Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line.
Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line.
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DOI：
10.1186/1743-422x-7-354
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发表时间：
2010-12-03
期刊：
影响因子：
4.8
通讯作者：
Ferguson MR
中科院分区：
文献类型：
--
作者：
Friedrich B;Li G;Dziuba N;Ferguson MR
整合是HIV生命周期的中间步骤，定义为HIV-1前病毒DNA插入宿主染色体。如果当HIV-1 cDNA进入细胞核时没有发生整合，则其自身环化并形成2-LTR环。监测不同原代细胞亚群中整合的HIV-1 cDNA的水平是非常重要的，特别是关于HAART在HIV-1感染个体中的效果。由于现有HIV-1整合试验的局限性，关于原代细胞亚群中整合水平和2-LTR环形成的数据有限，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，我们利用了一个定义明确，灵敏的两步定量实时PCR方法来检测HIV-1的整合，以及传统的实时PCR检测2-LTR环形成的人巨噬细胞和PBL分离自6个不同的健康供体，以及U373 CD 4+细胞感染HIV-1 SX（R5）或双嗜性分离株HIV-189.6（R5/X4）病毒株。我们使用FDA批准的整合酶抑制剂雷特格韦（raltegravir）来测定经和未经雷特格韦治疗的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，整合和2-LTR环的形成可以在原代巨噬细胞，PBL，和CD 4+细胞系通过这种方法进行评估。具体地说，我们的结果表明，这种两步实时PCR方法可以区分HIV-1整合的病毒cDNA和非整合的核HIV-1 2-LTR环与雷特格韦治疗受损的整合所造成的。这进一步证实了只有整合的HIV-1 cDNA可以通过两步PCR进行特异性扩增和定量，而不会非特异性检测非整合的病毒cDNA。这些结果一致表明，使用实验室适应性病毒株（而非假病毒），所使用的成熟实时PCR检测方法对于检测生理相关人巨噬细胞和PBL中的HIV-1整合和2-LTR环形成具有耐用性、灵敏度和定量性。用两步实时PCR，我们发现，未整合的，核HIV-1 cDNA在雷特格韦处理的细胞中未检测到，而在未处理的细胞中仅整合HIV-1 cDNA。这些方法可作为进一步监测HIV-1感染者特异性治疗的有用工具。
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.