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

项目总结/摘要 干扰素（IFN）反应引发的细胞固有抗病毒蛋白构成了第一道防线之一 抵抗病毒感染然而，IFN抑制的介质及其效力因病毒和细胞类型而异， 强调需要研究自然感染期间IFN的限制。像其他病毒一样，HIV-1触发IFN 在细胞培养物和慢性感染患者中显示IFN敏感性。而且， 艾滋病毒的传播是低效的，通常需要多次暴露， 病毒变种。总之，这些发现表明，抗病毒干扰素刺激基因（ISGs）可能构成了一个主要的干扰素受体。 艾滋病毒在传播和早期感染过程中的屏障。尽管如此，仍然不知道哪些ISG实际上是 这主要是由于在短暂的急性感染窗口期难以获得样本。 关于ISG在自然环境中限制HIV的数据也很有限，因为以前的工作主要使用永生化的 细胞系和实验室适应的病毒。更全面地了解急性HIV感染期间存在的ISG 以及它们在促进HIV传播的细胞中的抗病毒效力， 形成早期HIV感染。为了解决这一差距，该提案将细胞ISG表达的特征描述为珍贵的， 在体内早期感染期间收集的外周血单核细胞（PBMC），并定义ISG限制 在体外针对早期HIV分离物中的HIV靶细胞，CD 4 + T细胞。本研究将探讨假设， 一组HIV诱导的功能性抗病毒ISG负责IFN介导的对CD 4 + T细胞中HIV的抑制作用， 细胞首先，体内组将确定在体外培养期间在每种PBMC细胞类型中上调的ISG转录物的概况。 早期艾滋病毒感染通过单细胞方法。这支队伍利用罕见的急性艾滋病毒和后抗逆转录病毒治疗 (ART)来自蒙巴萨队列的PBMC，这是一个长期开放的HIV高危女性队列。通过比较 供体内急性感染和ART介导的病毒抑制后PBMC之间的表达水平 （n=10名供体），该方法将告知HIV感染在每种细胞类型中诱导哪些ISG， 艾滋病毒的传播。其次，本项目的体外研究将量化ISGs在CD 4 + T细胞中的抗病毒功能 来自多个捐赠者。选择的ISG将通过CRISPR失活，以测量它们对IFN介导的免疫应答的贡献。 IFN敏感的HIV分离株的限制。这种方法将评估HIV在CD 4 + T细胞中诱导的ISG， 通过体内组定义，并且还将通过细胞系研究验证描述为抗病毒的ISG。通过询问 ISG表达和HIV限制直接在原代人类样本中，该项目将告知ISG环境， 艾滋病毒在感染的最早阶段面临的风险，这些因素中哪些对艾滋病毒复制有意义。 进行这项研究将为我提供广泛适用的培训，尖端技术， 我的长期目标是领导一个研究实验室，研究病毒与宿主的相互作用。与 我的赞助商Julie Overbaugh博士和共同赞助商Catherine Blish博士的综合专业知识，我将获得 在这次培训和成功完成拟议工作中，我得到了出色的指导。

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.