Molecular Mechanisms of Alphavirus Entry and Exit

描述(申请人提供)：甲病毒和黄病毒是具有结构和功能相关的膜融合蛋白的小包膜正义RNA病毒。这些病毒包括重要的人类病原体，如脑炎甲型病毒和基孔肯雅病毒，以及黄病毒登革热、黄热病和西尼罗河病毒。这些病毒中的许多被归类为A、B或C类优先病原体，但尽管它们在医学上很重要，但目前还没有可用的抗病毒疗法。虽然甲病毒和黄病毒在结构上有很好的特征，但对病毒生命周期中涉及的宿主因子知之甚少。甲型病毒和黄病毒通过网状蛋白介导的进入和低pH在内吞途径中触发的膜融合反应来感染细胞。新的甲型病毒粒子通过宿主细胞质膜发芽，而新生的黄病毒则发芽进入内质网。这笔赠款的长期目标是确定甲病毒和黄病毒进出的分子机制，以及宿主蛋白在这些过程中的作用。我们的研究将集中在高度发达的甲型病毒实验系统塞姆利基森林病毒和辛德比斯病毒，以及新出现的甲型病毒病原体基孔肯雅病毒。将使用三种综合和互补的方法来解决甲病毒进入和退出的关键特征：1.我们将确定通过我们的全基因组siRNA筛查确定的细胞蛋白在甲病毒生命周期中涉及的新宿主因子中的作用。通过生物信息学分析确定命中的优先顺序，用几种字母病毒进行确认，并对病毒感染的关键步骤进行差异筛选。现在将确定这些宿主蛋白促进或抑制甲型病毒进入、复制和退出的机制。2.小病毒膜蛋白6K通过一种未知的机制促进甲型病毒的萌发，这种机制可能涉及直接作用和/或作用于细胞蛋白。我们的初步结果表明，细胞膜蛋白BST2/tetherin抑制甲型病毒的释放。我们将确定Tetherin抑制的机制，并确定Tetherin是否被6K或其他病毒蛋白拮抗。我们将测定6K在芽部位直接调节膜曲率的活性。3.将利用生化和成像方法研究甲型病毒退出的细胞生物学，特别是 病毒核衣壳核心的转运机制和组装位置，以及最终包膜步骤的性质和宿主和病毒蛋白的机制作用。这一结果将进一步加深我们对甲病毒进出分子机制的理解，为病毒病的发病机制和抗病毒治疗的新策略提供重要信息，并为黄病毒的研究提供富有成效的新途径。

DESCRIPTION (provided by applicant): Alphaviruses and flaviviruses are small enveloped plus-sense RNA viruses with structurally and functionally-related membrane fusion proteins. These viruses include important human pathogens such as the encephalitic alphaviruses and chikungunya virus, and the flaviviruses dengue, yellow fever, and West Nile virus. Many of these viruses are classified as category A, B or C priority pathogens, yet in spite of their medica importance there are no available antiviral therapies. Although alphaviruses and flaviviruses are well-characterized structurally, relatively little is known about host factors involved in the viru lifecycle. Alphaviruses and flaviviruses infect cells by clathrin-mediated entry and a membrane fusion reaction triggered by the low pH in the endocytic pathway. New alphavirus particles bud through the host cell plasma membrane, while nascent flaviviruses bud into the endoplasmic reticulum. The long-term goal of this grant is to define the molecular mechanisms of alphavirus and flavivirus entry and exit and the role of host proteins in these processes. Our studies will focus on the highly-developed alphavirus experimental systems Semliki Forest virus and sindbis virus, and the emerging alphavirus pathogen chikungunya virus. Three integrated and complementary approaches will be used to address key features of alphavirus entry and exit: 1. We will define the roles of cellular proteins identified by our genome-wide siRNA screen for novel host factors involved in the alphavirus lifecycle. Hits were prioritized by bioinformatics analysis, confirmed with several alphaviruses, and differentially screened for key steps in virus infection. The mechanisms by which these host proteins promote or inhibit alphavirus entry, replication and exit will now be determined. 2. The small viral membrane protein 6K promotes alphavirus budding by an unknown mechanism that may involve direct effects and/or effects on cellular proteins. Our preliminary results show that the cellular membrane protein BST2/tetherin inhibits alphavirus release. We will define the mechanism of tetherin inhibition and determine if tetherin is antagonized by 6K or other viral proteins. We will determine the activity of 6K in directly modulating membrane curvature at the bud site. 3. Biochemical and imaging approaches will be used to study the cell biology of alphavirus exit, focusing in particular on the transport mechanism and assembly site of the viral nucleocapsid core, and on the properties of the final envelopment step and the mechanistic roles of host and viral proteins. Results will further our understanding the molecular mechanisms of alphavirus entry and exit, provide critical information on viral disease mechanisms and novel strategies for anti-viral therapy, and suggest fruitful new avenues for research on the flaviviruses.