个密集的丝状基质，其中嵌入了许多组装和组装的病毒颗粒，并被？微管。矩阵的丝状性质被描述为？变态吗?细丝和细丝与波浪状的细丝区别开来。中间丝。一些作者认为？NS长丝和中间长丝可能形成异聚物。虽然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这一点？是NS形成了丝状基质还是证实了？2与微管相互作用。通过薄切片电镜，我注意到线粒体经常围绕在工厂周围。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与工厂基质接触。免疫电镜和三维电镜数据应该有助于阐明这些蛋白质和结构在工厂中的定位和组织。这将有助于我们了解这些工厂是如何促进病毒复制和组装的。我一直在做的一些工作是描述哪些病毒蛋白质被招募到工厂以及它们是如何被招募的。到目前为止我们所知道的是所有的病毒核心蛋白都被招募到病毒工厂或类似工厂的内含物中当你表达？细胞中的NS蛋白。非结构蛋白？NS，也被招募并集中在工厂内。吗?NS是一种非特异性的ssrna结合蛋白，我们认为它的功能是将被封盖的病毒mRNA保留在工厂内，以整合到新的颗粒中(复制的模板是每个被封盖的病毒mRNA，它们被分类然后包装成新的核心。我也有数据表明分子伴侣Hsp40和Hsp70集中在工厂内蛋白酶体在工厂内结合。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，使其不暴露于周围细胞质中的PKR。美国国立卫生研究院的约翰·巴顿的实验室使用轮状病毒提供了支持这一观点的证据；他们已经确定了两个病毒mRNA库——一个与抵抗siRNA敲低的工厂有关，另一个在细胞质中是自由的，可能被用作新蛋白质合成的模板。蛋白酶体与工厂的连接是有趣的，因为病毒粒子的外壳蛋白并不总是定位于工厂，并且在体外，¿NS蛋白抑制核心颗粒与外壳蛋白的重涂。我们的一种假设是，n - NS蛋白被动态地添加到工厂中，然后通过蛋白酶体降解去除，使新组装的核暴露于细胞质溶胶中游离的外衣壳蛋白。支持这一假设的证据是，¿NS蛋白是泛素化的，半衰期约为3-4小时。我们的工作模型是，10个基因组rna的分类和包装成组装核心颗粒是在病毒工厂内协调的。将外衣壳蛋白添加到核心可能需要去除？NS基质可能被蛋白酶体降解。我们假设这种情况发生在工厂内部。这意味着蛋白酶体也会嵌入到？NS矩阵。为了支持这一假设，我们可以通过中频显微镜定位蛋白酶体亚基到工厂。我们正在研究的这些病毒细胞生物学的另一个有趣的方面是外衣壳蛋白之一(？1)在细胞质中形成多个高度规则的环状结构。这些环状结构有时在中频显微镜下可以看到。这些结构也会在？1蛋白在细胞中单独表达。我们不知道这些环状结构来自于什么膜，尽管我有一些内质网和高尔基标记物的部分共定位数据。我们目前正在研究这些结构是否类似自噬体。同样，EM研究将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。我们有关于负责工厂形态发生的病毒蛋白质的数据以及被招募并集中在工厂内的病毒和细胞蛋白质的数据。工厂的功能尚不完全清楚，但它们是新病毒颗粒组装的地点，并保留病毒RNA。对工厂如何与细胞结构（如微管、中间细丝、线粒体和膜）相关联的更详细的超微结构理解，应该有助于我们理解这些复杂的分子机器是如何在细胞防御的情况下在细胞的细胞质内构建的。

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.