HIV-1 evolution in the female genital tract and trafficking to the blood

描述(申请人提供)：在20世纪90年代中期，S在病毒衰变模型中预测，抗逆转录病毒疗法(ART)可以在大约3年的治疗时间内消除艾滋病毒感染。然而，艾滋病毒被发现在抑制ART期间持续存在。据估计，感染的半衰期将延长许多年，因此预计病毒在正常寿命内不会被清除。我们和其他人对抑制ART期间持续存在的艾滋病毒的研究观察到，在少数人(~20%)中存在低水平的病毒复制。我们研究了艾滋病毒在血液、痰和生殖器组织中的持续存在，在有效的抗逆转录病毒治疗期间，所有这些都可能成为潜在的病毒库。利用单基因组扩增(SGA)，我们独立地从多个样本中获得并直接测序(以避免检测到PCR介导的突变)足够的病毒模板，以表征病毒种群及其在抑制ART期间的变化。我们在不同的细胞类型中检测到大量相同的病毒基因组，在这里被称为单型病毒。低水平血浆病毒斑点(&lt；500c/ml)的SGA显示，个体中存在单型病毒，系统发育和耐药性分析显示没有证据表明病毒复制。我们推断，单型病毒的斑点可能是由于病毒粒子的产生而没有整个复制周期，因为核苷和非核苷逆转录酶抑制剂(NRTI和NNRTI)可以防止感染额外的细胞。由于对病毒爆发大小的估计表明，单个艾滋病毒感染细胞不能产生足够的病毒粒子来产生50-500c/毫升的病毒载量，这进一步支持了正在形成的假设，即艾滋病毒前病毒在增殖的细胞中被放大，有时这些细胞在没有完整轮感染的情况下产生病毒粒子。如果我们的假设是正确的，细胞增殖可能是一种重要的机制，随着时间的推移，这一概念还没有被描述出来。这一提议的假设是：(1)HIV感染细胞的克隆性扩张(即增殖)导致多个细胞，每个细胞在宿主基因组的同一位置整合了相同的病毒序列。(2)在女性生殖器粘膜和血液中检测到的相当大比例的病毒来自艾滋病毒感染细胞的增殖，这些病毒在抑制ART期间变得更加突出。(3)单型前病毒序列包括具有复制能力的模板。我们的目标是：1：确定宫颈中单型(相同的)HIV env序列是由感染细胞的增殖还是病毒复制的爆发造成的2：确定整个单型HIV基因组是否包括具有复制能力的病毒我们拟议的研究探索细胞增殖在持续HIV感染中的作用可能会改变目前解释病毒持久性的范式，并形成治愈HIV所需的干预措施。 公共卫生相关性：我们观察到，艾滋病毒种群可能在生殖道中增长，因为携带病毒的细胞似乎增殖，可能是由于感染和创伤(例如，性交、卫生棉条等)。如果生殖道艾滋病毒的很大一部分来自感染细胞的增殖，这一现象可能会成为治愈艾滋病毒感染的主要障碍；除非能够开发新的方法来消除体内的艾滋病毒感染。

DESCRIPTION (provided by applicant): In the mid-1990's, modeling of viral decay predicted that antiretroviral therapy (ART) would result in clearance of HIV infection with ~3 years of treatment. However, HIV was found to persist during suppressive ART. The half-life of infection was estimated to extend for many years, such that viral clearance would not be expected to occur during a normal life span. Our and others' studies of HIV that persists during suppressive ART have observed low-level viral replication in a minority (~20%) of individuals. We have studied HIV persisting in blood, sputum and genital tissues, all of which could serve as potential viral reservoirs during effective ART. Utilizing single genome amplification (SGA), we independently-derived and directly sequenced (to avoid detection of PCR-mediated mutations) sufficient viral templates from multiple specimens to characterize viral populations and their changes during suppressive ART. We detected sizable populations of identical viral genomes, here termed monotypic virus, in various cell types. SGA of low-level plasma viral blips (<500c/mL) revealed monotypic viruses in individuals that phylogenetic and drug resistance analyses showed no evidence of viral replication. We reason that blips of monotypic virus could result from production of virions without full- cycles of replication, as nucleoside- and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) would prevent infection of additional cells. As estimates of viral burst size suggest that a single HIV infected cell could not produce sufficient virions to generate a viral load of 50-500c/mL, this gave further support to a developing hypothesis that HIV proviruses are amplified in proliferating cells, and that at times these cells produce virions without full rounds of infection. If our hypothesis is correct, cellular proliferation could be an important mechanism that over time perpetuates HIV infection, a concept that has not been previously characterized. The hypotheses of this proposal are that: (1) Clonal expansion (i.e., proliferation) of HIV infected cells results in multiple cells, each with an identical viral sequence integrated at the same site in the host's genome. (2) A substantial proportion of viruses detected in the female genital mucosa and blood are derived from the proliferation of HIV infected cells, and these viruses become more prominent during suppressive ART. (3) Monotypic proviral sequences include templates that are replication competent. We Aim to: 1: Determine if monotypic (identical) HIV env sequences in the uterine cervix result from proliferation of infected cells or from bursts of viral replication 2: Determine if whole monotypic HIV genomes include replication competent viruses Our proposed studies exploring the role of cellular proliferation in perpetuating HIV infection could alter current paradigms explaining viral persistence and shape interventions needed to cure HIV. PUBLIC HEALTH RELEVANCE: We have observed that HIV populations may grow in the genital tract because cells with virus appear to proliferate, probably due to infections and traumas (e.g., sexual intercourse, tampons, etc.). If a significant fraction of genital tract HIV is derived from proliferation of infected cells, this phenomenon may present a major obstacle to curing HIV infection; unless novel approaches can be developed to eliminate HIV infection from the body.