Antiviral activity of chemical compound isolated from Artemisia morrisonensis against hepatitis B virus in vitro Tsurng-Juhn Huang a , Shu-Heng Liu a , Yu-Cheng Kuo b,c , Chia-Wen Chen d,e , Shen-Chieh Chou a,⇑ a Graduate Institute of Ecology and Evolutionary Biology and Research Center for Biodiversity, China Medical University, Taichung, Taiwan b Department of Radiation Oncology, China Medical University Hospital, Taichung, Taiwan c Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung, Taiwan d Department of Anesthesiology, China Medical University Hospital, Taichung, Taiwan e School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan article info Article history: Received 31 May 2013 Revised 28 October 2013 Accepted 11 November 2013 Available online 20 November 2013 Keywords: Artemisia morrisonensis ER stress Hepatitis B virus HepG2 2.2.15 cell Huh7 cell p-Hydroxyacetophenone abstract The compound p-hydroxyacetophenone (PHAP) isolated from Artemisia morrisonensis was found to have potential anti-HBV effects in HepG2 2.2.15 cells. We clarified its antiviral mode further and HBV-trans- fected Huh7 cells were used as the platform. During viral gene expression, treatment with PHAP had no apparent effects on the viral precore/pregenomic RNA. However, the 2.4-kb preS RNA of viral surface gene increased significantly relative to the 2.1-kb S RNA with PHAP. Promoter activity analysis demon- strated that PHAP had a potent effect on augmenting the viral preS promoter activity. The subsequent increase in the large surface protein and induce endoplasmic reticular (ER) stress has been reported pre- viously. Interestingly, PHAP specifically reduced ER stress related GRP78 RNA/protein levels, but not those of GRP94, in treated Huh7 cells while PHAP also led to the significant intracellular accumulation of virus. Moreover, treatment with the ER chaperone inducer thapsigargin relieved the inhibitory effect of PHAP based on the supernatant HBV DNA levels of HBV-expressed cells. In conclusion, this study sug- gests that the mechanism of HBV inhibition by PHAP might involve the regulation of viral surface gene expression and block virion secretion by interference with the ER stress signaling pathway. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Hepatitis B virus (HBV) infections are a major global health problem despite the availability of an effective vaccine. Over two billion people are infected at present and about 400 million are chronically infected carriers of this virus (McMahon, 2005; Popper et al., 1987). These carriers are at risk of severe liver diseases including acute hepatitis, chronic active hepatitis, cirrhosis, and eventually hepatocellular carcinoma (Arbuthnot and Kew, 2001; Feitelson, 1999; Park et al., 2006). Although interferons and nucle- otide analogs have been used widely to treat chronically infected patients, the rapid development of resistance and their undesirable side-effects mean that the development of an antiviral drug is ur- gently needed to treat HBV. Previous studies have shown that several naturally occurring constituents of medicinal plants have anti-HBV activities and it was suggested that their antiviral activities were mediated via the regulation of endoplasmic reticulum (ER) homeostasis (Huang et al., 2006, 2009). The ER is a cytoplasmic compartment where proteins and lipids are synthesized. Chaperones have been proposed to facilitate correct protein folding in the ER and they also prevent protein unfolding, which causes the aggregation of proteins in the ER and induces ER stress (Lambert and Prange, 2003). Two well-characterized ER chaperone proteins are the 78- kDa glucose-regulated protein (GRP78 or BiP) and GRP94, which are known to be induced by ER stress (Boelens et al., 2013; Dey et al., 2006; Eletto et al., 2010; Kaufman, 1999; Lee, 2005; Patil and Walter, 2001) and they are involved with the ER stress signal- ing pathway. GRP78 was also demonstrated to function during HBV viral morphogenesis where it interacts with the large surface protein of HBV and regulates its posttranscriptional topological reorientation (Cho et al., 2003; Lambert and Prange, 2003). How- ever, the mechanisms by which natural products affect the ER sig- naling pathway and inhibit HBV propagation remain to be elucidated. Therefore, it is important to clarify the interaction be- tween HBV gene expression and ER stress to facilitate anti-HBV drug development. Artemisia morrisonensis is an endemic alpine species in Taiwan. Traditionally, A. morrisonensis was used to treat rheumatoid arthri- tis, allergic rhinitis, headache, and edema by the aboriginal people. Relatives of this species have long been used as traditional medi- cines with antiviral and antibacterial activities, while they have also been used to treat related liver disease (Tan et al., 1998). 0166-3542/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.antiviral.2013.11.007 ⇑ Corresponding author. Tel.: +886 4 22053366x8108; fax: +886 4 22071507. E-mail address: scc@mail.cmu.edu.tw (S.-C. Chou). Antiviral Research 101 (2014) 97–104 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral