Development of Novel Therapeutics by Targeting Vif-APOBEC3 Interactions

APOBEC3G （A3G）和APOBEC3F （A3F）抑制Delta vif HIV-1需要它们在病毒产生细胞中表达并结合到病毒粒子中。A3G和A3F脱胺化负链DNA中的胞苷，引起G-to-A超突变，抑制病毒DNA合成。HIV-1 Vif （Vif1）通过靶向A3G和A3F进行蛋白酶体降解来克服这些宿主防御。Vif1-A3G和Vif1-A3F的相互作用为开发干扰vif1介导的A3G和A3F降解的药物提供了两个潜在的靶点。为了鉴定这些小分子抑制剂，我们开发了基于细胞的高通量测定。我们正在与NIH化学基因组学中心（NCGC）合作，对33万种化合物进行定量高通量筛选（qHTS），以确定特异性抑制Vif1-A3G和Vif1-A3F相互作用的分子。Vif1与几种宿主蛋白酶体蛋白结合；我们将与NCGC合作，使用相同的基于细胞的检测方法进行小干扰rna （siRNA）筛选，以确定可能参与vif1介导的A3G和A3F降解的宿主因子。对HIV-1感染者进行基因治疗有可能提供一次性治疗，减少病毒载量，减少毒性抗病毒药物的使用，并保持或改善免疫功能。我们的目标是验证HIV-1靶细胞中抗vif1的A3G和A3F蛋白的表达会降低病毒载量并控制HIV-1复制的假设。我们将开发新的逆转录病毒载体，可以有效地将抗vif1的A3G和A3F传递到造血干细胞，并使用人源化小鼠模型系统来确定基因治疗对HIV-1病毒载量和CD4+ T细胞保存的影响。这些研究将提供概念上的证据，证明抗vif1的A3G和A3F基因治疗可以有效治疗HIV-1感染。【对应2011年10月HIV耐药项目实地考察报告中的Pathak Project 1】

Inhibition of Delta vif HIV-1 by APOBEC3G (A3G) and APOBEC3F (A3F) requires their expression in the virus producer cells and incorporation into virions. A3G and A3F deaminate cytidines in minus-strand DNA, causing G-to-A hypermutation and inhibiting viral DNA synthesis. HIV-1 Vif (Vif1) overcomes these host defenses by targeting A3G and A3F for proteasomal degradation. The Vif1-A3G and Vif1-A3F interactions provide two potential targets for development of pharmacological agents that interfere with Vif1-mediated degradation of A3G and A3F. To identify such small-molecule inhibitors, we have developed cell-based high-throughput assays. We are collaborating with the NIH Chemical Genomics Center (NCGC) to perform a quantitative high-throughput screen (qHTS) of a >330,000 compound collection to identify molecules that specifically inhibit the Vif1-A3G and Vif1-A3F interactions. Vif1 binds to several host proteasomal proteins; we will collaborate with the NCGC and use the same cell-based assays to carry out a small-interfering-RNA (siRNA) screen to identify host factors that may be involved in Vif1-mediated degradation of A3G and A3F. Gene therapy for HIV-1 infected individuals has the potential to provide a one-time treatment that will reduce viral load, lessen the use of toxic antiviral drugs, and preserve or improve immune function. Our goal is to test the hypothesis that expression of Vif1-resistant A3G and A3F proteins in HIV-1 target cells will reduce viral loads and control HIV-1 replication. We will develop novel retroviral vectors that can efficiently deliver the Vif1-resistant A3G and A3F to hematopoietic stem cells, and use a humanized mouse model system to determine the effect of the gene therapy on HIV-1 viral load and preservation of CD4+ T cells. These studies will provide the proof of concept that gene therapy with Vif1-resistant A3G and A3F can provide an effective treatment for HIV-1 infection.[Corresponds to Pathak Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]