Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIV^RNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA⁺/Gag protein⁺ cells per million CD4 T cells. Uniquely, the HIV^RNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.

中文翻译：

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使用RNA流FISH技术对罕见的HIV感染细胞进行多参数表征。

体内支持HIV复制的细胞的有限表征以及用于接受抗逆转录病毒治疗（ART）的个体对潜在病毒库的定量方法不足，都阻碍了治愈HIV的努力。我们描述了一种通过对原位RNA杂交结合抗体对HIV Gag蛋白染色同时检测细胞HIV Gagpol mRNA的基础上，对病毒库进行流式细胞术鉴定的协议。通过同时检测HIV RNA和蛋白质，可以鉴定出具有翻译能力的病毒的CD4 T细胞。HIV ^{RNA / Gag}方法的灵敏度比单独的Gag蛋白染色高1,000倍，检出限为0.5-1 Gagpol mRNA ⁺ / Gag蛋白⁺每百万个CD4 T细胞中的细胞数。独特的是，HIV ^{RNA / Gag}分析还可以对病毒库进行平行表型分析，包括临床样本中重新激活的潜伏库。该测定需要2天，需要对表面和细胞内标记物进行抗体标记，然后进行mRNA标记和多个信号放大步骤。