Understanding the Function of F13 as a Matrix Protein for Poxvirus Intracellular Envelopment

痘病毒是DNA病毒的一个大家族，其中有几个成员能够感染人类并引起人类疾病。虽然最臭名昭著的成员天花病毒是天花的病原体，并已从自然感染中根除，但仍有人担心在生物攻击期间秘密释放。此外，该家族的其他成员，如猴痘病毒，引起了人们对能够引起流行病的动物流行病感染的关注。由于这些原因，NIH/NIAID将几种痘病毒列为A类优先病原体。痘病毒产生两种感染形式，细胞内成熟病毒（IMV）和细胞外病毒（EV）。EV是由IMV的细胞内增殖形成的，并且对于细胞间传播、全身感染和发病至关重要。我们研究的长期目标是了解正痘病毒包封、运输和释放传染性EV的分子机制。已知只有9种病毒蛋白是EV形式所特有的。虽然这9种蛋白质具有某些功能，但它们都不是基质样蛋白质，与病毒的IMV形式直接相关以协调表达。本申请的直接目标是更好地确定推定的基质蛋白F13在EV细胞内表达中的作用及其与其他EV糖蛋白的关系。为了更好地理解F13的功能，我们提出了3个目标：1）询问F13和IMV表面蛋白之间的相互作用。我们假设F13作为一种基质蛋白，并提供了外部EV膜和内部IMV颗粒之间的联系，并促进与IMV在细胞内增殖，TGN的网站的相互作用。在这个目标中，我们将进一步表征F13和IMV表面蛋白之间的相互作用。2)发现与假定的基质蛋白F13相互作用的细胞和病毒蛋白。我们将使用BioID来识别在特定的表达阶段与F13相互作用的病毒和细胞蛋白。3）。确定F13、糖蛋白含量、EV细胞结合和病毒进入的非融合溶解之间的关系。我们将利用一组重组病毒来确定F13如何控制糖蛋白含量以及这如何影响EV的细胞结合和进入。从这些研究中获得的结果将为痘病毒用于产生感染性EV，细胞间传播和在宿主中引起疾病的分子机制提供更深入的了解。这些信息将反过来为设计用于疫苗和溶瘤平台的重组痘病毒载体提供明智的决策。

Poxviruses are a large family of DNA viruses with several members capable of infecting and causing disease in humans. Whereas the most notorious member, variola virus, is the causative agent of smallpox and was eradicated from natural infection, there are still concerns about a clandestine release during a biological attack. In addition, other members of the family, such as monkeypox virus, have raised concern about epizootic infections that are capable of causing epidemics. For these reasons several poxviruses are listed as Category A priority pathogens by NIH/NIAID. Poxviruses produce two infectious forms, intracellular mature virus (IMV) and extracellular virus (EV). EV are formed by the intracellular envelopment of IMV and are critical for cell-to-cell spread, systemic infection, and pathogenesis. The long-term goal of our research is to understand the molecular mechanisms employed by orthopoxviruses to envelope, transport, and release infectious EV. Only 9 viral proteins are known to be unique to the EV form. Whereas some functions have be assigned to these 9 proteins, none of them have been shown to be a matrix- like protein and make a direct connection with the IMV form of the virus to coordinate envelopment. The immediate goal of this application is to better define the role of the putative matrix protein F13 in intracellular envelopment of EV and its relationship with the other EV glycoproteins. We propose 3 aims to better understand the function of F13: 1) Interrogate interactions between F13 and IMV surface proteins. We hypothesize that F13 acts as a matrix protein and provides a link between the outer EV membrane and the inner IMV particle and facilitates interactions with IMV at the site of intracellular envelopment, the TGN. In this aim we will further characterize interactions between F13 and IMV surface proteins. 2) Uncover cellular and viral proteins that interact with the putative matrix protein F13. We will use BioID to identify viral and cellular proteins that interact with F13 during specific stages of envelopment. 3). Determine the relationship between F13, glycoprotein content, EV cell binding, and non fusogenic dissolution for virus entry. We will utilize a panel of recombinant viruses to determine how F13 controls glycoprotein content and how this effects cell binding and entry of EV. The results obtained from these studies will provide greater insight into the molecular mechanism poxviruses use to produce infectious EV, spread cell-to-cell, and cause disease in their hosts. This information will in turn inform intelligent decisions in designing recombinant poxvirus vectors for both vaccines and oncolytic platforms.