Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0435, USA.
Virol J. 2010 Dec 3;7:354. doi: 10.1186/1743-422X-7-354.
Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL).
In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4+ cells by infecting with HIV-1SX (R5) or dual-tropic isolate HIV-189.6 (R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA.
These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.
整合是 HIV 生命周期中的一个中间步骤，定义为 HIV-1 前病毒 DNA 插入宿主染色体。如果 HIV-1 cDNA 进入细胞核时没有发生整合，它就会自身环化并形成 2-LTR 环。监测不同原代细胞亚群中整合的 HIV-1 cDNA 水平非常重要，特别是在 HIV-1 感染者接受 HAART 治疗的情况下。由于之前 HIV-1 整合检测方法的限制，关于原代细胞亚群中整合和 2-LTR 环形成的水平的数据有限，特别是在人单核细胞衍生的巨噬细胞和外周血淋巴细胞（PBL）中。
在这项研究中，我们利用一种定义明确、敏感的两步实时定量 PCR 方法来检测 HIV-1 整合，以及常规实时 PCR 来检测从六位不同健康供体分离的人巨噬细胞和 PBL 以及 U373 CD4+细胞中形成的 2-LTR 环，这些细胞通过感染 HIV-1SX（R5）或双嗜性分离株 HIV-189.6（R5/X4）病毒株而感染。我们使用了 FDA 批准的整合酶抑制剂拉替拉韦，以确定在有和没有拉替拉韦治疗的 HIV-1 感染细胞中整合的 HIV 病毒 cDNA 的定量差异。我们的结果表明，通过这种方法可以在原代巨噬细胞、PBL 和 CD4+细胞系中评估整合和 2-LTR 环形成。具体来说，我们的结果表明，两步实时 PCR 方法可以区分拉替拉韦治疗引起的整合受损的 HIV-1 整合病毒 cDNA 和非整合的核 HIV-1 2-LTR 环。这进一步证实，只有整合的 HIV-1 cDNA 可以通过两步 PCR 特异性扩增和定量，而不会非特异性地检测非整合的病毒 cDNA。
这些结果一致表明，所使用的成熟的实时 PCR 检测方法在使用实验室适应的病毒株而不是假病毒时，对于检测生理相关的人巨噬细胞和 PBL 中的 HIV-1 整合和 2-LTR 环形成是稳健、敏感和定量的。通过两步实时 PCR，我们表明在拉替拉韦处理的细胞中未检测到未整合的核 HIV-1 cDNA，而在未处理的细胞中仅特异性检测到整合的 HIV-1 cDNA。这些方法可作为 HIV-1 感染者特定治疗监测的有用工具。
