Biogenesis of the Poxvirus Membrane

描述(申请人提供)：痘病毒在DNA病毒中是非常不寻常的，因为它只在受感染细胞的细胞质内复制。这种脱离细胞核的物理独立性伴随着遗传的复杂性：大约200种病毒基因产物调节病毒生命周期的不同方面。尽管有这种遗传自主性，但细胞之间的密切关系 生物过程和病毒生命周期的进程越来越清楚。在细胞质中新生病毒粒子的形态发生过程中，这种相互作用最为明显。痘病毒膜的生物发生是该领域最耐人寻味和最令人困惑的问题之一。遗传分析已经确定F10蛋白激酶和A6、A11、A30.5、H7和L2调节蛋白是新月生物发生的基本调节蛋白。在没有它们的情况下，膜转移和新月的形成是有缺陷的，但关于它们是如何协调和诱导膜从内质网外流的知之甚少。同样，当主要的膜蛋白A14或A17被抑制时，新月体不形成，但细胞质中积累了大量的~25 nm小泡。因此，它们对新生细胞膜的成形、稳定性和延伸性至关重要。它们在调解这些进程中的作用尚不清楚。我们的长期目标是全面了解痘病毒膜的生物发生。我们目前的目标是确定使病毒ER出口位点组装和输出的宿主蛋白，了解病毒调节蛋白介导这一过程的机制，并评估A14和A17与膜的动态相互作用。我们将通过使用遗传、细胞生物学和生化方法来实现这些目标，以实现三个相辅相成的目标。目的I：分析宿主蛋白和膜转运系统在病毒形态发生中的作用。我们将鉴定和分析作为F10蛋白激酶底物的宿主蛋白，与病毒膜中间体相关，或对病毒膜生物发生重要。目的II：分析A6、A11、A30.5、H7和L2蛋白及其在新月生物发生中的作用。这一目标的重点是表征这5种调节蛋白的生化特性，评估它们彼此之间、与膜和与宿主蛋白的相互作用，并深入了解它们如何指导和促进膜的生物发生。目的：评价A14和A17在体内和体外与细胞膜的关系。使用各种创新的体外和体内技术，我们将探索这些蛋白质在膜内的拓扑结构，并验证它们介导膜管形成或异型融合的假设。

DESCRIPTION (provided by applicant): Poxviruses are highly unusual among DNA viruses in replicating solely within the cytoplasm of the infected cell. This physical autonomy from the nucleus is accompanied by genetic complexity: ~200 viral gene products regulate diverse aspects of the viral life cycle. Despite this genetic autonomy, the close relationship between cell biological processes and the progression of the viral life cycle is increasingly clear. Nowhere is this interplay more evident than during the morphogenesis of nascent virions in the cytoplasm. The biogenesis of the poxvirus membrane is one of the most intriguing and puzzling questions in the field. Genetic analyses have identified the F10 protein kinase, and the A6, A11, A30.5, H7 and L2 regulatory proteins as essential mediators of crescent biogenesis. In their absence, membrane diversion and crescent formation are defective, but little is known about how they coordinate and induce membrane egress from the ER. Likewise, when the major membrane proteins A14 or A17 are repressed, crescents do not form, but large numbers of ~25 nm vesicles accumulate in the cytoplasm. Thus, they are essential for the shaping, stability and elongation of nascent membranes. Their role in mediating these processes remains unknown. Our long-term goal is to fully understand poxviral membrane biogenesis. Our current objectives are to identify host proteins that enable the assembly and egress of viral ER exit sites, to understand the mechanism by which the viral regulatory proteins mediate this process, and to evaluate the dynamic interaction of A14 and A17 with membranes. We will accomplish these objectives by using genetic, cell biological and biochemical approaches to pursue three complementary Aims. Aim I: Analysis of the role of host proteins and membrane trafficking systems in viral morphogenesis. We will identify and analyze host proteins that are either substrates of the F10 protein kinase, associated with viral membrane intermediates, or important for viral membrane biogenesis. Aim II: Analysis of the A6, A11, A30.5, H7 and L2 proteins and their contributions to crescent biogenesis. This aim is focused on characterizing the biochemical properties of these 5 regulatory proteins, assessing their interaction with each other, with membranes, and with host proteins, and gaining insight into how they direct and facilitate membrane biogenesis. Aim III: Assessment of the association of A14 and A17 with membranes in vivo and in vitro. Using a variety of innovative in vitro and in vivo techniques, we will probe the topology of these proteins within membranes and test the hypothesis that they mediate membrane tubulation or heterotypic fusion.