title: Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3.
abstract: An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2',3'-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37 °C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37 °C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.
authors.affiliation: Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Frezza
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Sandro
authors.initials: S
authors.lastname: Grelli
authors.affiliation: Division of Pathogenesis of Retroviruses, National AIDS Center, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Federico
authors.affiliation: Department of Biological and Environmental Sciences, University of Messina, Messina, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Marino-Merlo
authors.affiliation: Department of Biological and Environmental Sciences, University of Messina, Messina, Italy.|Institute of Translational Pharmacology, CNR, Rome, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Mastino
authors.affiliation: Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Macchi
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D019829:Nevirapine / Q000494:pharmacology*; D014162:Transfection; D015215:Zidovudine / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D015215:Zidovudine; D019829:Nevirapine
keywords: anti-HIV assay; antiviral drugs; drug resistance; green fluorescent protein (GFP); human immunodeficiency virus type 1 (HIV-1)