The Role of HIV-1 Evolution in Neuroadaptation

描述(由申请人提供)：HIV-1病毒表现出许多逃避宿主免疫的策略。这些策略包括在避难所持续存在，避难所被定义为尽管进行了高效抗逆转录病毒治疗(HAART)，病毒仍可能在其中继续复制的解剖或特权细胞部位。中枢神经系统(CNS)是HIV-1的重要避难所。艾滋病毒-1对中枢神经系统的渗透发生在感染早期，可导致广泛的病理和临床表现--所有这些都极大地促进了发病率和死亡率。因此，重要的是要更好地了解HIV-1在中枢神经系统中的适应行为。这项研究的首要目标是利用从脑脊液中测序的HIV-1变异体，通过深入、综合、系统发育和功能的方法，阐明HIV-1中枢神经系统的进化途径，并探索神经适应的遗传决定因素。为了执行拟议的研究，我们将使用具有良好特征的CNS HIV抗逆转录病毒治疗效果研究(CHECURE)研究中的患者样本。使用克隆序列的标准分析和异源双链跟踪分析(HTA)，观察到了同时存在的患者内CNS和血源性病毒的系统发育区划。在这项研究中，我们将利用单基因组扩增(SGA)技术来产生HIV-1变异序列。与更传统的测序方法相比，SGA已被证明减少了Taq诱导的重组、模板重采样、核苷酸误掺和克隆偏见，并更准确地表示体内HIV-1准种。我们假设，通过对HIV-1包膜(Env)和聚合酶(Poll)的纵向特征观察到的CNS特有的进化模式反映了病毒在CNS内适应和持续的尝试。此外，使用SGA来产生这些变异体将提供比目前文献报道的更准确的病毒准种表示。这些变异的系统发育分析和体外特征分析有望揭示中枢神经系统特有进化的新模式和所观察到的基因变化的功能后果。因此，这项建议的具体目的是：1)确定HAART初学者队列中HIV-1 env的CNS区划模式2)确定抗逆转录病毒耐药突变的CNS区划模式以及对HAART经验人群中HIV-1 pol1和env基因的影响，以及3)确定SGA来源的HIV-1 env序列中基因变异的功能后果。 了解艾滋病毒-1为在病毒储存库和避难所持续存在而采取的适应措施，对于制定改进病毒抑制和在受感染宿主中根除艾滋病毒-1的战略至关重要。据我们所知，目前的科学文献不包含新的SGA方法在表征中枢神经系统中HIV-1变异的应用。重要的是，这项提议的成功执行将使我获得必要的技术技能和经验，成为翻译HIV-1发病机制研究中的一名创新和富有成效的独立研究员。

DESCRIPTION (provided by applicant): HIV-1 exhibits numerous strategies to evade host immunity. These strategies include persistence in sanctuary sites, defined as anatomical or privileged cellular sites in which viral replication may continue despite highly active antiretroviral therapy (HAART). The central nervous system (CNS) is an important sanctuary site for HIV-1. Penetration of the CNS by HIV-1 occurs early in infection, and can result in a wide range of pathological and clinical manifestations - all of which contribute significantly to morbidity and mortality. It is therefore important to develop an improved understanding of the adaptive behavior of HIV-1 in the CNS. The overriding goal of the proposed research is to use HIV-1 variants sequenced from cerebrospinal fluid (CSF), to elucidate HIV-1 CNS evolutionary pathways and explore genetic determinants of neuroadaptation through an in-depth, integrated, phylogenetic and functional approach. In order to perform the proposed research, we will use patient samples from the well-characterized CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study. Using standard analysis of clonal sequences and heteroduplex tracking assays (HTA), phylogenetic compartmentalization of contemporaneous intra-patient CNS- and blood-derived viruses has been observed. For this research, we will utilize the single genome amplification (SGA) technique to generate HIV-1 variant sequences. When compared to more conventional sequencing methods, SGA has been shown to decrease taq- induced recombination, template resampling, nucleotide misincorporation and cloning bias and produces a more accurate representation of in-vivo HIV-1 quasispecies. We hypothesize that CNS-specific patterns of evolution observed through longitudinal characterization of HIV-1 envelope (env) and polymerase (pol) reflect attempts by the virus to adapt and persist within the CNS. Furthermore, the use of SGA to generate these variants will provide a more accurate representation of viral quasispecies than currently reported in the literature. Phylogenetic analysis and in-vitro characterization of these variants is expected to reveal novel patterns of CNS-specific evolution and functional consequences of the observed genetic changes. The specific aims of this proposal are therefore: 1) To determine patterns of CNS compartmentalization of HIV-1 env in a cohort of HAART naive individuals 2) To determine patterns of CNS compartmentalization of antiretroviral drug resistance mutations and effects on the HIV-1 pol and env genes in a cohort of HAART experienced individuals, and 3) To determine the functional consequences of genetic variation in SGA-derived HIV-1 env sequences. Understanding adaptive measures undertaken by HIV-1 to persist in viral reservoirs and sanctuary sites are of paramount importance in defining strategies to improve viral suppression and obtain eradication of HIV-1 in the infected host. To our knowledge, current scientific literature contains no application of the novel SGA method to the characterization of HIV-1 variants in the CNS. Importantly, the successful execution of this proposal will allow me to acquire the technical skill and experience necessary to become an innovative and productive independent investigator in translational HIV-1 pathogenesis research.