ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances. AIM OF THE STUDY: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity. MATERIALS AND METHODS: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4+/CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC50 values in the nanomolar range. RESULTS: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 µM (IC50: 0.49 µM, CC50: 3089 µM) and 1343 µM (IC50: 2.3 µM, CC50: 3089 µM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC50 values of 11.11 µg/ml and 16.86 µg/ml, respectively. CONCLUSION: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Plant Preparations/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Anti-HIV Agents/administration & dosage , Dose-Response Relationship, Drug , HEK293 Cells , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/enzymology , Humans , Inhibitory Concentration 50 , Nevirapine/administration & dosage , Nevirapine/pharmacology , Plant Preparations/administration & dosage , Plants, Medicinal/chemistry , Reproducibility of Results , Reverse Transcriptase Inhibitors/administration & dosage , Zidovudine/administration & dosage , Zidovudine/pharmacologyABSTRACT
There is paucity of data on the genetic landscape of HIV-1 viruses circulating in the Limpopo Province of northeastern South Africa. Here, we examine the genetic diversity of viruses from Bela-Bela and Musina, two towns with high HIV prevalence. Between June 2007 and March 2008, blood samples were collected from antiretroviral-drug-naïve individuals. Viruses were analyzed for genetic subtypes and drug resistance mutations. All of the viruses in these samples were shown by phylogenetic analysis based on gag p17, gag p24, reverse transcriptase, protease and envelope C2-C3 gene regions to belong to HIV-1 subtype C. Two of 44 reverse transcriptase sequences (4.5%) contained N rather than the consensus K at position 103. The K103N mutation is normally associated with resistance to NNRTIs. No major mutations were observed in the protease gene. However, several polymorphisms and amino acid changes normally considered to be minor drug resistance mutations were observed in the protease sequences. These results suggest that HIV-1 subtype C remains the predominant variant responsible for the epidemic in northeastern South Africa and that the prevalence of drug-resistant viruses among the naïve population is low.
