Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

Abstract

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.

Figures

Figure 1. Ivermectin inhibits Impα/β1- but not Impβ1-dependent nuclear import, whereas mifepristone specifically inhibits IN nuclear accumulation

(A) Typical CLSM images of HeLa cells expressing the indicated GFP-fusion proteins 24 h after transfection, treated with or without 25 μM ivermectin or 50 μM mifepristone as indicated for 1 h before imaging. (B) Results (mean±S.E.M., n>89) for quantitative analysis of images such as those in (A) to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c); *P<0.01.

Figure 2. Ivermectin is a broad-spectrum Impα/β1 inhibitor that does not affect other nuclear import pathways

HeLa cells transfected to express the indicated GFP-fusion proteins were treated with or without 25 μM ivermectin for 1 h before live-cell imaging 24 h after transfection. Results (mean±S.E.M., n>68) were determined as described in Figure 1(B); **P<0.001.

Figure 3. Ivermectin can inhibit HIV-1 and DENV infection

(A) HeLa cells were infected with 200 ng (capsid protein-equivalent) of VSV-G-pseudotyped NL4-3.Luc.R-E- HIV, treated with or without the indicated agents (concentration in parentheses, μM) 2 h after infection for 6 h, then medium was removed, and cells were harvested for measurement of luciferase reporter activity. LD50 values for ivermectin and mifepristone in 50% confluent HeLa cells incubated for 24 h with each compound were 150 μM and 33 mM respectively. Results are means±S.E.M. for an average of four repeats; *P<0.05, **P<0.01. (B) AlphaScreen binding inhibition curves for DENV NS5 and the indicated Imps. Assays were performed as described in the Materials and methods section using 30 μM His6-tagged NS5 protein and 30 μM biotinylated Impα/β1, in the presence of the indicated concentrations of ivermectin, mifepristone or DMSO (vehicle) control. (C) Vero cells were treated with or without 25 μM (left) or 50 μM (right) ivermectin or mifepristone (as indicated) for 3 h before infection with DENV-2. Cell culture medium was collected and viral titres were analysed at various times after infection by plaque assay. PFU, plaque-forming units.