Structure function studies of a molecular complex for generating viral membrane

包膜病毒复制的基本步骤是病毒包膜的产生。虽然大多数包膜病毒通过从细胞隔室出芽获得其包膜，但核质大DNA病毒（NCLDV），包括痘病毒、asfarvirus和拟病毒，通过源自内质网（ER）的开放末端新月形膜的组装获得其初级包膜。这种极不寻常的膜生物发生过程在半个多世纪里一直是个谜。最近对牛痘病毒（VACV）的研究已经确定了五种病毒蛋白对这一过程至关重要。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，但其作用机制尚不清楚。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，包括鉴定VACV A6为VMAP以及确定A6和另一种VACV VMAP H7的结构。我们发现H7结合磷脂酰肌醇-3-磷酸（PI3P）和磷脂酰肌醇-4-磷酸（PI4P），并且A6 C-末端结构域（A6-C）捕获具有膜双层样构型的多个脂质，揭示了用于封闭脂质双层的新的分子形态。此外，我们通过采用一种新的实验病毒进化方法，发现了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，协同工作以产生和/或稳定开放式膜片。我们的长期目标是充分阐明痘病毒膜生物发生过程，目前的目标是测试我们的新假设，并确定A6和H7协调膜断裂和重塑的分子机制。目标1.确定A6与H7结合脂质的机制。目标二。确定A6和H7在膜断裂中的作用。对痘病毒VMAPs的拟议研究不仅将阐明抗病毒开发的关键病毒复制步骤，还将为细胞膜断裂和重塑过程提供见解。

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.