Genotypic and functional properties of HIV-1 Nef clinical isolates in PAH-HIV

描述(由申请人提供)：随着艾滋病毒感染者的年龄不断增长，非传染性并发症的频率增加。尽管1987年首次描述了患者病例，但对艾滋病毒相关性肺动脉高压(PAH-HIV)的发病机制知之甚少。慢性暴露于HIV-1 Nef等病毒产物和HIV诱导的肺部免疫失调可能会导致肺血管疾病，特别是通过它们对肺内皮细胞(EC)的影响。我们小组首次将HIV-1nef蛋白与PAH-HIV血管重塑的发病机制联系起来。我们纵向跟踪了34名PAH-HIV患者，收集了连续的临床和超声心动图；我们创建和管理了血浆、支气管灌洗液和细胞库。我们从PAH患者、肺动脉收缩压升高的患者和非PAH HIV感染患者的血液和肺样本中对nef基因进行了测序。我们发现Nef蛋白中的氨基酸替换在统计学上与PAH表型相关；这些替换聚集在Nef功能结构域周围，可能会干扰Nef适配器的功能。在本申请中初步报道的进一步研究表明，特定的氨基酸特征 与外周相比，肺脏占优势。肺可能是病毒逃避免疫反应的保护性环境；此外，特定的nef等位基因会增强T细胞反应，导致肺血管内皮细胞增殖/凋亡，扰乱信号转导途径，导致血管重构。我们假设NEF的等位基因变异将对T细胞和肺内皮细胞功能产生影响。我们建议1)用含有初级nef等位基因的分子克隆病毒粒子感染T细胞，并测量T细胞受体密度；2)将人的肺动脉内皮细胞与感染的T细胞共培养或转染nef分子克隆，检测内皮细胞的基因表达、凋亡和增殖情况；3)确定肺是否是nef序列进化的保护室。这些研究将检验来自PAH-HIV个体的包含这些氨基酸替代的nef等位基因的功能特性。 本申请中提出的研究将使用现有的生物标本和克隆的nef等位基因来研究HIV-nef在PAH-HIV发病机制中影响肺血管重构的机制。我们的研究项目，与基础和临床科学家相结合，很好地解决了nef病毒蛋白和免疫失调是如何导致艾滋病毒肺部并发症的因素。

DESCRIPTION (provided by applicant): As HIV-infected individuals continue to age, non-infectious complications increase in frequency. Despite the first descriptions of patient cases in 1987, little is known about the pathogenesis of pulmonary hypertension associated with HIV (PAH-HIV). Chronic exposure to viral products such as HIV-1 Nef and HIV-induced immune deregulation in the lung may contribute to pulmonary vascular disease, particularly through their impact on pulmonary endothelial cells (EC). Our group was the first to associate the HIV-1 nef protein with the pathogenesis of vascular remodeling PAH-HIV. We longitudinally followed 34 individuals with PAH-HIV and collected sequential clinical and echocardiographic; we created and curated a repository of plasma, bronchial lavage fluid and cells. We sequenced the nef gene from blood and lung samples from patients with PAH, elevated pulmonary artery systolic pressures and from non- PAH HIV infected. We found amino acid substitutions in the Nef protein statistically associated with the PAH phenotype; these substitutions clustered around Nef functional domains that potentially interfere with Nef adaptor functions. Further studies, which are preliminarily reported in this application, suggest that particular amino acid signatures predominate in the lungs compared with the periphery. The lung may be a protected environment that allows the virus to evade immune responses; furthermore, particular nef alleles will enhance T cell responses and result in pulmonary vascular endothelial cell proliferation/apoptosis, disrupt signal transduction pathways and result in vascular remodeling. We hypothesize that allelic variants of nef will have an impact on T cell and pulmonary endothelial cell function. We propose to 1) infect T cells with molecularly cloned virions containing primary nef alleles and measure T cell receptor density; 2) human pulmonary artery endothelial cells will be co-cultured with infected T cells or transfected with nef molecular clone and endothelial cell gene expression, apoptosis and proliferation measured and 3) determine whether the lung is a protected compartment for evolution of nef sequences. These studies will examine the functional properties of nef alleles containing these amino acid substitutions from PAH-HIV individuals. The studies proposed in this application will use existing biospecimens and cloned nef alleles to examine the mechanisms whereby HIV-nef influences pulmonary vascular remodeling in the pathogenesis of PAH-HIV. Our research tem, with a combination of basic and clinician scientists is well poised to address how the nef viral protein and immune dysregulation are contributing factors to this lung complication of HIV.