FIG 3.

Characterizing SERM-mediated inhibition of viral growth. (A) Schematic representation of the time course of viral infection and treatment with SERM compounds. (B to D) Vero cells were treated with compounds at different time points (in hours) before, during, or after virus infection, and the viral titer (PFU/mL) was determined for CHIKV (B and C) and SINV (B and D). For cotreatment studies, compounds were added simultaneously with the virus inoculum. For the pretreatment assay, Vero cells were treated with the inhibitors 2 h prior to viral adsorption. For the posttreatment assay, cells were incubated at 37°C with viral infection medium, which was replaced by maintenance medium at different time points containing 5 μM 4-OHT or 7.5 μM tamoxifen/clomifene. Supernatants were collected 24 hpi for the quantification of viral titer by plaque assay; 0.1% DMSO was used as a vehicle control. For all the experimental groups, Vero cells were infected with CHIKV or SINV at an MOI of 1. (E) SINV minigenome replicon assay capturing the luciferase activity in lysate derived from BHK21 cells transfected with capped RNA encoding the SINV-REP reporter replicon. Cells were subjected to 6 h of treatment with VC or 2.5 μM of the indicated SERMs. A schematic diagram of SINV-REP has also been presented (top). (F and G) Changes in vRNA levels in CHIKV- and SINV-infected cells at 6 hpi. Vero cells were infected with CHIKV and SINV at an MOI of 5 followed by treatment with 5 μM compound. vRNA levels were assessed by qRT-PCR, and the fold reduction in vRNA relative to VC-treated cells is plotted; actin, endogenous control; E1, target gene. (H) Quantitative determination of extracellular SINV vRNA levels at 24 hpi. Vero cells were infected with SINV at an MOI of 1 and were treated with indicated concentrations of the SERM compounds. Quantification of the vRNA in the culture supernatant was done using qRT-PCR as discussed in Materials and Methods. Statistical significance of the difference in viral titer/luciferase activity/vRNA levels between treated and vehicle control-treated cells was assessed by a one-way ANOVA test and Dunnett’s posttest; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Values are means, and error bars are standard deviation values from triplicate experiments.