Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line

Abstract

Background: 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).

Results: 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.

Conclusion: 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.

Figure 1

Quantitation of HIV-1 integration and 2-LTR circle formation in CD4+ U373 cells. U373-MAGI-CCR5 cells were plated in 6-well plates with or without raltegravir treatment 24 h prior to infection and during infection (MOI = 0.1). Two days after infection, (A) β-galactosidase activity (expressed as RLU = Relative Light Units) was analyzed for determination of HIV-1_SX infection; (B) cellular genomic DNA was extracted from U373 cells 48 h after infection and HIV-1_SX integration was detected using two-step quantitative PCR, and (C) 2-LTR circle formation was measured by real-time PCR. (**p < 0.01)

Figure 2

Quantitation of HIV-1 integration and 2-LTR circle formation in human PBL. PBL were plated in 6-well plates with or without raltegravir treatment 24 h prior to infection with HIV-1_89.6, during infection and 48 h after infection (MOI = 0.1). (A) Seven days after infection, supernatant was assessed for p24 level of each group by p24 capture ELISA; (B) Six days after infection, cellular genomic DNA was extracted from PBLs and HIV-1 integration was measured by two-step quantitative PCR, and (C) 2-LTR circle formation was measured by real-time PCR. (**p < 0.01)

Figure 3

Quantitation of HIV-1 integration and 2-LTR circle formation in human macrophages. Macrophages were plated in 6-well plates with or without raltegravir treatment 24 h prior to infection, during infection and 48 h after infection (MOI = 0.1). (A) Seven days after infection, supernatant was assessed for p24 level of each group by p24 capture ELISA; (B) Six days after infection, cellular genomic DNA was extracted from macrophages, and HIV-1_SX integration was measured by two-step quantitative PCR, and (C) 2-LTR circle formation was measured by real-time PCR. (**p < 0.01)