Amantadine inhibits hepatitis A virus internal ribosomal entry site-mediated translation in human hepatoma cells q Tatsuo Kanda a,b, * , Osamu Yokosuka b , Fumio Imazeki b , Keiichi Fujiwara b , Keiichi Nagao a , Hiromitsu Saisho b a Safety and Health Organization, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan b Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-0856, Japan Received 25 March 2005 Available online 9 April 2005 Abstract The effect of six drugs (amantadine, glycyrrhizin, ribavirin, ursodeoxycholic acid, alcohol, and IFN) on HAV RNA translation from the HAV internal ribosomal entry site (IRES) was investigated using a bicistronic reporter construct containing HAV IRES as intragenic spacer. Huh-7 cells and derivatives were transfected with in vitro transcripts, and the reporter gene activity was deter- mined. IFN suppressed both cap-dependent and HAV IRES-dependent translation, while amantadine specifically inhibited HAV IRES-dependent translation. In contrast to IFN, by reporter assay, amantadine did not activate the interferon-stimulated response element (ISRE) or interferon c-activated sequence (GAS)-associated pathways. Immunoblot analysis revealed that amantadine had no effect on PKR and on IFN-regulatory factor-1 (IRF-1) expression. These findings demonstrated a novel antiviral effect of aman- tadine against HAV with or without HCV infection. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Hepatitis A; Hepatitis C; IRES; Replicon; Fulminant hepatitis Hepatitis A virus (HAV) is a picornavirus, a common causative agent of acute self-limited hepatitis that some- times leads to fulminant hepatic failure. The rate of acute liver failure due to hepatitis A virus (HAV) has not de- creased [1], and treatment of severe infections is still an issue of major concern. Hepatitis A infection is a public health dilemma of worldwide proportions, and new ther- apeutic options are urgently required for acute illness [2]. HAV has a positive sense RNA genome of approximately 7500 bases that includes the 5 0 non-translated region (NTR), one open reading frame encoding both structural and non-structural proteins, and the 3 0 NTR. The down- stream part of 5 0 NTR presents an internal ribosomal en- try site (IRES) with a pyrimidine-rich tract that allows HAV RNA translation by a cap-independent mechanism [3–5]. A number of in vitro studies have suggested that several cellular proteins, including polypyrimidine tract- binding protein (PTB) [6,7] and glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) [7,8], may interfere with HAV translation. First, in HAV, the nucleotide sequence of 5 0 NTR was more conserved than those of other sites [9]. Second, siRNAs against 5 0 NTR suppress not only HAV translation but also HAV replication [10]. 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.212 q Abbreviations: HAV, hepatitis A virus; IFN, interferon; IRES, internal ribosomal entry site; HCV, hepatitis C virus; NTR, non- translated regions; UDCA, ursodeoxycholic acid; GAPDH, glyceral- dehyde-3-phosphate dehydrogenase; hnRNP I , heterogeneous nuclear ribonucleoprotein I; PTB, polypyrimidine tract-binding protein; IRF- 1, interferon-regulatory factor-1; PKR, double-stranded-specific RNA-activated serine/threonine kinase; ISRE, interferon-stimulated response element; GAS, interferon c-activated sequence; NFAT, nuclear factor of activated T cells; NFjB, nuclear factor jB; AP-1, activator protein 1; SRE, serum response element; SRF, serum response factor; MTS, dimethylthiazol carboxymethoxyphenyl sulfo- phenyl tetrazolium; PBS, phosphate-buffered saline. * Corresponding author. Fax: +81 43 226 2088. E-mail address: kandat-cib@umin.ac.jp (T. Kanda). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 331 (2005) 621–629 BBRC