Figure 3
Cistus incanus extract blocks invasion of cells by HIV.
(A) Antiviral activity of Ci extract inhibits a very early phase of the HIV replication cycle. Time-of-addition (TOA) assays were performed with Cistus incanus (Ci) extract or the reference inhibitors T20 (virus-cell fusion) and Efavirenz (reverse transcription). Each time point was assayed in triplicate wells and infection levels normalized to those of untreated cultures (=100%). Symbols indicate mean values and error bars standard deviation of the mean. (B) Ci extract prevents delivery of genomic HIV RNA to cells. LC5-CD4 cultures were exposed to HIV-1LAI in the absence (no Ci) or in the presence of 100 μg/ml whole Ci extract (Ci) or a Ci-derived polyphenol enriched fraction (CiPP) for 4 hours. Efavirenz was included to block HIV reverse transcription in target cells. RNA was isolated from cultures after 4 hours of virus exposure, cDNA generated and used for quantitative PCR analysis. Levels of HIV-1 sequences were related to RNA polymerase II reference sequences. The data is expressed as relative levels of HIV-RNA genomes in cultures exposed to HIV-1, normalized to background (i.e. no virus). Each bar represents the mean results of 3 virus exposure experiments Error bars indicate the standard deviation of the mean. (C,D) Ci extract prevents attachment of virus particles to cells. Spinning disc confocal microscopy was used to analyse the influence of Ci or CiPP on the attachment of GFP-labelled HIV-1 virus particles to LC5-RIC cells. Cells were exposed to virus for 4 hours. The fusion inhibitor T20 was included to block entry of virus into target cells. Controls consisted of cells exposed to virus without Ci (attachment control) and unexposed cells (background). Cell nuclei were visualized by DAPI staining. (C) Quantitative analysis of cell-associated GFP-spots. GFP-signals associated with 60 cells from 4 images were analysed for each sample. The bars represent the mean values of GFP signals per cell and the error bars indicate the standard deviation of the mean. (D) Representative images for each sample. Scale bars: 20 μm.
