The Role of HIV-1 Evolution in Neuroadaptation

描述（由申请人提供）：HIV-1表现出多种逃避宿主免疫的策略。这些策略包括在庇护所位点持续存在，庇护所位点被定义为解剖或特权细胞位点，在这些位点上，尽管高活性抗逆转录病毒治疗（HAART），病毒仍可继续复制。中枢神经系统（CNS）是HIV-1的重要庇护所。HIV-1侵入中枢神经系统发生在感染早期，可导致广泛的病理和临床表现——所有这些都是导致发病率和死亡率的重要因素。因此，有必要进一步了解HIV-1在中枢神经系统中的适应性行为。该研究的首要目标是利用从脑脊液（CSF）中测序的HIV-1变异，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。为了进行拟议的研究，我们将使用来自具有良好特征的中枢神经系统HIV抗逆转录病毒治疗效果研究（CHARTER）研究的患者样本。利用克隆序列的标准分析和异源双工跟踪测定（HTA），观察了同时期患者体内中枢神经系统和血液源性病毒的系统发育区隔。在本研究中，我们将利用单基因组扩增（SGA）技术产生HIV-1变异序列。与更传统的测序方法相比，SGA已被证明可以减少taq诱导的重组、模板重采样、核苷酸错误掺入和克隆偏差，并能更准确地表示体内HIV-1准种。我们假设，通过HIV-1包膜（env）和聚合酶（pol）的纵向特征观察到的CNS特异性进化模式反映了病毒在CNS内适应和持续存在的尝试。此外，使用SGA生成这些变异将比目前文献中报道的更准确地表示病毒准种。系统发育分析和这些变异的体外表征有望揭示cns特异性进化的新模式和所观察到的遗传变化的功能后果。因此，本建议的具体目的是：1)确定HAART新手队列中HIV-1 env的中枢神经系统区域化模式；2)确定HAART经验个体队列中抗逆转录病毒耐药突变的中枢神经系统区域化模式及其对HIV-1 pol和env基因的影响；3)确定sga衍生的HIV-1 env序列遗传变异的功能后果。

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. PUBLIC HEALTH RELEVANCE: Infection of the central nervous system (CNS) by the human immunodeficiency virus-1 (HIV-1) can result in a wide range of pathological and clinical manifestations. The widespread use of highly active antiretroviral therapy (HAART) has led to a clear reduction in the incidence of HIV-associated dementia (HAD), one of the most severe manifestations of HIV-1 CNS infection. Despite this decrease, the prevalence of minor HIV-1 associated cognitive impairment appears to be on the rise. Although more subtle, these impairments can be disabling and have been demonstrated to be independently associated with an increased risk for mortality in those with HIV-1. An improved understanding of HIV-1 neuroadaptation will be important for promoting the development of targeted therapies against HIV-1 in this unique sanctuary site.