Mechanism of Action of Methotrexate Against Zika Virus.

Sungjun Beck,Jair L Siqueira-Neto,Zhe Zhu,Jean A Bernatchez,Jeremy N Rich,Davey M Smith,Michelli F Oliveira

doi:10.3390/v11040338

Abstract

Zika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barré syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to 10-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DHFR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

Highlights

Mosquito-borne flaviviruses, such as Dengue virus (DENV), Yellow Fever virus (YFV), WestNile virus (WNV), Japanese Encephalitis virus (JEV), and Zika virus (ZIKV), are well characterized as human pathogenic viruses [1]
Envelope protein synthesis in Vero cells was observed from 5 μM MTX treatment at 48 h PI (Figure 1A)
The fluorescence signal difference was measured between 5 μM MTX treatment and DMSO control (Figure 1E)

Summary

Introduction

Mosquito-borne flaviviruses, such as Dengue virus (DENV), Yellow Fever virus (YFV), WestNile virus (WNV), Japanese Encephalitis virus (JEV), and Zika virus (ZIKV), are well characterized as human pathogenic viruses [1]. ZIKV has become a global public health threat that has brought substantial economic burden to affected countries during the recent outbreak in Latin America [2]. There are no specific antivirals or vaccines to treat ZIKV infection. High-throughput screening (HTS) of various compound libraries against ZIKV have been published, and several small molecules active against ZIKV were identified [5,6,7,8]. One active compound was methotrexate (MTX), used to treat a variety of diseases including leukemia, psoriatic arthritis, and rheumatoid arthritis [9,10,11]. MTX has been reported to interfere with a variety of cellular mechanisms, such as oxidative stress or cellular differentiation via methylation [14,15]

Methods

Results

Conclusion