Effects of host chaperone inhibitors on RVFV levels and timing of HSP effects.

<p>(A) Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control), 17-AGG (10 µM), BAPTA (10 µM), or KNK437 (10 µM), prior to infection with the MP-12 strain of RVFV (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. Twenty-four hours later, supernatants were collected and viral RNA was measured by qRT-PCR as described in the Methods section. Percent viral RNA is expressed as the percent of the DMSO control. The viability of the cells in the presence of 10 µM inhibitor or in DMSO (vehicle) was assessed by MTT assay, as described in the Methods section. Data are average values from three independent assays. (B) Time of addition studies were performed using 17-AAG and BAPTA-AM inhibitors to analyze the timing of HSP effects during RVFV infection. Vero cells were treated with HSP inhibitors, or with vehicle only (DMSO), at the indicated time points relative to MP-12 infection (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. qRT-PCR was performed to measure viral RNA levels in the supernatants collected 24 hours post-infection. Data are average values from three independent assays. (C), and (D) Viral inhibition following HSP inhibitor treatment was measured in real time. One hour prior to infection with rMP12-rLuc (MOI 0.1), HepG2 cells were treated with HSP inhibitors 17-AAG, or BAPTA-AM, or a combination of both inhibitors. Controls included no inhibitor treatment and no infection [Untreated (UT)], no inhibitor treatment but with infection using either the regular MP-12 strain that has no luciferase expression (MP12; UT) or with the luciferase expressing rMP12-rluc strain (Mp12-Luc; UT), and treatment with vehicle only, either without any infection (DMSO) or with infection using the rMP12-rluc strain (Mp12-Luc; DMSO). All samples were analyzed either at 4 hours p.i. (C) or at 8 hours p.i. (D), using the Renilla Luciferase assay. Data are average values from three independent assays. Asterisks designate significant changes in luminescence levels (P<0.05).