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. 2023 Sep 16:5:100203.
doi: 10.1016/j.crmicr.2023.100203. eCollection 2023.

A cysteine protease inhibitor GC376 displays potent antiviral activity against coxsackievirus infection

Yongkang Chen  1 , Xiaohong Li  1 , Min Wang  1 , Yuan Li  1 , Jun Fan  1 , Jingjing Yan  1 , Shuye Zhang  2 , Lu Lu  1 , Peng Zou  1
Affiliations

A cysteine protease inhibitor GC376 displays potent antiviral activity against coxsackievirus infection

Yongkang Chen et al. Curr Res Microb Sci. .

Abstract

Infection with coxsackievirus A10 (CV-A10) can cause hand-foot-mouth disease and is also associated with severe complications, including viral pneumonia, aseptic and viral meningitis. Coxsackievirus infection may also play a role in the pathogenesis of acute myocardial infarction and in the increased risk of type 1 diabetes mellitus in adults. However, there are no approved vaccines or direct antiviral agents available to prevention or treatment of coxsackievirus infection. Here, we reported that GC376 potently inhibited CV-A10 infection in different cell lines without cytotoxicity, significantly suppressed production of viral proteins, and strongly reduced the yields of infectious progeny virions. Further study indicated that GC376, as viral 3C protease inhibitor, had the potential to restrain the cleavage of the viral polyprotein into individually functional proteins, thus suppressed the replication of CV-A10. Furthermore, the drug exhibited antiviral activity against coxsackieviruses of various serotypes including CV-A6, CV-A7 and CV-A16, suggesting that GC376 is a broad-spectrum anti-coxsackievirus inhibitor and the 3C protease is a promising target for developing anti-coxsackievirus agents.

Keywords: 3C protease; Antiviral; Coxsackievirus; Cysteine protease; GC376; Protease inhibitor.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image, graphical abstract
Graphical abstract
Fig 1
Fig. 1
The inhibitory activity of GC376 against CV-A10 infection in RD cells. (A) Antiviral effects of GC376 against CV-A10 infection was measured by plaque reduction assay and IC50 value was calculated (B). GC376 suppressed the expression of viral 3A proteins (C) and the yields of progeny virions (D) following CV-A10 infection. Data are represented as means ± SD. NS, not significant; **** p < 0.0001.
Fig 2
Fig. 2
GC376 effectively restrained viral protein expression in CV-A10 infected-RD cells. RD cells were infected by CV-A10 at a multiplicity of infection (MOI) of 2, and then treated with serially diluted GC376. After 8 h of culturation, viral 3A proteins expressed in the cells were stained by anti-3A mouse monoclonal antibody (green); nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 25 µm.
Fig 3
Fig. 3
The inhibitory activity of GC376 against CV-A10 infection in Vero and HEK-293T cells and its toxicity to these cell lines. (A) Anti-CV-A10 effects of GC376 was measured by plaque reduction assay in Vero cells. (B) GC376 significantly inhibited the yield of progeny virions in Vero cells infected by CV-A10. (C) GC376 restricted viral 3A proteins expression in HEK-293T cells following CV-A10 infection at MOI of 0.5. The RD (D), Vero (E) and HEK-293T cells (F) were treated with different concentrations of GC376, and the cell viability was detected by Cell Counting Kit-8. Data are represented as means ± SD. NS, not significant; **** p < 0.0001.
Fig 4
Fig. 4
GC376 inhibited CV-A10 infection in the late stage. (A) Outline of the time-of-addition experiment that was utilized to identify at which stage the CV-A10 lifecycle was blocked by GC376. hpi means hours post infection. (B) GC376 was added at different time points as indicated, and then cytopathic effect (CPE) were observed under microscope. Scale bar: 400 µm. (C) The virus titers in the supernatants were also measured at 12 hpi by plaque assays. (D) For the entry assay, RD cells were treated with CV-A10 and 5 µM of GC376 at 37 °C for 1 h, and then unbound viruses and drug were removed. For the attachment assay, RD cells were infected by CV-A10 at 4 °C for 1 h in the presence of 5 µM of GC376, followed by removing unbound viruses and drugs. For the internalization assay, RD cells were infected with CV-A10 at 4 °C for 1 h to allow virus adsorption and washed twice with cold DMEM to remove unbound viruses, and then RD cells were incubated at 37 °C for 1 h to allow virus internalization in the presence of 5 µM of GC376, followed by removal of the drug. For the post-entry experiment, RD cells were infected with CV-A10 at 37 °C for 1 h to allow viral entry, and then the inoculum was removed. The overlay medium, supplemented with GC376 for post-entry assay or without GC376 for the other three assays, was then added to conduct plaque assay. Data are represented as means ± SD. NS, not significant; **** p < 0.0001.
Fig 5
Fig. 5
GC376 potently suppressed the replication of the CV-A10 subgenomic viral RNA. (A) Single round infectious particles (SRIPs), which encapsulated the luciferase reporter gene-containing CV-A10 subgenomic RNA, was utilized to infect RD cells with or without GC376 treatment. The luciferase activity of cell lysate was detected at 24 hpi, showing the suppression of CV-A10 viral RNA replication by GC376 treatment. (B) RD cells were transfected with in vitro transcribed CV-A10 replicon RNA bearing luciferase reporter and simultaneously treated with GC376, followed by detection of luciferase activity of the cell lysate at 24 hpi. Data are represented as means ± SD. NS, not significant; **** p < 0.0001.
Fig 6
Fig. 6
GC376 effectively inhibited the proteolytic activity of CV-A10 3C protease. CV-A10 3C protease together with the MBP-TATVQGPSLD-EGFP-His6 protein, which contained the 3C protease cleavage site TATVQGPSLD as the substrate, were treated with or without GC376 at 30 °C for 4 h. Then the proteins in each sample were subsequently separated and visualized by SDS-PAGE and Coomassie brilliant blue staining.
Fig 7
Fig. 7
The activities of GC376 against different serotypes of coxsackieviruses. Inhibitory effects of GC376 against infection of CV-A16 (A), CV-A6 (B) and CV-A7 (C) were measured by plaque reduction assay. Data are represented as means ± SD.
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