Determining the Role of APOBEC3 Proteins in HIV-1 Drug Resistance

描述(由申请人提供)：人APOBEC3蛋白通过逆转录过程中病毒cdna的突变抑制人类免疫缺陷病毒1型(HIV-1)的复制。在足够数量的情况下，这些突变会导致前病毒的失活。在患者中，APOBECs介导的限制无法控制病毒复制，因为最有效的APOBEC3蛋白被病毒辅助蛋白病毒感染性因子(Vif)中和。然而，可能会有一种中间情景，在这种情景中，一些APOBEC3蛋白逃避降解，在不影响病毒生长的情况下突变HIV-1。有趣的是，许多导致抗逆转录病毒药物耐药的HIV-1点突变发生在APOBEC3突变热点，这增加了病毒实际上颠覆APOBEC3介导的突变以增强其抵抗治疗能力的可能性。因此，我们假设APOBEC3蛋白引入的亚致死性突变水平有助于获得HIV-1耐药性。如果得到证实，这将为合理设计抗逆转录病毒方案提供基础，这些方案通过避免易受APOBECs介导的耐药性的药物来最大限度地减少治疗失败。这样的战略有可能极大地改善对药物滥用使治疗复杂化的人的护理，并且符合国家药物滥用研究所2008年艾滋病研究方案资金优先事项的“重点领域3：药物使用和艾滋病毒/艾滋病”。为了检验我们的假设，我们提出了两个具体目标。具体目标1-确定能够突变Vif熟练的HIV-1的APOBEC3蛋白：这些实验将定量确定激活的CD4T细胞中存在哪些APOBEC3蛋白，以及这些蛋白在感染早期受到抑制的程度。进一步的实验将量化APOBEC3蛋白在Vif熟练的病毒粒子中的包裹和突变活性。特定目的2-确定APOBEC3蛋白是否影响耐药性：该目的侧重于在感染的细胞培养模型中，在APOBEC3蛋白存在的情况下，HIV-1的生长是否影响耐药性的获得。其他实验将确定APOBEC3蛋白的缺失是否会降低HIV-1产生抗药性的能力。公共卫生相关性：抗病毒药物的成功受到病毒获得对这些药物产生抗药性的突变的能力的影响。这些实验将确定被称为APOBEC3的天然人类蛋白质是否增强了HIV对用于治疗HIV感染的药物产生抗药性的能力。这将对优化抗HIV治疗具有重要意义。

DESCRIPTION (provided by applicant): Human APOBEC3 proteins inhibit the replication of human immunodeficiency virus type 1 (HIV-1) via the mutation of viral cDNA during reverse transcription. In sufficient numbers, these mutations lead to the inactivation of proviruses. In patients, APOBECS-mediated restriction is incapable of controlling viral replication because the most potent APOBEC3 proteins are neutralized by the viral accessory protein virion infectivity factor (Vif). Nevertheless, there may be an intermediate scenario in which some APOBEC3 proteins escape degradation and mutate HIV-1 without affecting viral growth. Interestingly, many HIV-1point mutations conferring resistance to antiretroviral drugs occur inAPOBEC3 mutational hotspots, raising the possibility that the virus actually subverts APOBEC3-mediated mutation to enhance its ability to resist treatment. Thus, we hypothesize that sublethal levels of mutation introduced by APOBEC3 proteins contribute to the acquisition of HIV-1 drug resistance. If proven, this will provide a basis for the rational design of antiretroviral regimens that minimize treatment failure by avoiding drugs susceptible to APOBECS-mediated resistance. Such strategies carry the potential to greatly improve the care of people for whom drug abuse complicates treatment and are consistent with "Area of Emphasis 3: Drug Use and HIV/AIDS" of the 2008 funding priorities of the AIDS Research Program at the National Institute on Drug Abuse. To test our hypothesis, we propose two specific aims. Specific Aim 1 - Define the APOBEC3 Proteins Capable of Mutating Vif-proficient HIV-1: These experiments will quantitatively determine which APOBEC3 proteins are present in activated CD4+ T cells and to what extent these proteins are suppressed early in infection. Further experiments will quantify the encapsidation and mutational activity of APOBEC3 proteins in Vif-proficient virions. Specific Aim 2 - Determine Whether APOBEC3 Proteins Impact Drug Resistance: This aim focuses on whether growth of HIV-1in the presence of APOBEC3 proteins affects the acquisition of drug resistance in a cell culture model of infection. Additional experiments will determine whether the depletion of APOBEC3 proteins decreases the ability of HIV-1to develop resistance. PUBLIC HEALTH RELEVANCE: The success of antiviral drugs is tempered by the ability of viruses to acquire mutations conferring resistance to those drugs. These experiments will determine whether natural human proteins known as APOBEC3 enhance the ability of HIV to evolve resistance to the medications used to treat HIV infection. This will have implications for the optimization of anti-HIV therapy.