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Evolution of the Vif E3 Ubiquitin Ligase

Vif E3 泛素连接酶的演变

基本信息

批准号：
9233738
负责人：
Jennifer Binning
金额：
$ 5.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9233738
关键词：
AIDS prevention APOCEC3G gene Address Affect Animals Antiviral Agents Binding Biochemical Biochemistry Biological Biological Assay Case Study Cells Cercopithecidae Chronic Complex Cullin Proteins Data Development Enzymatic Biochemistry Enzymes Event Evolution Fluorescence Polarization HIV HIV-1 Hominidae Human Immune Immune Evasion Immune response Immunity In Vitro Individual Interferometry Link Literature Measures Mediating Modeling Molecular Monkeys Mutation Analysis Negative Staining Pan Genus Pathogenesis Phenotype Polyubiquitination Population Primates Proteins Race Reaction Recording of previous events Research Resolution Role SIV Species Specificity Specificity Structural Models Structure Subfamily lentivirinae Techniques Testing Therapeutic Thermodynamics Ubiquitination Viral Viral Genome Viral Pathogenesis Viral Proteins Virion Virus Diseases X-Ray Crystallography Zoonoses arm base design experimental study factor C global health immunoregulation in vivo innovation insight novel pathogen pressure protein degradation public health relevance stoichiometry structural biology transmission process ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Evolutionary pressures placed upon lentiviruses, such as HIV, and their affected hosts have resulted in a molecular "arms race" which has shaped both host immunity and pathogen immune evasion strategies. Central to this race is the HIV protein Virion Infectivity Factor (Vif), an immunomodulatory protein critical to the replicatio cycle of nearly all lentiviruses. The primary function of Vif is to counteract the antiviral effect of the host APOBEC3 (A3) innate immune proteins. A3 proteins are restriction factors that inhibit lentiviral replication by inducing hypermutation of the viral genome. Vif antagonizes A3 by hijacking a cellular Cullin-RING ubiquitin ligase, resulting in the ubiquitination and subsequent targeting of APOBEC3 for proteasomal degradation. Despite the central role of Vif in the HIV replication cycle and an extensive body of literature describing HIV pathogenesis, we still do not have a clear molecular understanding of how Vifs bind and ubiquitinate A3. This research plan seeks to combine biochemistry, enzymology, and structural biology to understand how Vif inhibits A3 at a molecular level, and determine how A3 inhibition relates to the evolutionary events that allowed HIV to "jump" from monkeys to humans. Lentiviral Vifs have evolved to specifically counteract the effects of the A3 from their animal hosts. To determine the biochemical basis for species-specific A3 recognition I will use a panel of HIV and SIV Vif E3 ligases and their respective A3G proteins to carry out binding and in vitro ubiquitination assays and address the question: is A3G species-specificity conferred during the ground state, binding to Vif, or the transition state, the catalytic step, of the enzyme catalyzed ubiquitination reactio? To provide the molecular determinants that govern Vifs' ability to confer species-specific recognition of A3G, I will utilize low- and high-resolution structural techniques that allow me to visualize the A3G-Vif interface at atomic resolution. Mutational analyses, using in vitro ubiquitination assays and cell-base infectivity assays, will be used to link structure to phenotype and validate the biological relevance of the structural model. The successful completion of these studies will provide "snapshots" of Vif from different stages of lentivirial evolution, and our combined structural and functional approach will provide detailed insight into the Vif- A3 interaction. Together these studies will allow us to "see" the Vif-A3 interaction, characterize the evolutionary steps required for species-species transmission into human populations, and ultimately aid in the development of innovative therapeutics to counter viral infection.