Emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type 1 yields in cell cultures.

British Journal of Pharmacology (2008) 155, 227–235 & 2008 Macmillan Publishers Limited All rights reserved 0007– 1188/08 $32.00 www.brjpharmacol.org RESEARCH PAPER Emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type 1 yields in cell cultures C-Y Hsiang1 and T-Y Ho2 1 Department of Microbiology, China Medical University, Taichung, Taiwan and 2Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan Background and purpose: Most antiviral therapies directed against herpes simplex virus (HSV) infections are limited to a small group of nucleoside analogues that target the viral polymerase. Extensive clinical use of these drugs has led to the emergence of resistant viral strains, mainly in immunocompromised patients. This highlights the need for the development of new antiherpesviral drugs with novel targets. Herein the effects of a plant anthraquinone, emodin, on the HSV-1 alkaline nuclease activity and virus yields were investigated. Experimental approach: HSV-1 alkaline nuclease activity was examined by nuclease activity assay. Inhibition of virus yields was measured by plaque reduction assay and immunohistochemical staining. Interaction between emodin and alkaline nuclease was analysed by docking technology. Key results: Emodin specifically inhibited the nuclease activity of HSV-1 UL12 alkaline nuclease in a biochemical assay. Plaque reduction assay revealed that emodin reduced the plaque formation with an EC50 of 21.5±4.4 mM. Immunohistochemical staining using the anti-nucleocapsid protein antibody demonstrated that emodin induced the accumulation of viral nucleocapsids in the nucleus in a dose-dependent manner. Docking analysis further suggested that the inhibitory effect of emodin on the UL12 activity may result from the interaction between emodin and critical catalytic amino acid residues of UL12. Conclusions and implications: Our findings suggest that emodin is a potent anti-HSV agent that inhibits the yields of HSV-1 via the suppression of a novel target, UL12. British Journal of Pharmacology (2008) 155, 227–235; doi:10.1038/bjp.2008.242; published online 16 June 2008 Keywords: herpes simplex virus type 1; emodin; alkaline nuclease Abbreviations: CK2, casein kinase 2; DMSO, dimethyl sulphoxide; FITC, fluorescein; HSV, herpes simplex virus; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PFU, plaque-forming unit Introduction Herpes simplex virus (HSV) causes herpes labialis, herpes keratitis, genetic herpes and life-threatening herpes encephalitis. HSV infections are more severe in immunocompromised patients, which are characterized by chronic and extensive lesions of the mucous membranes (Whitley, 2001). Most therapies directed against HSV infections are nucleotides, nucleosides or pyrophosphate analogues, such as acyclovir, valacyclovir, penciclovir and famciclovir. After uptake by virus-infected cells, these drugs are phosphory- Correspondence: Professor T-Y Ho, Graduate Institute of Chinese Medical Science, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. E-mail: Received 15 February 2008; revised 18 April 2008; accepted 7 May 2008; published online 16 June 2008 lated by virus-encoded thymidine kinase, compete with the nucleotides to inhibit the viral DNA polymerase and subsequently cause the termination of growing viral DNA chains (Field, 2001). Although these drugs are effective in the treatment of many acute infections, the intensive use of these drugs has led to the emergence of resistant viral strains, mainly in immunocompromised patients (Field, 2001). Therefore, there is a need to provide other drugs with distinct mechanisms as alternatives to existing therapies. Alkaline nuclease, which is encoded by the UL12 gene of HSV-1, possesses both endonuclease and exonuclease activities under alkaline pH conditions (Hoffmann and Cheng, 1978; McGeoch et al., 1986; Knopf and Weisshart, 1990). Null mutants incapable of expressing UL12 are able to synthesize near wild-type levels of viral DNA, suggesting that UL12 is not essential for viral DNA replication in culture 228 Emodin inhibits HSV-1 yields in vitro C-Y Hsiang and T-Y Ho (Weller et al., 1991). Although UL12 is not essential for viral DNA synthesis, UL12 mutant viral yields are 0.1–1% of wildtype yields (Shao et al., 1993; Martinez et al., 1996a). The analysis of UL12 null mutants has shown that the decrease in virus yield results from the reduction of capsids exiting from the nucleus (Martinez et al., 1996b; Goldstein and Weller, 1998). Analysis of replicating DNA from UL12 mutantinfected cells has shown that UL12 is implicated in resolving branched structures of HSV-1 replicative intermediates prior to encapsidation (Martinez et al., 1996a; Porter and Stow, 2004a, b). Therefore, these results indicate that, even though UL12 is not essential for either viral DNA synthesis or packaging, UL12 is required for full efficiency of these processes. Additionally, these findings suggest that HSV-1 UL12 can be a novel target for anti-herpes viral drugs. Increasing the emergence of resistant viral strains has highlighted the crucial need for the development of new anti-herpes virus drugs with different mechanisms. Several potential viral targets, such as helicase–primase complex and DNA polymerase, have been known to be involved in HSV-1 infection and for which specific inhibitors with anti-HSV activity, at least in cell cultures, have been identified (Crute et al., 2002; Thomsen et al., 2003; Greco et al., 2007). In the present study, we analysed the potent inhibitor of HSV-1 that targeted viral UL12. Our findings indicated that emodin (1,3,8-trihydroxy-6-methylanthraquinone), the naturally occurring anthraquinone present in the root and bark of numerous plants of the genera Rheum and Polygonum, inhibited HSV-1 UL12 activity, leading to the accumulation of nucleocapsids in the nucleus and the subsequent reduction of HSV-1 yields in Vero cells. Methods Materials All chemicals, except where indicated, were purchased from Sigma (St Louis, MO, USA). Plant materials were purchased from Sun Ten Pharmaceutical Corporation (Taipei, Taiwan). Plant samples were ground to fine powders with homogenizers and extracted with methanol, as described previously (Chen et al., 2007a). Emodin and its analogues were dissolved in dimethyl sulphoxide (DMSO). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in phosphate-buffered saline (PBS) (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2). Bovine pancreatic DNase I was purchased from New England BioLabs (Beverly, MA, USA). Mouse anti-HSV-1 nucleocapsid protein monoclonal antibody and fluorescein (FITC)-conjugated goat anti-mouse antibody were purchased from USBiological (Swampscott, MA, USA) and Jackson ImmunoResearch Laboratories (West Grove, PA, USA), respectively. Cells and viruses A (...truncated)