Virus and cell regulation of reovirus escape from translation inhibition

描述（由申请人提供）：呼肠孤病毒科的分节dsRNA病毒包括许多具有相当健康和经济重要性的成员。此外，哺乳动物正肠病毒（MRV）是选择性杀死肿瘤细胞的溶瘤病毒。该病毒家族的许多成员诱导翻译起始因子eIF2的磷酸化。，导致mRNA翻译受到抑制。MRV mrna逃避细胞翻译关闭。蛋白质合成的抑制也导致细胞mrna和翻译起始因子被隔离到称为应激颗粒（SGs）的细胞质结构中。最近的数据表明MRV在病毒进入时诱导SGs。随着病毒感染的进行，即使eIF2？尽管仍被磷酸化，但SGs以依赖于蛋白质合成的方式被破坏。SG破坏还与称为病毒工厂（VFs）的病毒编码结构的形成有关。MRV阻止亚砷酸钠（SA）诱导eIF2？磷酸化和MRV菌株关闭宿主细胞翻译的能力不同，在SA存在的情况下都能够翻译高水平的mRNA。这表明MRV诱导的宿主细胞翻译关闭和MRV从翻译关闭中逃脱是相互独立的，此外，从关闭中逃脱与eIF2的抑制无关。磷酸化。这些发现支持了一种假设，即SG破坏在MRV感染和逃避细胞关闭蛋白质合成中起重要作用。本提案中的实验将通过实现两个具体目标来检验这一假设。通过定义VF和SGs之间的关系，以及确定VF的形成是否为SG破坏所必需，mrv诱导SG破坏的机制将得到阐明。进一步的实验将确定SG破坏所需的MRV蛋白，并确定MRV感染是否改变SG形成所需的细胞途径。此外，将通过分离eIF2的影响来研究宿主细胞关闭环境中MRV翻译的机制。磷酸化和SG形成对MRV翻译的影响，以及在磷酸化eIF2?存在下鉴定MRV mRNA翻译所需的病毒蛋白和mRNA序列。阐明这种复杂的病毒-宿主细胞相互作用背后的机制将增加我们对这些病毒用来克服宿主细胞防御的基本机制的理解，并可能导致开发使用靶向抗病毒药物阻断该家族病原体的策略，或者增强MRV的溶瘤特性。

DESCRIPTION (provided by applicant): The Reoviridae family of segmented dsRNA viruses includes many members that are of considerable health and economic importance. Additionally, mammalian orthoreoviruses (MRV) are oncolytic viruses that selectively kill tumor cells. Many members of this virus family induce the phosphorylation of translation initiation factor eIF2?, which leads to inhibition of mRNA translation. MRV mRNAs escape cellular translation shutoff. Inhibition of protein synthesis also leads to sequestration of cellular mRNAs and translation initiation factors into cytoplasmic structures termed stress granules (SGs). Recent data suggests that MRV induces SGs during viral entry. As viral infection proceeds, even though eIF2? remains phosphorylated, SGs are disrupted in a manner dependent on protein synthesis. SG disruption also correlates with the formation of virally encoded structures called virus factories (VFs). MRV prevents sodium arsenite (SA) induction of SGs downstream of eIF2? phosphorylation, and strains of MRV that differ in their ability to shutoff host cell translation are all able to translate high levels of mRNA in the presence of SA. This suggests MRV-induced host cell translation shutoff and MRV escape from translational shutoff are independent of each other, and additionally, that escape from shutoff is independent of inhibition of eIF2? phosphorylation. These findings support a hypothesis that SG disruption plays an important role in MRV infection and escape from cellular shutoff of protein synthesis. Experiments in this proposal will test this hypothesis by accomplishing two specific goals. The mechanism of MRV-induced SG disruption will be elucidated by defining the relationship between VFs and SGs, and by determining if VF formation is necessary for SG disruption. Additional experiments will identify MRV proteins required for SG disruption, and determine if MRV infection alters cellular pathways necessary for SG formation. Additionally the mechanism(s) used for MRV translation in the host cell shutoff environment will be investigated by separating the impact of eIF2? phosphorylation and SG formation on MRV translation, and identifying viral proteins and mRNA sequences that are necessary for MRV mRNA translation in the presence of phosphorylated eIF2?. Elucidation of the mechanisms behind this complex virus-host cell interaction will increase our understanding of the basic mechanisms used by these viruses to overcome host cell defenses, and may lead to the development of strategies to either interdict the pathogens in this family using targeted anti-virals, or alternatively, to enhance the oncolytic properties of MRV. PUBLIC HEALTH RELEVANCE: Mammalian orthoreoviruses are members of a family of viruses that includes important human pathogens, and are also oncolytic viruses that selectively kill tumor cells alone or synergistically when combined with radiation or other chemotherapeutic treatments. This proposal is designed to elucidate the mechanisms used by these viruses to escape an important aspect of the host cell defense system that was recently found to play a role in tumor resistance to chemotherapy and radiation. Understanding this complex virus-host cell interaction may lead to the development of targeted anti-virals against the pathogens in this family, or allow educated manipulation of the virus to enhance its individual or synergistic oncolytic properties.