Downloaded from www.microbiologyresearch.org by IP: 54.147.215.157 On: Fri, 09 Dec 2016 05:44:26 A bifunctional anti-enterovirus compound that inhibits replication and the early stage of enterovirus 71 infection Minetaro Arita, 1 Yutaka Takebe, 2 Takaji Wakita 1 and Hiroyuki Shimizu 1 Correspondence Minetaro Arita minetaro@nih.go.jp Received 5 May 2010 Accepted 14 July 2010 1 Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan 2 Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Enviroxime is an anti-enterovirus compound that targets viral protein 3A and/or 3AB and suppresses a replication step of enterovirus by an unknown mechanism. To date, a number of anti-enterovirus compounds that have little structural similarity to enviroxime but induce common resistance mutations in the 3A-encoding region have been identified. The present study identified a novel type of functionally enviroxime-like compound, AN-12-H5. This compound had no structural similarity to enviroxime or to known enviroxime-like compounds, including TTP-8307, GW5074 and Flt3 Inhibitor II. A resistance phenotype of poliovirus (PV) to these compounds was conferred by a major enviroxime-resistance mutation of PV (G5318A, 3A-Ala70Thr), but not by resistance mutations to guanidine hydrochloride and brefeldin A. AN-12-H5 had a common structure with the anti-enterovirus 71 (EV71) compound AN-23-F6. AN-12-H5 and AN-23-F6 inhibited an early stage of EV71 infection after virus binding to the cells. Mutations in capsid proteins (G3112A, VP1-Ala224Thr, and G2396A, VP3-Arg227Lys mutations) were determined as resistant mutations to AN-12-H5 and AN-23-F6 in the early stage of EV71 infection. These results suggest that AN-12-H5 is a bifunctional anti-enterovirus compound that belongs to a novel class of enviroxime-like compounds and targets both a replication step and an early stage of EV71 infection. INTRODUCTION The genus Enterovirus, family Picornaviridae, consists of at least ten species of non-enveloped virus with a single- stranded, positive-sense genomic RNA. Enterovirus infec- tion is mostly asymptomatic, but sometimes causes severe neurological symptoms, as exemplified by poliomyelitis. Among the enteroviruses, poliovirus (PV) and enterovirus 71 (EV71) are known to be highly neurotropic entero- viruses. In infection by PV, which is the causative agent of poliomyelitis and belongs to Human enterovirus species C, the motor neurons are the major target for neurovirulence (Bodian, 1949). EV71, which belongs to Human enterovirus species A, is a causative agent of hand, foot and mouth disease and herpangina, but sometimes causes severe neurological diseases such as brainstem encephalitis and poliomyelitis-like paralysis (Chumakov et al., 1979; McMinn, 2002; Wang et al., 2003). Live attenuated oral PV vaccine and inactivated PV vaccine have been established for PV and used for global eradication of poliomyelitis (Sabin, 1965; Salk et al., 1954); however, currently no vaccine is available for EV71. With available effective vaccines for PV, the role of antivirals for PV infection is limited in the current situation. In this post-eradication era of PV, anti-PV compounds are instead anticipated to have a role in the control of a circulating vaccine-derived PV along with inactivated PV vaccine, and for the treatment of patients chronically infected with PV and for persons exposed to PV (Collett et al., 2008; Committee on Development of a Polio Antiviral and Its Potential Role in Global Poliomyelitis Eradication, N. R. C., 2006). For EV71 infection, antivirals could be an effective therapeutic means during the course of the rapid progression of symptoms in fatal cases (¡12 h after hospitalization) (Chang et al., 1999). To date, several anti-enterovirus compounds that target viral or cellular factors have been identified (reviewed by Barnard, 2006; Chen et al., 2008; Rotbart, 2002). Among the compounds with anti-PV activity, guanidine hydrochloride (GuHCl), brefeldin A and enviroxime inhibit replication steps with known targets. GuHCl belongs to a group of 2C inhibitors that inhibit initiation of negative-strand RNA synthesis by targeting viral proteins 2C and/or 2BC (Barton & Flanegan, 1997; Bienz et al., 1990; Caliguiri & Tamm, 1968). Some benzimidazole derivatives, including A supplementary figure is available with the online version of this paper. Journal of General Virology (2010), 91, 2734–2744 DOI 10.1099/vir.0.023374-0 2734 023374 G 2010 SGM Printed in Great Britain