Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral MNK1 inhibitor as an antiviral agent suppresses buffalopox virus protein synthesis Ram Kumar a,b,1 , Nitin Khandelwal a,1 , Yogesh Chander a,1 , Thachamvally Riyesh a , Bhupendra N. Tripathi a , Sudhir Kumar Kashyap b , Sanjay Barua a,** , Sunil Maherchandani b,*** , Naveen Kumar a,* a National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, India b Department of Veterinary Microbiology and Biotechnology, Rajasthan University of Veterinary and Animal Sciences, Bikaner, India ARTICLE INFO Keywords: MNK1 CGP57380 Antiviral efficacy Protein synthesis Drug resistance ABSTRACT A small molecule chemical inhibitor CGP57380 that blocks activation of MAPK interacting kinase 1 (MNK1) was found to significantly suppress buffalopox virus (BPXV) replication. BPXV infection was shown to induce MNK1 activation. Depletion of MNK1 by small interfering RNA (siRNA), blocking activation of extracellular regulated kinase (ERK, an upstream activator of MNK1) and disruption of eIF4E/eIF4G interaction (downstream substrate of MNK1 which plays a central role in cap-dependent translation initiation), resulted in reduced BPXV re- plication, suggesting that ERK/MNK1/eIF4E signaling is a prerequisite for BPXV replication. With the help of time-of-addition and virus step-specific assays, CGP57380 treatment was shown to decrease synthesis of viral genome (DNA). Disruption of ERK/MNK1/eIF4E signaling resulted in reduced synthesis of viral proteins, sug- gesting that BPXV utilizes cap-dependent mechanism of translation initiation. Therefore, we concluded that decreased synthesis of viral genome in presence of MNK1 inhibitor is the result of reduced synthesis of viral proteins. Furthermore, BPXV was sequentially passaged (P = 40) in presence of CGP57380 or vehicle control (DMSO). As compared to P0 and P40-control viruses, P40-CGP57380 virus replicated at significantly higher (∼10-fold) titers in presence of CGP57380, although a complete resistance could not be achieved. In a BPXV egg infection model, CGP57380 was found to prevent development of pock lesions on chorioallantoic membrane (CAM) as well as associated mortality of the embryonated chicken eggs. We for the first time demonstrated in vitro and in ovo antiviral efficacy of CGP57380 against BPXV and identified that ERK/MNK1 signaling is a prerequisite for synthesis of viral proteins. Our study also describes a rare report about generation of drug- resistant viral variants against a host-targeting antiviral agent. 1. Introduction Buffalopox is caused by an orthopoxvirus that belongs to the family Poxviridae. The natural host for buffalopox virus (BPXV) is domestic buffalo (Bubalus bubalis), but it can also infect cattle and humans and hence is considered as a potential zoonotic threat (Gujarati et al., 2018; Lewis-Jones, 2004; Singh et al., 2006, 2007). Buffalopox outbreaks have been reported in India, Egypt, Indonesia and Italy (Gujarati et al., 2018; Marinaik et al., 2018; Singh et al., 2007). Buffalopox virus (BPXV) infection in humans have been frequently reported in India (Bera et al., 2012; Dumbell and Richardson, 1993; Gujarati et al., 2018; Gurav et al., 2011; Marinaik et al., 2018; Riyesh et al., 2014; Singh et al., 2006). Buffalopox is characterized by development of pustular skin lesions, which may be localized or disseminated, depending upon the disease severity. Disease outbreaks in buffaloes often result in decreased milk production (Singh et al., 2007; Venkatesan et al., 2010). To minimize the economic impact, blocking virus transmission during epidemics is of utmost importance for which rapid control tools are urgently re- quired (Goris et al., 2008). Despite adverse effect on animal industry and severe consequences to human health (Singh et al., 2007), antiviral drugs that can be used to treat buffalopox or other viral infections of https://doi.org/10.1016/j.antiviral.2018.10.022 Received 14 July 2018; Received in revised form 22 October 2018; Accepted 24 October 2018 * Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: sbarua06@gmail.com (S. Barua), smchandani86@gmail.com (S. Maherchandani), naveenkumar.icar@gmail.com (N. Kumar). 1 Equal contribution. Antiviral Research 160 (2018) 126–136 Available online 28 October 2018 0166-3542/ © 2018 Elsevier B.V. All rights reserved. T