Figure 5

REV-ERB agonist SR9009 has pan-subtype antiviral activity. (a) Consensus plot illustrating the conserved nature of the E-box in the HIV-1 LTR based on the 897 HIV-1 sequences available in the LANL repository (analyzed with LANL QuikAlign/AnalyzeAlign software, coordinates are from the HXB2 referent). The level of conservation is reflected by the height of the bases (y axis 0–100%)^39,40, with the consensus E-box motif (JASPAR database)⁴⁰ shown below. (b) Conserved E-box motifs in the HIV-1 strains chosen to study, where HIV LAI has a variant, G, at position 8. Jurkat cells were transfected with HIV-1 promoter reporter constructs for 24 h. Transfected cells were treated with SR9009 at 20 µM for 24 h and LTR activity measured by quantifying luciferase activity (mean ± S.E.M., n = 4, Two-way ANOVA). (c) E-box mutation reduces HIV-LTR activity. Jurkat cells were transfected with wild type (LAI-B) or E-box mutated HIV-LTR constructs (where Δ represents a deletion and M represents a mutation). LTR activity was measured 48 h later and data are expressed relative to the wild type control. (mean ± S.E.M., n = 8–11, One-way ANOVA analysis). (d) Jurkat or activated CD4 T cells were infected with HIV-1 for 72 h. Infected cells were cultured in medium with or without SR9009 and viral RNA measured 48 h later. Data are expressed relative to the control untreated cells (mean ± S.E.M., n = 5–6, Mann–Whitney analysis). (e) CD8 depleted Human peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 and cultured in medium with or without SR9009. Extracellular p24 levels were measured at intervals across a 10-day time period. Mean values from n = 3 biological repeats are shown, with error bars reflecting the geometric standard deviation. The first biological repeat employed cells from a single HIV-seronegative donor, whilst repeats two and three employed cells pooled from three healthy donors. The statistical significance of differences in mean extracellular p24 values between SR9009 and control cells at each time point was assessed using a paired t test.