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，非传染性并发症的发生频率增加。尽管1987年首次对患者病例进行了描述，但对与HIV相关的肺动脉高压（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，特别是通过它们对肺内皮细胞（EC）的影响。我们的研究小组首次将HIV-1 nef蛋白与血管重塑PAH-HIV的发病机制联系起来。我们对34名PAH-HIV患者进行了纵向随访，并收集了序贯临床和超声心动图；我们创建并管理了血浆、支气管灌洗液和细胞的储存库。我们从多环芳烃患者、肺动脉收缩压升高患者和非多环芳烃HIV感染者的血液和肺样本中对nef基因进行了测序。我们发现Nef蛋白的氨基酸替换与PAH表型有统计学相关性；这些替换聚集在可能干扰Nef适配器功能的Nef功能域周围。本应用程序中初步报道的进一步研究表明，特定的氨基酸特征

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.