Comprehensive genetic dissection of poxvirus membrane assembly and function

痘病毒膜组装和功能的全面基因解剖

• 批准号: 10575027
• 负责人: Kartik Chandran
• 金额: $ 25.2万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10575027
• 关键词: Actins; Amino Acids; Behavior assessment; Binding; Biochemical; Biological Assay; Biology; C-Type Lectins; C-terminal; Cell surface; Cells; Collaborations; Complement; Complex; Cowpox virus; Cytoplasm; Cytoplasmic Tail; Data; Defect; Development; Dissection; GPA33 gene; Gene Combinations; Generations; Genetic; Genetic Screening; Genetic study; Growth; Human; Immunity; Individual; Infection; Internet; Intracellular Membranes; Libraries; Mammals; Mass Spectrum Analysis; Mediating; Membrane Proteins; Molecular; Monkeypox virus; Morphogenesis; Mutagenesis; Mutate; Mutation; Nature; Oncolytic; Orthopoxvirus; Persons; Phenotype; Point Mutation; Population; Poxviridae; Proteins; Proteomics; Public Health; Reporting; Risk; Role; Sensitivity and Specificity; Serial Passage; Series; Site; Smallpox; Surface; Therapeutic; Therapeutic antibodies; Vaccinated; Vaccination; Vaccines; Vaccinia virus; Viral; Viral Proteins; Virion; Virulence; Virus; Work; Zoonoses; cancer therapy; cell motility; extracellular; fitness; genomic locus; improved; in vivo; loss of function; member; membrane assembly; membrane biogenesis; mutant; mutation screening; new epidemic; next generation sequencing; novel; oncolytic vector; particle; poxvirus vectors; prevent; protein protein interaction; prototype; reverse genetics; tissue tropism; transmission process; virus genetics; virus host interaction; virus virus interaction

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.

感染哺乳动物的痘病毒属(正痘病毒)成员的人畜共患病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在其表面携带8种编码病毒的膜蛋白的独特互补，这些蛋白影响组织趋向性、在体内的远程传播，并最终影响毒力；为疫苗诱导的抗体和治疗性抗体提供关键靶点；是溶瘤痘病毒载体治疗潜力的重要决定因素。尽管EVS很重要，但对EVS的全面理解受到了病毒-宿主和病毒-病毒相互作用的复杂网络的挑战，这些网络是EVS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相结合的方法来解开这些功能蛋白质相互作用网络。使用一个新的平台对原型正痘病毒(VACV)中的任何基因组位点进行无偏见和深度突变，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适合性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，我们将进行遗传修饰和蛋白质组筛选，以揭示A33/A34与病毒和宿主蛋白的新的功能相互作用。总而言之，这些研究将提供有关EV如何在感染期间组装、出口和传播到新细胞的新信息。EV对正痘病毒的毒力和作为溶瘤载体的效用都是至关重要的。