Comprehensive genetic dissection of poxvirus membrane assembly and function

哺乳动物感染痘病毒的正痘病毒属成员具有人畜共患的潜力，对全球公共卫生构成持续威胁。正痘病毒在宿主细胞质中增殖的复杂生物学特征是产生两种不同的感染形式——细胞内成熟病毒粒子（IMV）和包膜病毒粒子（EV），前者主要与细胞相关，后者对细胞外病毒传播至关重要。ev在其表面具有八种独特的病毒编码膜蛋白补体，这些蛋白影响组织趋向性、体内远距离传播并最终影响毒力；提供疫苗诱导抗体和治疗性抗体的关键靶点；并且是溶瘤痘病毒载体治疗潜力的重要决定因素。尽管它们很重要，但对ev的全面理解受到病毒-宿主和病毒-病毒相互作用的复杂网络的挑战，这些网络是其形态发生和功能的基础。我们的总体目标是通过遗传学和蛋白质组学方法的结合来揭示这些功能性蛋白质相互作用网络。利用一个对原型正痘病毒——牛痘病毒（VACV）的任何基因组位点进行无偏深突变的新平台，我们在感染性病毒颗粒的背景下对两种EV蛋白A33和A34进行了深度突变扫描（DMS），并发现了增强EV适应度的新的区域特异性突变。我们将阐明这些突变体的作用机制。同时，我们将进行基因修饰和蛋白质组学筛选，以发现A33/A34与病毒和宿主蛋白的新的功能相互作用。总的来说，这些研究将提供新的信息，了解ev -对正痘病毒的毒力及其作为溶瘤载体的效用至关重要-在感染期间如何组装，出口和传播到新细胞。

The zoonotic potential of members of the genus Orthopoxvirus (orthopoxviruses) of mammal- infecting poxviruses represent a continued threat to global public health. A hallmark of the complex biology of orthopoxvirus multiplication in the host cell cytoplasm is the generation of two distinct infectious forms—intracellular mature virions (IMV), which remain largely cell- associated, and enveloped virions (EV), which are critical for extracellular viral spread. EVs bear a unique complement of eight virus-encoded membrane proteins at their surface that influence tissue tropism, long-range in vivo dissemination, and ultimately, virulence; provide key targets for vaccine-induced and therapeutic antibodies; and are important determinants of the therapeutic potential of oncolytic poxvirus vectors. Despite their importance, a comprehensive understanding of EVs is challenged by the complex networks of virus-host and virus-virus interactions that underlie their morphogenesis and function. Our overall objective is to unravel these functional protein interaction networks through a combination of genetic and proteomic approaches. Using a new platform for unbiased and deep mutagenesis of any genomic locus in the prototypic orthopoxvirus, vaccinia virus (VACV), we conducted deep-mutational scans (DMS) of two EV proteins, A33 and A34, in the context of infectious viral particles and identified novel regiospecific mutations that enhance EV fitness. We will elucidate the mechanisms of action of these mutants. In parallel, we will perform genetic modifier and proteomic screens to uncover new functional interactions of A33/A34 with viral and host proteins. Collectively, these studies will provide new information on how EVs—critical for both the virulence of orthopoxviruses and their utility as oncolytic vectors—assemble, egress, and spread to new cells during infection.