Jung Ulrike, Takahashi Mayumi, Rossi John J, Burnett John C
Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Fox North, 1001A, 1500 Duarte Rd., Duarte, CA, 91010, USA.
Methods Mol Biol. 2016;1354:255-64. doi: 10.1007/978-1-4939-3046-3_17.
Persistent latent HIV-1 reservoirs pose a major barrier for combinatorial antiretroviral therapy (cART) to achieve eradication of the virus. A variety of mechanisms likely contribute to HIV-1 persistence, including establishment of post-integration latency in resting CD4+ T-lymphocytes, the proliferation of these latently infected cells, and the induced or spontaneous reactivation of latent virus. To elucidate the mechanisms of latency and to investigate therapeutic strategies for reactivating and purging the latent reservoir, investigators have developed in vitro models of HIV-1 latency using primary CD4+ T-lymphocytes and CD4+ T-cell lines. Several types of in vitro latency models range from replication-competent to single-round, replication-deficient viruses exhibiting different degrees of viral genomic deletion. Working under the hypothesis that HIV-1 post-integration latency is directly linked to HIV-1 promoter activity, which can be obscured by additional proteins expressed during replication, we focus here on the creation of latently infected primary human T-cells and cell lines through the single-round, replication deficient HIV-1 LGIT model. In this model the long terminal repeat (LTR) of the HIV-1 virus drives a cassette of GFP-IRES-Tat that allows testing of reactivating components and initiates a positive feedback loop through Tat expression.
持续存在的潜伏性HIV-1病毒库是联合抗逆转录病毒疗法（cART）实现病毒根除的主要障碍。多种机制可能导致HIV-1持续存在，包括在静息CD4+ T淋巴细胞中建立整合后潜伏期、这些潜伏感染细胞的增殖以及潜伏病毒的诱导或自发重新激活。为了阐明潜伏期机制并研究重新激活和清除潜伏病毒库的治疗策略，研究人员利用原代CD4+ T淋巴细胞和CD4+ T细胞系建立了HIV-1潜伏期的体外模型。几种类型的体外潜伏期模型从具有复制能力的病毒到单轮、复制缺陷型病毒不等，这些病毒表现出不同程度的病毒基因组缺失。基于HIV-1整合后潜伏期与HIV-1启动子活性直接相关这一假设，而复制过程中表达的其他蛋白质可能会掩盖这种活性，我们在此重点介绍通过单轮、复制缺陷型HIV-1 LGIT模型创建潜伏感染的原代人T细胞和细胞系。在该模型中，HIV-1病毒的长末端重复序列（LTR）驱动一个GFP-IRES-Tat盒，该盒允许测试重新激活成分，并通过Tat表达启动正反馈回路。
