Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication

Abstract

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Keywords: chikungunya virus; deubiquitinase inhibitor; deubiquitinating enzymes; infection; replication; ubiquitination.

Figure 1

Cytotoxic effect of deubiquitinating enzyme inhibitors. HEK293, Huh-7, and Vero cells were exposed to PR-619 or WP1130 at the indicated concentrations, DMSO 0.1% was used as a control. After 24 h of treatment, cell viability was analyzed using the resazurin assay method. Data are represented as the mean + SD, and statistically significant differences are indicated as * p < 0.05, ** p < 0.01. n.s no significant differences.

Figure 2

Inhibition of deubiquitinating enzymes impairs chikungunya virus replication. Cells were pre-treated with the indicated concentrations of WP1130 (W), PR-619 (P), or DMSO for 1 h. After that, cells were infected with CHIKV for 1 h, the inoculum was then removed and replaced with fresh media supplemented with inhibitors. After 24 h of infection, supernatants and cells were collected. (A) Released viral progeny was measured using the plaque assay method. (B) Infected cells were analyzed using flow cytometry. Data are represented as the mean + SD, and statistically significant differences are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Deubiquitinating activity is required at an early stage of infection. (A) Schematic representation of the experiment. (B) Cells were treated with WP1130 at the indicated concentrations 1 h before infection or 4 h post-infection. Cells were collected 24 h later and analyzed using flow cytometry. (C) Infection rates normalized to control (DMSO). Data are represented as the mean + SD, and statistically significant differences are indicated as * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4

Inhibition of deubiquitinating enzymes impairs protein and viral RNA synthesis. Cells were pre-treated with the inhibitors WP1130 (W) or PR-619 (P) at the indicated concentrations, then infected with CHIKV for 1 h; after this time, the inoculum was removed and replaced with fresh media supplemented with inhibitors. After 24 h of infection, cells were collected. (A) Viral protein levels were evaluated using Western blot with antibodies against E1 and nsP1. β-actin was used as a loading control. (B) Levels of intracellular viral RNA were measured using RT-qPCR. Data are represented as the mean + SD, and statistically significant differences are indicated as * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 5

Expression of deubiquitinating enzymes throughout CHIKV infection. Cells were infected with CHIKV for 1 h; the inoculum was removed and replaced with fresh media; then, cells were collected at the indicated times. Expression of different DUB proteins was assessed using Western blot with antibodies against HAUSP, USP10, A20, STAMBP, and UCHL1, whereas viral protein levels were evaluated using antibodies against E1 and nsP1. β-actin was used as a loading control.