In vitro modeling of HIV proviral activity in microglia
In vitro modeling of HIV proviral activity in microglia
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DOI：
10.1111/febs.14293
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发表时间：
2017-12-01
期刊：
影响因子：
5.4
通讯作者：
Harvey, Brandon K.
中科院分区：
文献类型：
--
作者：
Campbell, Lee A.;Richie, Christopher T.;Harvey, Brandon K.
小胶质细胞是大脑中常驻的巨噬细胞，由于其可被 HIV 感染，因此在 HIV 相关神经认知障碍 (HAND) 的发病机制中发挥着关键作用。这导致神经毒性病毒蛋白和促炎化合物的释放，对周围神经元的功能产生负面影响。由于大脑内 HIV 感染的模型有限，我们的目标是创建一种新型小胶质细胞系，其具有整合的 HIV 原病毒，能够重现 HIV 感染的多种特征。我们利用成簇规则间隔短回文重复序列 (CRISPR)/Cas9 基因编辑技术，将改良的 HIV 原病毒整合到 CHME-5 永生化小胶质细胞中，以创建 HIV-NanoLuc CHME-5。在改良的原病毒中，Gag-Pol 区域被纳米荧光素酶 (NanoLuc) 的编码区域取代，从而可以使用发光底物快速检测 HIV 长末端重复活性，同时仍含有产生已确定的神经毒性病毒蛋白（例如 tat、nef、gp120）所需的遗传物质。我们证实 HIV-NanoLuc CHME-5 小胶质细胞表达 NanoLuc 以及 HIV 病毒蛋白 Nef。随后，我们将这些细胞暴露于一系列实验中，以调节原病毒的活性。通过用促炎因子脂多糖（LPS）和肿瘤坏死因子α处理细胞以及过表达病毒调节蛋白Tat来增强前病毒活性。相反，通过 CRISPR/Cas9 对 Toll 样受体 4 基因进行遗传修饰，减少了 LPS 介导的原病毒激活，并且 NF-B 抑制剂柳氮磺胺吡啶的药理应用同样降低了原病毒活性。总体而言，这些数据表明 HIV-NanoLuc CHME-5 可能是研究 HIV 介导的神经病理学和前病毒调节的有用工具。
Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-B inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.