title: Development of a new methodology for screening of human immunodeficiency virus type 1 microbicides based on real-time PCR quantification.
abstract: Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.
authors.affiliation: Laboratório de Virologia Molecular, Departamento de Genética, Universidade Federal do Rio de Janeiro, CCS-Bloco A2, Sala 121, Cidade Universitária, Ilha do Fundão, 21944-970, Rio de Janeiro-RJ, Brazil.
authors.forename: Renato S
authors.initials: RS
authors.lastname: Aguiar
authors.forename: Luciana J
authors.initials: LJ
authors.lastname: Costa
authors.forename: Helena S
authors.initials: HS
authors.lastname: Pereira
authors.forename: Rodrigo M
authors.initials: RM
authors.lastname: Brindeiro
authors.forename: Amilcar
authors.initials: A
authors.lastname: Tanuri
mesh_terms: D000480:Alkynes; D048588:Benzoxazines / Q000378:metabolism / Q000494:pharmacology; D001681:Biological Assay; D002474:Cell-Free System; D003521:Cyclopropanes; D005260:Female; D015497:HIV-1 / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D019829:Nevirapine / Q000378:metabolism / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription / Q000187:drug effects; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
chemical_list: D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; C098320:efavirenz