Protein Interactions Involved in Orthopoxvirus Envelopment

描述（由申请人提供）：痘病毒科的正痘病毒属包括天花的病原体，天花病毒和用于根除天花的疫苗，牛痘病毒。最近的事件重新引起人们对正痘病毒的兴趣，因为人们担心天花病毒或其近亲之一可能被用作生物武器。正痘病毒产生包膜和非包膜病毒粒子，两种类型都具有传染性。虽然非包膜病毒粒子构成了大多数子代病毒粒子，但包膜形式（EV）是细胞间传播、全身感染和毒力所必需的。工作的主要目的是确定包膜病毒特异性蛋白协调正痘病毒细胞内包膜的分子机制。我们的假设是，三种EV特异性蛋白A33、A34和B5的管腔结构域相互作用，并且这种相互作用是退出内质网和随后将B5（中和抗体的主要靶点）适当地靶向到细胞内包膜位点并随后进入感染性包膜病毒粒子所必需的。具体目的是：1)定位A33、A34和B5的残基，以实现合适的靶向和相互作用。这里提出的研究将使用一种创新的活反互补显微镜分析和联合ip来绘制相互作用区域。2)电动汽车生产中A33、A34和B5的关系表征。将创建表达Aim1中定义的A3R、A3R和BR突变的重组病毒，以表征相互作用在病毒形态发生过程中的作用。3) A33、A34和B5相互作用的时空特征。创新的荧光测定法FRET和BiFC将用于跟踪活细胞中的相互作用。绘制这些蛋白质上相互作用的位点将有助于定义功能域，并为更好地理解它们的结构和功能提供帮助。B5R蛋白是包膜痘病毒形成所必需的。我们将研究这种重要的蛋白质是如何被纳入并协调新病毒的形成的。更好地了解这一过程将为针对痘病毒的抗病毒药物提供新的靶点。

DESCRIPTION (provided by applicant): The Orthopoxvirus genus of the family Poxviridae includes both the causative agent of smallpox, variola virus, and the vaccine used in its eradication, vaccinia virus. Recent events have renewed interest in Orthopoxviruses because of concerns that variola virus or one of its close relatives could be used as a biological weapon. Orthopoxviruses produce both enveloped and unenveloped virions and remarkably both types are infectious. While non-enveloped virions make up the majority of progeny virions the enveloped form (EV) is required for cell-to-cell spread, systemic infection and virulence. The major objective of work is to determine the molecular mechanism employed by envelope virus specific proteins to coordinate the intracellular envelopment of Orthopoxviruses. Our hypothesis is that the lumenal domain of three EV specific proteins, A33, A34 and B5, interact and that interaction is required for exit from the ER and subsequently proper sub-cellular targeting of B5, which is the main target of neutralizing antibodies, to the site of intracellular envelopment and subsequently into infectious enveloped virions. The specific aims are: 1) Mapping residues of A33, A34 and B5 required for proper targeting and interaction. The studies proposed here will use an innovative live trans complementation microscopy assay and co-IP to map regions of interaction. 2) Characterization of the relationship between A33, A34 and B5 in EV production. Recombinant viruses will be created that express A3R, A3R and BR mutations defined in Aim1 to characterize the role the interaction has during viral morphogenesis. 3) Temporal and spatial characterization of A33, A34 and B5 interaction. The innovative fluorescent assays FRET and BiFC will be used to track the interaction in live cells. Mapping sites of interaction on these proteins will help define functional domains and provide for a better understanding of their structure and function. The B5R protein is required for the formation of enveloped poxviruses. We will investigate how this important protein is incorporated into and coordinates the formation of newly made viruses. A better understanding of this process will provide new targets for antivirals directed against poxviruses.