Simian Virus 40 (SV40) uncoating and penetration

描述（由申请人提供）：该提案的总体目标是阐明以猿猴病毒40 （SV40）为模型病毒的非包膜DNA病毒的病毒脱壳机制。病毒利用宿主细胞机制在活细胞内复制。它们通过与特定宿主细胞表面受体结合并破坏已建立的内吞途径而内化。在内化过程中，病毒必须摆脱在内吞过程中获得的膜包封。为了实现这一点，病毒进化出了穿透或渗透内吞膜的机制，或者通过膜运输它们的基因组。包括SV40在内的一些病毒被转运到内质网（ER）。在这个过程中，病毒失去了它的内吞膜外衣，并被释放到内质网腔中，在那里它获得了更大的细胞器膜屏障。最近的数据表明，病毒在内质网内分解，其基因组被运送到细胞质/细胞核。这有利于基因组复制、病毒蛋白的胞质合成和病毒组装。病毒脱衣和基因组渗透的机制在很大程度上都是未知的。内质网是一个专门的成熟室，支持成千上万的膜嵌入和可溶性分泌蛋白的折叠和组装。内质网还具有质量控制活性，确保只有正确折叠和组装的蛋白质通过分泌途径输出。用于分泌货物成熟和质量控制的常驻内质网蛋白很可能被几种非包膜病毒篡夺，以剥去其二十面体衣壳的涂层。内质网是一种受严格控制的细胞器，能够通过未折叠的蛋白质反应信号转导途径重塑自身以改变成熟和质量控制之间的平衡。我们最近确定SV40的次要结构蛋白VP2和VP3能够翻译后插入内质网膜。这些蛋白似乎具有病毒孔蛋白活性，因为它们可以穿透细菌膜，支持VP2和VP3在病毒基因组穿透内质网膜中的潜在作用。我们假设病毒在进入急诊室时，在住院伴侣和质量控制因素的帮助下游离。它的基因组和病毒蛋白随后从内质网逆转录到细胞质/细胞核，这一过程涉及内质网驻留蛋白和插入膜的VP2和VP3， VP2和VP3在颗粒解体或脱壳后被释放。本课题的具体目的是：(1)探讨SV40感染对内质网生理或功能的影响；(2)确定病毒组分（基因组、主要和次要结构蛋白）进入内质网后的状态；(3)利用新开发的体外反转录易位试验研究基因组穿透内质网膜的机制。总之，这些研究将为非包膜病毒的剥膜和渗透过程提供有价值的见解。公共卫生相关性：这些研究将对非包膜病毒生命周期中的两个重要步骤提供有价值的见解；剥离和渗透。利用猿猴病毒40 （SV40）作为模型病毒，表征该病毒在细胞内分解并将其基因组转移到复制位点的机制，将使我们对这种小DNA肿瘤病毒及其相关的人类病毒BK、JC、新发现的KI和WU病毒以及与宫颈癌相关的乳头状瘤病毒的生物学有更深入的了解。此外，了解病毒介导的DNA传递过程对于提高基因治疗中DNA传递载体的效率也有潜在的应用。

DESCRIPTION (provided by applicant): The overall goal of the proposal is to elucidate the mechanism of viral uncoating for nonenveloped DNA viruses using Simian Virus 40 (SV40) as a model virus. Viruses replicate within living cells by exploiting the host cellular machinery. They are internalized by binding to their specific host cell surface receptor and pirating established endocytic pathways. Upon internalization, the virus must shed its membrane encapsulation acquired during endocytic entry. To accomplish this, viruses have evolved mechanisms to penetrate or permeabilize the endocytic membrane, or to transport their genome across membranes. Some viruses, including SV40 are transported to the endoplasmic reticulum (ER). During this process, the virus loses its endocytic membrane coat and is released into the ER lumen where it acquires the larger organelle membrane barrier. Recent data suggests that within the ER the virus disassembles and its genome is delivered to the cytosol/nucleus. This facilitates genome replication, the cytosolic synthesis of viral proteins, and viral assembly. Both the mechanisms of viral uncoating and genome penetration are largely unknown. The ER is a specialized maturation compartment that supports the folding and assembly of thousands of membrane embedded and soluble secretory proteins. The ER also possesses a quality control activity that ensures that only properly folded and assembled proteins are exported through the secretory pathway. Resident ER proteins dedicated to the maturation and quality control of secretory cargo are likely usurped by several nonenveloped viruses for the uncoating of their icosohedral capsids. The ER is a tightly controlled organelle that is able to remodel itself to alter the balance between maturation and quality control through the unfolded protein response signal transduction pathway. We have recently determined that the SV40 minor structural proteins VP2 and VP3 are capable of post- translationally inserting into ER membranes. These proteins appear to possess viroporin activity since they permeabilize bacterial membranes supporting a potential role for VP2 and VP3 in viral genome penetration of the ER membrane. We hypothesize that during entry the virus disassociates in the ER with the help of resident chaperones and quality control factors. Its genome along with viral proteins are then retrotranslocated from the ER to the cytosol/nucleus in a process that involves ER resident proteins and the membrane inserted VP2 and VP3, which are liberated following particle disassembly or uncoating. The specific aims of this proposal are: (1) to explore the affect of SV40 infection on ER physiology or function; (2) to determine the status of the viral components (genome, major and minor structural proteins) after entry into the ER; and (3) to investigate the mechanism of genome penetration of the ER membrane using a newly developed in vitro retrotranslocation assay. Together, these studies will provide valuable insight into the uncoating and penetration processes for nonenveloped viruses. PUBLIC HEALTH RELEVANCE: These studies will provide valuable insight into two essential steps in the life cycle of nonenveloped viruses; uncoating and penetration. The employment of Simian Virus 40 (SV40) as a model virus to characterize the mechanisms utilized by the virus to disassemble within the cell and translocate its genome to its site of replication will provide us with a deeper understanding of the biology for this small DNA tumor virus and its related human viruses BK, JC, the newly discovered KI and WU viruses, as well as papilloma viruses, which are associated with cervical cancer. Furthermore, understanding the viral-mediated DNA delivery process also has potential applications for increasing the efficiency of DNA delivery vehicles for gene therapy.