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Báo cáo y học: " Quantitative PCR used to Assess HIV-1 Integration and 2-LTR Circle Formation in Human Macrophages"

Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành y học dành cho các bạn tham khảo đề tài: Quantitative PCR used to Assess HIV-1 Integration and 2-LTR Circle Formation in Human Macrophages, | Friedrich et al. Virology Journal 2010 7 354 http www.virologyj.eom content 7 1 354 VIROLOGY JOURNAL RESEARCH Open Access Quantitative PCR used to Assess HIV-1 Integration and 2-LTR Circle Formation in Human Macrophages Peripheral Blood Lymphocytes and a CD4 Cell Line Brian Friedrichf Guangyu Li Natallia Dziuba Monique R Ferguson 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 .