Inhibition of heat-shock protein 90 reduces Ebola virus replication

Abstract

Ebola virus (EBOV), a negative-sense RNA virus in the family Filoviridae, is known to cause severe hemorrhagic fever in humans and other primates. Infection with EBOV causes a high mortality rate and currently there is no FDA-licensed vaccine or therapeutic treatment available. Recently, heat-shock protein 90 (Hsp90), a molecular chaperone, was shown to be an important host factor for the replication of several negative-strand viruses. We tested the effect of several different Hsp90 inhibitors including geldanamycin, radicicol, and 17-allylamino-17-demethoxygeldanamycin (17-AAG; a geldanamycin analog) on the replication of Zaire EBOV. Our results showed that inhibition of Hsp90 significantly reduced the replication of EBOV. Classic Hsp90 inhibitors reduced viral replication with an effective concentration at 50% (EC(50)) in the high nanomolar to low micromolar range, while drugs from a new class of Hsp90 inhibitors showed markedly more potent inhibition. These compounds blocked EBOV replication with an EC(50) in the low nanomolar range and showed significant potency in blocking replication in primary human monocytes. These results validated that Hsp90 is an important host factor for the replication of filoviruses and suggest that Hsp90 inhibitors may be therapeutically effective in treating EBOV infection.

Figure 1

Effect of Hsp90 inhibitors geldanamycin, 17-AAG, and radidicol on the replication of EBOV-GFP. The dose response curves of geldanamycin (closed circles) 17-AAG (closed squares) and radicicol (closed triangles) at A) 16-h PI, B) 24-h PI, C) 40-h PI, and D) 48-h PI are plotted as percent decrease of a fluorescent signal compared to dimethylsulfoxide-treated control infection. Results show a decrease in the percent GFP reduction at increasing concentrations of compound.

Figure 2

Toxicity and time-course of geldanamycin, 17-AAG, and radidicol during EBOV-GFP infection. A) Cell toxicity was determined by evaluating the viable cells by crystal violet stain. Percentage of surviving cells 48 h after drug treatment is plotted. B) % reduction of viral replication at 1.6 µM was plotted over the 48-h time-course.

Figure 3

Compounds tested at 12.5 µM, 1 µM, 37 nM, and 1 nM by yield-reduction assay and supernatant analyzed by plaque assay (A, C, and E) or real-time RT-PCR (B, D, and F). PFU=plaque-forming units; PFUe=plaque-forming unit equivalents

Figure 4

Structure of the Serenex compounds AV-1-3 and AV-81. A) Core structure and B) side-groups.

Figure 5

Effect of novel Hsp90 inhibitors geldanamycin, 17-AAG, and radidicol on the replication of EBOV-GFP. The dose response curves of AV-1, AV-2, AV-3, and AV-81 at A) 16-h PI, B) 24-h PI, C) 40-h PI, and D) 48-h PI.

Figure 6

Compounds were tested at 12.5 µM and 37 nM by yield-reduction assay in A) Vero or B) monocytes and supernatant analyzed by real-time RT-PCR.