Cell-intrinsic effectors of the antiviral interferon response against HIV in primary human samples

项目摘要/摘要 干扰素反应所激发的细胞固有抗病毒蛋白构成了第一道防线。 对抗病毒感染。然而，干扰素抑制的介体及其效力因病毒和细胞类型而异。 强调在自然感染期间研究干扰素限制的必要性。像其他病毒一样，HIV-1会触发干扰素 并在细胞培养和慢性感染患者中显示干扰素的敏感性。更重要的是，它很好 特点是艾滋病毒传播效率低下，通常需要多次接触，从而导致瓶颈 病毒变种。综上所述，这些发现表明，抗病毒干扰素刺激基因(ISGs)可能是 在传播和早期感染过程中对艾滋病毒的屏障。然而，目前还不清楚ISG到底是什么 由艾滋病毒引起的，主要是由于在急性感染的短暂窗口期间很难获得样本。 关于自然环境中艾滋病毒ISG限制的数据也是有限的，因为以前的工作在很大程度上使用了不朽的 细胞系和实验室适应的病毒。对急性HIV感染期间存在的ISGs有更全面的了解 而它们在助长艾滋病毒传播的细胞中的抗病毒效力需要确定干扰素如何反应 塑造了早期的艾滋病毒感染。为了解决这一差距，这一提议将细胞内ISG的表达定性为 体内早期感染时采集的外周血单个核细胞(PBMC)及其对ISG的限制 体外抗HIV早期分离的HIV靶细胞，即CD4+T细胞。这项研究将探索这样一种假设 一组HIV诱导的功能性抗病毒ISG负责干扰素介导的CD4+T细胞中HIV的抑制 细胞。首先，体内的ARM将确定在每种PBMC细胞类型中上调的ISG转录本的图谱 通过单细胞方法早期感染艾滋病毒。这种手臂使用罕见的急性艾滋病毒和抗逆转录病毒后治疗 (抗逆转录病毒药物)来自蒙巴萨队列的PBMC，这是一个长期开放的艾滋病毒高危妇女队列。通过比较 急性感染和ART病毒抑制后供者外周血单核细胞的表达水平 (n=10名捐赠者)，这种方法将告知哪些ISGs是由艾滋病毒感染在每种类型的细胞中引起的 艾滋病病毒的传播。其次，该项目的体外ARM将量化ISGs在CD4+T细胞中的抗病毒功能 来自多个捐赠者。选定的ISG将通过CRISPR被灭活，以衡量它们对干扰素介导的贡献 对干扰素敏感的艾滋病毒分离株的限制。这一方法将评估HIV在CD4+T细胞中诱导的ISGs，因为 由体内ARM定义，并将通过细胞系研究验证被描述为抗病毒的ISGs。通过审问 直接在原生人类样本中进行ISG表达和HIV限制，该项目将告知ISG环境 艾滋病毒在感染的最早阶段面临哪些因素，以及这些因素中哪些对艾滋病毒复制有意义。 开展这项研究将为我提供广泛适用的尖端技术方面的培训 对于我领导一个研究实验室研究病毒与宿主相互作用的长期目标来说，肯定是有用的。使用 我的赞助商Julie Overbaugh博士和共同赞助商Catherine Blish博士的专业知识结合在一起，我将获得 特别的指导，在这次培训中支持我，并成功地完成了拟议的工作。

PROJECT SUMMARY/ABSTRACT Cell-intrinsic antiviral proteins elicited by the interferon (IFN) response comprise one of the first lines of defense against viral infection. However, the mediators of IFN inhibition and their potency vary by virus and cell type, underscoring the need to study IFN restriction during natural infection. Like other viruses, HIV-1 triggers the IFN response and demonstrates IFN sensitivity in cell culture and in chronically infected patients. Moreover, it is well characterized that HIV transmission is inefficient, typically requiring several exposures that result in a bottleneck of viral variants. Together, these findings suggest that antiviral IFN-stimulated genes (ISGs) may pose a major barrier to HIV during transmission and early infection. Nevertheless, it is still unknown which ISGs are actually induced by HIV, primarily due to the difficulty of obtaining samples during the brief window of acute infection. Data on ISG restriction of HIV in natural settings are also limited, as previous work has largely used immortalized cell lines and lab-adapted viruses. A more complete understanding of the ISGs present during acute HIV infection and their antiviral potency in the cells that fuel HIV dissemination are needed to determine how the IFN response shapes early HIV infection. To address this gap, this proposal characterizes cellular ISG expression in precious peripheral blood mononuclear cells (PBMCs) collected during early infection in vivo and defines ISG restriction in vitro against an early HIV isolate in HIV target cells, CD4+ T cells. This study will explore the hypothesis that a set of HIV-induced, functionally antiviral ISGs are responsible for IFN-mediated inhibition of HIV in CD4+ T cells. First, the in vivo arm will determine the profile of ISG transcripts upregulated in each PBMC cell type during early HIV infection through a single-cell approach. This arm utilizes rare acute HIV and post-antiretroviral therapy (ART) PBMCs from the Mombasa Cohort, a long-term open cohort of women at high risk for HIV. By comparing expression levels between PBMCs from acute infection and after ART-mediated viral suppression within donors (n=10 donors), this approach will inform which ISGs are induced by HIV infection in each cell type that contributes to HIV spread. Second, the in vitro arm of this project will quantify the antiviral function of ISGs in CD4+ T cells from multiple donors. Chosen ISGs will be inactivated via CRISPR to measure their contribution to IFN-mediated restriction of an IFN-sensitive HIV isolate. This approach will assess ISGs induced by HIV in CD4+ T cells, as defined by the in vivo arm, and will also validate ISGs described as antiviral by cell line studies. By interrogating ISG expression and HIV restriction directly in primary human samples, this project will inform the ISG milieu that HIV faces during the earliest stages of infection and which of these factors meaningfully impact HIV replication. Carrying out this research will provide me with training in widely applicable, cutting-edge techniques that are sure to be useful for my long-term goal of leading a research laboratory studying virus-host interactions. With the combined expertise of my Sponsor Dr. Julie Overbaugh and Co-Sponsor Dr. Catherine Blish, I will receive exceptional mentorship to support me in this training and in successfully completing the proposed work.