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: 7598370
• 负责人: John S Parker
• 金额: $ 1.72万
• 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/7598370
• 关键词: 3-Dimensional; Autophagosome; Binding Proteins; Caliber; Capsid; Capsid Proteins; Cell Nucleus; Cells; Cellular Structures; Cellular biology; Child; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Cytoskeleton; Cytosol; Data; Deposition; Dimensions; Double-Stranded RNA; Electron Microscopy; Excision; Face; Filament; Funding; GDF15 gene; Gastroenteritis; Genetic Transcription; Genome; Genomics; Golgi Apparatus; Grant; Half-Life; Human; In Situ; In Vitro; Institution; Intermediate Filaments; Link; Localized; Lung diseases; Membrane; Messenger RNA; Microfilaments; Microscopy; Microtomy; Microtubule-Associated Proteins; Microtubules; Mitochondria; Modeling; Molecular Chaperones; Molecular Genetics; Molecular Machines; Morphogenesis; Mus; Nature; Nonstructural Protein; Nucleosome Core Particle; Organelles; PLAB Protein; Pathogenesis; Penetration; Process; Production; Protein Biosynthesis; Proteins; Recruitment Activity; Reovirus; Research; Research Personnel; Resistance; Resources; Retroviridae; Rotavirus; Site; Small Interfering RNA; Source; Structure; Thick; United States National Institutes of Health; Viral; Viral Core Proteins; Viral Proteins; Virion; Virus; Work; abstracting; cancer cell; design; gastrointestinal; interest; killings; knock-down; multicatalytic endopeptidase complex; neoplastic; particle; protein structure; viral RNA

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. ABSTRACT: 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 mm. 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 mNS 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 mNS 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 mNS forms the filamentous matrix or confirming that m2 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 mNS 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 mNS 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 mNS 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 (m1) 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 m1 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.