Structure and Function of APOBEC Proteins

宿主胞苷脱胺酶APOBEC3G （A3G）和APOBEC3F （A3F）在病毒粒子感染因子（Vif）表达缺失的情况下被包装到HIV-1病毒粒子中。Vif与APOBEC蛋白结合并诱导其降解，从而阻止病毒粒子结合。我们的长期目标是了解APOBEC蛋白的结构和功能，阐明APOBEC蛋白和Vif相互作用的结构决定因素，并确定干扰这种相互作用的小分子抑制剂，这将形成一类新的抗逆转录病毒药物的基础。通过突变分析，我们发现单个氨基酸取代（D128K）是A3G蛋白的物种特异性的原因。为了深入了解A3G抑制HIV-1复制的机制，我们开发了一种敏感的胞苷脱胺实验，并证明了与包装的病毒和非病毒rna的相互作用足以使A3G病毒粒子结合，而与病毒蛋白的相互作用则不是必需的。为了阐明A3G在感染的自然靶细胞中抑制HIV-1复制的机制，我们量化了激活的原代CD4+ T细胞中产生的vif缺陷病毒粒子中A3G的数量；这些研究表明，在自然感染过程中，很少有分子（74）被包装成病毒粒子。在目前的研究中，我们对HIV-1 Vif进行了广泛的突变分析，发现了与A3F和A3G相互作用的两种不同的决定因素。我们还使用遗传和生化方法鉴定了A3G和A3F与Vif相互作用的结构域，并通过生化和结构方法表征了这些相互作用。取代D128附近的氨基酸，最显著的是W127，可以消除A3G病毒粒子的结合。我们将描述这种突变体的生化和细胞特性，以了解A3G被纳入病毒粒子的机制。为了阐明A3G抑制病毒复制的机制，我们正在通过实时荧光定量PCR检测其对病毒DNA代谢的影响。这些研究表明，在A3G存在的情况下，逆转录过程中引物tRNA的去除是异常的和低效的，导致病毒DNA整合和合成的缺陷。对A3G和A3F的抗病毒抑制机制的比较表明，这两种蛋白都抑制病毒DNA整合，但它们的机制不同。与A3G不同，A3F通过抑制HIV-1整合酶对病毒DNA末端的3个加工来抑制原病毒的形成。我们正在研究A3G和A3F的核酸结合特性，以了解为什么这两种蛋白质对3加工反应有不同的影响。利用一种新的双分子荧光互补（BiFC）方法，我们研究了A3G蛋白之间以及A3G与病毒蛋白和RNA之间的细胞内相互作用。这些研究表明，A3G在依赖RNA结合的过程中形成多聚体。我们正在扩展这些研究，以分析A3G/A3F与Vif蛋白之间的相互作用以及Vif的多聚性。我们还在开发Vif-A3G和Vif-A3F相互作用的体内试验，用于高通量筛选阻断这些相互作用的小分子抑制剂。我们正在研究最近发现的A3G积累位点P小体在A3G功能和病毒复制中的作用。我们正在提纯A3G，以进行生化研究和x射线晶体学的结构研究。我们将确定纯化的A3G对核酸底物和其他病毒蛋白（如Gag和Vif）的亲和力。这些研究将用于识别和表征A3G-Vif相互作用域。[对应于2007年4月HIV耐药项目现场访问报告中的Pathak Project 1]APOBEC3G （A3G）和APOBEC3F (A3F)，宿主胞苷脱胺酶，在没有病毒粒子感染因子（Vif）表达的情况下被包装到HIV-1病毒粒子中。Vif与APOBEC蛋白结合并诱导其降解，从而阻止病毒粒子结合。我们的长期目标是了解APOBEC蛋白的结构和功能，阐明APOBEC蛋白和Vif相互作用的结构决定因素，并确定干扰这种相互作用的小分子抑制剂，这将形成一类新的抗逆转录病毒药物的基础。通过突变分析，我们发现单个氨基酸取代（D128K）是A3G蛋白的物种特异性的原因。为了深入了解A3G抑制HIV-1复制的机制，我们开发了一种敏感的胞苷脱胺实验，并证明了与包装的病毒和非病毒rna的相互作用足以使A3G病毒粒子结合，而与病毒蛋白的相互作用则不是必需的。为了阐明A3G在感染的自然靶细胞中抑制HIV-1复制的机制，我们量化了激活的原代CD4+ T细胞中产生的vif缺陷病毒粒子中A3G的数量；这些研究表明，在自然感染过程中，很少有分子（74）被包装成病毒粒子。在目前的研究中，我们对HIV-1 Vif进行了广泛的突变分析，发现了与A3F和A3G相互作用的两种不同的决定因素。我们还使用遗传和生化方法鉴定了A3G和A3F与Vif相互作用的结构域，并通过生化和结构方法表征了这些相互作用。取代D128附近的氨基酸，最显著的是W127，可以消除A3G病毒粒子的结合。我们将描述这种突变体的生化和细胞特性，以了解A3G被纳入病毒粒子的机制。为了阐明A3G抑制病毒复制的机制，我们正在通过实时荧光定量PCR检测其对病毒DNA代谢的影响。这些研究表明，在A3G存在的情况下，逆转录过程中引物tRNA的去除是异常的和低效的，导致病毒DNA整合和合成的缺陷。对A3G和A3F的抗病毒抑制机制的比较表明，这两种蛋白都抑制病毒DNA整合，但它们的机制不同。与A3G不同，A3F通过抑制HIV-1整合酶对病毒DNA末端的3个加工来抑制原病毒的形成。我们正在研究A3G和A3F的核酸结合特性，以了解为什么这两种蛋白质对3加工反应有不同的影响。利用一种新的双分子荧光互补（BiFC）方法，我们研究了A3G蛋白之间以及A3G与病毒蛋白和RNA之间的细胞内相互作用。这些研究表明，A3G在依赖RNA结合的过程中形成多聚体。我们正在扩展这些研究，以分析A3G/A3F与Vif蛋白之间的相互作用以及Vif的多聚性。我们还在开发Vif-A3G和Vif-A3F相互作用的体内试验，用于高通量筛选阻断这些相互作用的小分子抑制剂。我们正在研究最近发现的A3G积累位点P小体在A3G功能和病毒复制中的作用。我们正在提纯A3G，以进行生化研究和x射线晶体学的结构研究。我们将确定纯化的A3G对核酸底物和其他病毒蛋白（如Gag和Vif）的亲和力。这些[摘要被截断为7800个字符]

APOBEC3G (A3G) and APOBEC3F (A3F), host restriction factor cytidine deaminases,are packaged into HIV-1 virions in the absence of virion infectivity factor (Vif) expression. Vif binds to the APOBEC proteins and induces their degradation, thereby preventing virion incorporation. Our long-term goals are to understand the structure and function of the APOBEC proteins, to elucidate the structural determinants of APOBEC proteins and Vif that interact with each other, and to identify small-molecule inhibitors interfering with this interaction that will form the basis of a new class of antiretroviral drugs. Using mutational analysis, we showed that a single amino acid substitution (D128K) is responsible for the species specificity of A3G proteins. To gain insight into the mechanism by which A3G inhibits HIV-1 replication, we developed a sensitive cytidine deamination assay and demonstrated that interactions with packaged viral and nonviral RNAs are sufficient for A3G virion incorporation, whereas interactions with viral proteins are not essential. To elucidate the mechanism by which A3G inhibits HIV-1 replication in natural target cells of infection, we quantified the amounts of A3G in Vif-deficient virions produced in activated primary CD4+ T cells; these studies indicated that very few molecules (7 4) are packaged into virions during natural infection. In current studies, our extensive mutational analysis of HIV-1 Vif has identified two different determinants that interact with A3F and A3G. We have also used genetic and biochemical approaches to identify domains of A3G and A3F that interact with Vif and characterize these interactions by biochemical and structural approaches. Substitution of amino acids adjacent to D128, most prominently W127, abrogates A3G virion incorporation. We will characterize biochemical and cellular properties of this mutant to understand the mechanism by which A3G is incorporated into virions. To elucidate the mechanism by which A3G inhibits viral replication, we are examining its effects on viral DNA metabolism by quantitative real-time PCR assays. These studies indicate that removal of the primer tRNA during reverse transcription is aberrant and inefficient in the presence of A3G, leading to defects in viral DNA integration and synthesis. A comparison of the mechanism of antiviral inhibition of A3G and A3F has revealed that both proteins inhibit viral DNA integration, but they do so by different mechanisms. Unlike A3G, A3F inhibits provirus formation by inhibiting the 3 processing of viral DNA ends by HIV-1 integrase. We are examining the nucleic acid binding properties of A3G and A3F to understand why these two proteins have different effects on the 3 processing reaction. Using a novel bimolecular fluorescence complementation (BiFC) approach, we have examined the intracellular interactions between A3G proteins, and between A3G and viral proteins and RNA. These studies show that A3G forms multimers in a process that is dependent on RNA binding. We are extending these studies to analyze the interactions between A3G/A3F and Vif proteins, and Vif multimerization. We are also developing in vivo assays for the Vif-A3G and Vif-A3F interactions for high-throughput screening of small molecule inhibitors that block these interactions. We are examining the role of P bodies, recently identified sites of A3G accumulation, in the function of A3G and viral replication. We are purifying A3G to carry out biochemical studies and structural studies using X-ray crystallography. We will determine the affinity of the purified A3G for nucleic acid substrates and other viral proteins such as Gag and Vif. These studies will be used to identify and characterize A3G-Vif interacting domains. [Corresponds to Pathak Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]APOBEC3G (A3G) and APOBEC3F (A3F), host restriction factor cytidine deaminases,are packaged into HIV-1 virions in the absence of virion infectivity factor (Vif) expression. Vif binds to the APOBEC proteins and induces their degradation, thereby preventing virion incorporation. Our long-term goals are to understand the structure and function of the APOBEC proteins, to elucidate the structural determinants of APOBEC proteins and Vif that interact with each other, and to identify small-molecule inhibitors interfering with this interaction that will form the basis of a new class of antiretroviral drugs. Using mutational analysis, we showed that a single amino acid substitution (D128K) is responsible for the species specificity of A3G proteins. To gain insight into the mechanism by which A3G inhibits HIV-1 replication, we developed a sensitive cytidine deamination assay and demonstrated that interactions with packaged viral and nonviral RNAs are sufficient for A3G virion incorporation, whereas interactions with viral proteins are not essential. To elucidate the mechanism by which A3G inhibits HIV-1 replication in natural target cells of infection, we quantified the amounts of A3G in Vif-deficient virions produced in activated primary CD4+ T cells; these studies indicated that very few molecules (7 4) are packaged into virions during natural infection. In current studies, our extensive mutational analysis of HIV-1 Vif has identified two different determinants that interact with A3F and A3G. We have also used genetic and biochemical approaches to identify domains of A3G and A3F that interact with Vif and characterize these interactions by biochemical and structural approaches. Substitution of amino acids adjacent to D128, most prominently W127, abrogates A3G virion incorporation. We will characterize biochemical and cellular properties of this mutant to understand the mechanism by which A3G is incorporated into virions. To elucidate the mechanism by which A3G inhibits viral replication, we are examining its effects on viral DNA metabolism by quantitative real-time PCR assays. These studies indicate that removal of the primer tRNA during reverse transcription is aberrant and inefficient in the presence of A3G, leading to defects in viral DNA integration and synthesis. A comparison of the mechanism of antiviral inhibition of A3G and A3F has revealed that both proteins inhibit viral DNA integration, but they do so by different mechanisms. Unlike A3G, A3F inhibits provirus formation by inhibiting the 3 processing of viral DNA ends by HIV-1 integrase. We are examining the nucleic acid binding properties of A3G and A3F to understand why these two proteins have different effects on the 3 processing reaction. Using a novel bimolecular fluorescence complementation (BiFC) approach, we have examined the intracellular interactions between A3G proteins, and between A3G and viral proteins and RNA. These studies show that A3G forms multimers in a process that is dependent on RNA binding. We are extending these studies to analyze the interactions between A3G/A3F and Vif proteins, and Vif multimerization. We are also developing in vivo assays for the Vif-A3G and Vif-A3F interactions for high-throughput screening of small molecule inhibitors that block these interactions. We are examining the role of P bodies, recently identified sites of A3G accumulation, in the function of A3G and viral replication. We are purifying A3G to carry out biochemical studies and structural studies using X-ray crystallography. We will determine the affinity of the purified A3G for nucleic acid substrates and other viral proteins such as Gag and Vif. These stu [summary truncated at 7800 characters]