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年龄的增长，非感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HIV相关肺动脉高压（PAH-HIV）的发病机制知之甚少。慢性暴露于病毒产物如HIV-1 Nef和HIV诱导的肺免疫失调可能导致肺血管疾病，特别是通过其对肺内皮细胞（EC）的影响。我们的研究小组是第一个将HIV-1 nef蛋白与PAH-HIV血管重塑的发病机制联系起来的。我们纵向跟踪了34名PAH-HIV患者，并收集了连续的临床和超声心动图;我们创建并管理了一个血浆、支气管灌洗液和细胞库。我们从PAH、肺动脉收缩压升高和非PAH HIV感染患者的血液和肺样本中测序nef基因。我们发现Nef蛋白中的氨基酸取代与PAH表型统计学相关;这些取代聚集在Nef功能结构域周围，可能干扰Nef适配器功能。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 与周围相比，在肺中占主导地位。肺可能是一个受保护的环境，允许病毒逃避免疫应答;此外，特定的nef等位基因将增强T细胞应答并导致肺血管内皮细胞增殖/凋亡，破坏信号转导途径并导致血管重塑。我们假设nef的等位基因变异对T细胞和肺内皮细胞功能有影响。我们提出1）用含有主要nef等位基因的分子克隆的病毒体感染T细胞并测量T细胞受体密度; 2）将人肺动脉内皮细胞与感染的T细胞共培养或用nef分子克隆转染，并测量内皮细胞基因表达、凋亡和增殖; 3）确定肺是否是nef序列进化的受保护区室。这些研究将检查来自PAH-HIV个体的含有这些氨基酸取代的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等位基因来检查HIV-nef在PAH-HIV发病机制中影响肺血管重塑的机制。我们的研究项目，与基础和临床科学家的结合，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.