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

描述（由申请人提供）：在20世纪90年代中期，病毒衰变模型预测抗逆转录病毒治疗（ART）将在约3年的治疗后导致HIV感染的清除。然而，在抑制性抗逆转录病毒治疗期间发现艾滋病毒持续存在。据估计，感染的半衰期可延长许多年，因此，在正常寿命期间，病毒清除预计不会发生。我们和其他人对在抑制性抗逆转录病毒治疗期间持续存在的艾滋病毒的研究发现，在少数（约20%）的个体中存在低水平的病毒复制。我们研究了存在于血液、痰和生殖组织中的艾滋病毒，所有这些都可能在有效的抗逆转录病毒治疗期间成为潜在的病毒储存库。利用单基因组扩增（SGA），我们从多个标本中独立衍生并直接测序（避免检测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性抗逆转录病毒治疗期间的变化。我们在不同的细胞类型中检测到相当数量的相同病毒基因组，这里称为单型病毒。低水平血浆病毒点（<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未显示病毒复制的证据。我们推断，单型病毒的突变可能是由于病毒粒子的产生而没有完整的复制周期，因为核苷和非核苷逆转录酶抑制剂（NRTI和NNRTI）可以防止其他细胞的感染。由于对病毒爆发大小的估计表明，单个HIV感染细胞不能产生足够的病毒粒子以产生50-500c/mL的病毒载量，这进一步支持了一种正在发展的假设，即HIV前病毒在增殖细胞中被扩增，并且有时这些细胞在没有完整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，随着时间的推移使HIV感染永久化，这是一个以前没有被描述过的概念。该建议的假设是：(1)HIV感染细胞的克隆扩增（即增殖）导致多个细胞，每个细胞在宿主基因组的同一位点整合相同的病毒序列。(2)在女性生殖器粘膜和血液中检测到的大量病毒来源于HIV感染细胞的增殖，这些病毒在抑制性抗逆转录病毒治疗期间变得更加突出。(3)单型原序列包括具有复制能力的模板。我们的目标是：1：确定子宫颈内的单型（相同）HIV环境序列是由感染细胞的增殖还是病毒复制的爆发引起的2：确定整个单型HIV基因组是否包括复制能力强的病毒。我们提出的研究探索细胞增殖在持续HIV感染中的作用，可以改变目前解释病毒持久性的范式，并形成治疗HIV所需的干预措施。

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.