Structure function studies of a molecular complex for generating viral membrane

复制被囊膜病毒的一个基本步骤是产生病毒包膜。大多数被囊膜病毒的包膜是从细胞室发芽获得的，而核质大DNA病毒(NCLDV)，包括痘病毒、阿斯法病毒和拟态病毒，则是通过内质网(ER)来源的开放的新月形膜组装获得其初级包膜的。半个多世纪以来，这种非常不寻常的膜生物发生过程一直是个谜。最近对痘苗病毒(VACV)的研究已经确定了五种病毒蛋白在这一过程中各自必不可少。这些所谓的病毒膜组装蛋白(VMAP)被认为参与产生和/或稳定ER膜的断裂，但它们的作用机制尚不清楚。十多年来，我们对VACV膜生物发生过程的理解做出了持续的贡献，包括VACV A6作为VMAP的鉴定以及A6和另一种VACV VMAP H7的结构的确定。我们发现，H7结合了磷脂酰肌醇-3-磷酸(PI3P)和磷脂酰肌醇-4-磷酸(PI4P)，A6 C末端结构域(A6-C)以膜双层结构捕获多种脂质，揭示了一种新的包裹脂双层的分子方式。此外，我们采用了一种新的实验性病毒进化方法，揭示了A6和H7之间的重要相互作用。这些发现导致了我们的创新假设，即H7和A6分别与磷脂的亲水性头部和疏水性酰基尾部结合，共同作用产生和/或稳定开放的膜片。为了全面阐明痘病毒膜的生物发生过程，我们目前的目标是检验我们的新假说，并确定A6和H7在膜断裂和重塑中协调的分子机制。目的1.确定A6与H7结合脂类的作用机制。目的2.确定A6和H7在膜脱落形成中的作用。对痘病毒VMAP的研究不仅将阐明抗病毒发展的关键病毒复制步骤，而且还将为细胞膜断裂和重塑过程提供深入的见解。

A fundamental step in replication of enveloped viruses is the generation of viral envelope. While most enveloped viruses obtain their envelope by budding from cellular compartments, nucleocytoplasmic large DNA viruses (NCLDVs), including poxvirus, asfarvirus and mimivirus, acquire their primary envelope through assembly of open-ended, crescent membranes derived from endoplasmic reticulum (ER). This highly unusual process of membrane biogenesis has been enigmatic for over half a century. Recent studies with vaccinia virus (VACV) have identified five viral proteins to be individually essential for this process. These so-called viral membrane assembly proteins (VMAPs) are thought to be involved in generating and/or stabilizing scission of ER membranes, but their mechanisms of action are unknown. We have made sustained contributions to the understanding of VACV membrane biogenesis process for over a decade, including the identification of VACV A6 as a VMAP and the determination of the structures of A6 and another VACV VMAP, H7. We found that H7 binds phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P) and that A6 C- terminal domain (A6-C) traps multiple lipids with a membrane bilayer-like configuration, revealing a novel molecular modality for enclosing the lipid bilayer. Moreover, we uncovered an essential interaction between A6 and H7 by employing a novel experimental viral evolution approach. These findings led to our innovative hypothesis that H7 and A6 bind respectively to the hydrophilic head and the hydrophobic acyl tail of phospholipids, working in concert to generate and/or stabilize open-ended membrane sheets. With a long-term goal of fully elucidating the poxvirus membrane biogenesis process, our current objective is to test our novel hypothesis and determine the molecular mechanism by which A6 and H7 coordinate in membrane scission and remodeling. Aim 1. To determine the mechanism by which A6 coordinates with H7 in binding lipids. Aim 2. To determine the roles of A6 and H7 in generating membrane scissions. The proposed study on poxvirus VMAPs will not only elucidate a key viral replication step for antiviral development but also provide insights into the process of cellular membrane scission and remodeling.