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


CONCLUSIONS
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.
Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0435, USA.
Brian
B
Friedrich
Guangyu
G
Li
Natallia
N
Dziuba
Monique R
MR
Ferguson
D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008264:Macrophages / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D016662:Virus Integration
D018076:DNA, Complementary; D004279:DNA, Viral