HIV-1 evolution driven by intracellular defenses

描述(申请人提供)：逆转录病毒已经设计了一些策略来逃避旨在预防逆转录病毒感染的细胞机制。HIV-1表达Vif，这是一种可以抵消胞苷脱氨酶APOBEC3G和APOBEC3F的抗病毒活性的蛋白质。然而，HIV-1基因组的核苷酸组成表明，对宿主介导的病毒cDNA脱氨基的保护可能不是绝对的。在初步研究中，我们发现在体内可以检测到编码不能降解APOBEC3G和/或APOBEC3F的蛋白的vif基因。Vif功能的丧失被映射为单核苷酸替换。这些研究表明，Vif功能的自然变异可能会深刻影响HIV-1感染者体内病毒序列进化的程度和方向。 本文提出的实验将确定旨在预防逆转录病毒感染的宿主机制在多大程度上实际上有助于病毒的多样化和致病。我们将分析表达Vif蛋白的病毒的适合性，这些Vif蛋白密切相关，但在中和APOBEC3G或APOBEC3F活性的能力上存在差异。我们还将通过确定APOBEC3G或APOBEC3F的胞苷脱氨基是否选择某些耐药突变来测试在某些情况下(例如，在存在抗逆转录病毒药物的情况下)Vif功能的变化是否有利于病毒适应。与部分保护免受胞苷脱氨基作用相关的超突变模式将与HIV的适合性相关。由于APOBEC3酶在不同的二核苷酸环境中诱导超突变，了解针对一种酶而不是另一种酶的活性是如何保持的，这与病毒进化有关。我们将进行结构功能研究，以确定Vif SoCs盒基序以外的结构域是否对于Vif介导的APOBEC3酶的特异性中和是必要和必要的。最后，我们将评估反转录和APOBECs驱动的突变对Vif功能丧失的影响。我们将使用基于荧光标记的APOBEC3G降解的分析来确定由于逆转录酶或胞苷脱氨基诱导的单细胞水平突变而导致的VIF失活的比率。Vif功能的变化可能通过使其基因组对脱氨酶活性产生或多或少的抵抗力来影响HIV-1的致病性，这些研究有可能揭示Vif介导的对胞苷脱氨的保护是如何在体内调节的。

DESCRIPTION (provided by applicant): Retroviruses have devised a number of strategies to evade cellular mechanisms aimed at preventing retroviral infection. HIV-1 expresses Vif, a protein that counteracts the antiviral activity of the cytidine deaminases APOBEC3G and APOBEC3F. The nucleotide composition of the HIV-1 genome suggests, however, that protection from host-mediated viral cDNA deamination may not be absolute. In preliminary studies, we showed that vif genes encoding proteins that fail to degrade APOBEC3G, APOBEC3F or both can be detected in vivo. The loss of Vif function was mapped to single nucleotide substitutions. These studies indicate that natural variation in Vif function may profoundly impact the extent and direction of viral sequence evolution within HIV-1 infected individuals. The experiments proposed herein will determine the extent to which host mechanisms aimed to prevent retroviral infection, in fact, contribute to viral diversification and pathogenesis. We will analyze the fitness of viruses expressing Vif proteins that are closely related but differ in their ability to neutralize APOBEC3G or APOBEC3F activities. We will also test if variation in Vif function may be beneficial for viral adaptation under certain circumstances (e.g., in the presence of antiretroviral drugs) by determining whether cytidine deamination by APOBEC3G or APOBEC3F selects for certain drug resistance mutations. The pattern of hypermutations associated with partial protection from cytidine deamination will be correlated to HIV fitness. Since APOBEC3 enzymes induce hypermutations in different dinucleotide contexts the understanding of how activity against one enzyme but not the other is maintained is relevant for viral evolution. We will conduct structure function studies to determine whether domains other than the Vif SOCS box motif are necessary and essential for Vif mediated specific neutralization of APOBEC3 enzymes. Finally, we will assess the impact of reverse transcription and APOBECS-driven mutagenesis on loss of Vif function. We will determine the rate of Vif inactivation as result of either reverse transcriptase or cytidine deamination induced mutations on a single cell level using assays based on fluorescence tagged APOBEC3G degradation. Variation in Vif function may influence the pathogenicity of HIV-1 by rendering its genome more or less resistant to deaminase activity and these studies have the potential to reveal how Vif mediated protection from cytidine deamination is modulated in vivo.