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，病毒衰减模型预测抗逆转录病毒治疗（ART）将导致约3年治疗的HIV感染清除。然而，发现HIV在抑制性ART期间持续存在。据估计，感染的半衰期可延长多年，因此在正常寿命期间预计不会发生病毒清除。我们和其他人对抑制性ART期间持续存在的HIV的研究观察到少数（约20%）个体中的低水平病毒复制。我们已经研究了HIV在血液、痰和生殖器组织中的持续存在，所有这些都可以在有效的ART期间作为潜在的病毒储存库。利用单基因组扩增（SGA），我们独立推导并直接测序（避免检测到PCR介导的突变）从多个标本中获得足够的病毒模板，以表征病毒群体及其在抑制性ART期间的变化。基因组，这里称为单型病毒，在各种细胞类型中。低水平血浆病毒斑点（<500 c/mL）的SGA显示个体中的单型病毒，系统发育和耐药性分析显示没有病毒复制的证据。我们推断，单型病毒的光点可能是由于病毒体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）可以防止感染其他细胞。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体以产生50- 500 c/mL的病毒载量，这进一步支持了一种发展中的假设，即HIV前病毒在增殖细胞中扩增，并且有时这些细胞产生病毒体而没有完整的感染。如果我们的假设是正确的，细胞增殖可能是一个重要的机制，随着时间的推移，使艾滋病毒感染，一个概念，以前没有特征。该建议的假设是：（1）克隆扩张（即，HIV感染的细胞的增殖）导致多个细胞，每个细胞具有整合在宿主基因组中相同位点的相同病毒序列。(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源于HIV感染细胞的增殖，并且这些病毒在抑制性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：一曰：确定子宫颈中的单型（相同）HIV env序列是否是由感染细胞的增殖或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的病毒我们提出的探索细胞增殖在维持HIV感染中的作用的研究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：我们已经观察到，HIV群体可能在生殖道中生长，因为带有病毒的细胞似乎增殖，这可能是由于感染和创伤（例如，性交、卫生棉条等）。如果生殖道HIV的很大一部分来自受感染细胞的增殖，这种现象可能会成为治愈HIV感染的主要障碍;除非能够开发出新的方法来消除体内的HIV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.