1 2 Inhibition of norovirus replication by the nucleoside analogue 2 0 -C-methylcytidine 3 J. Rocha-Pereira a,b Q1 , D. Jochmans c , K. Dallmeier c , P. Leyssen c , R. Cunha a , I. Costa a , M.S.J. Nascimento a,b , 4 J. Neyts c,⇑ 5 a Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal 6 b Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal 7 c Rega Institute for Medical Research, University of Leuven (KU Leuven), Minderbroedersstraat 10, B-3000 Leuven, Belgium 8 9 11 article info 12 Article history: 13 Received 1 September 2012 14 Available online xxxx 15 Keywords: 16 Norovirus 17 Antiviral activity 18 2 0 -C-methyl nucleoside analogues 19 RNA-dependent RNA polymerase 20 21 abstract 22 We here report on the activity of 2 0 -C-methylcytidine (2CMC) [a nucleoside polymerase inhibitor of the 23 hepatitis C virus (HCV)] on the in vitro replication of (murine) norovirus (MNV). 2CMC inhibits (i) virus- 24 induced CPE formation, (ii) viral RNA synthesis and (iii) infectious progeny formation with EC 50 values of 25 2 lM. 2CMC acts at a time-point that coincides with the onset of viral RNA synthesis. Even following 30 26 passages of selective pressure no MNV-resistant virus was selected, which is in line with the high barrier 27 to resistance of the nucleoside analogue for HCV. When combined with the broad-spectrum RNA virus 28 inhibitor ribavirin, a marked antagonistic activity was observed indicating that these molecules should 29 not be combined for the treatment of norovirus infections. Our results suggest that 2 0 -C-methyl nucleo- 30 side analogues should be further explored for the treatment and prophylaxis of norovirus infections. 31 Ó 2012 Published by Elsevier Inc. 32 33 34 1. Introduction 35 Noroviruses are today recognized as the leading cause of food- 36 borne outbreaks of gastroenteritis worldwide, affecting millions of 37 individuals and resulting in heavy health and economic burden 38 every year [1,2]. However, no vaccine or specific antiviral therapy 39 is available today for treatment or prevention of norovirus illness. 40 Noroviruses are positive-sense single stranded (ss) RNA viruses 41 belonging to the genus Norovirus, family of the Caliciviridae [3]. The 42 six/seven nonstructural proteins of norovirus include a viral prote- 43 ase [4], a NTPase/helicase [5] and a RNA-dependent RNA polymer- 44 ase (RdRp) [6,7], responsible for the synthesis and amplification of 45 the genomic RNA. The crystal structure of the Norwalk virus RdRp 46 has been resolved and overall showed catalytic and structural 47 elements highly conserved among RdRps of other (+)ssRNA viruses 48 [8,9]. Therefore, the norovirus RdRp is a critical enzyme for viral 49 replication and an important antiviral target [3,10]. 50 Insights into the norovirus life cycle and thus potential antiviral 51 targets have long been hampered by the lack of efficient cell cul- 52 ture systems, given human noroviruses are not cultivable [11]. Sig- 53 nificant progress has been made with surrogate viruses (murine 54 norovirus (MNV) and others) and a Norwalk virus replicon-bearing 55 cell line [9,12–14]. 56 There are very few reports of antiviral activity of small molecule 57 inhibitors of noroviruses [15–17]. However, a substantial number 58 of selective inhibitors of other ssRNA(+) viruses, such as picornav- 59 iruses, have been described [18]. Given the similarities of the rep- 60 lication strategies between noroviruses and such (+)ssRNA viruses, 61 compounds with activity against these viruses could serve as scaf- 62 folds for the development of antivirals for norovirus. We therefore 63 wanted to evaluate the potential anti-norovirus activity of a selec- 64 tion of such molecules. To that end a rapid in vitro antiviral assay 65 was elaborated using the infectious MNV as a surrogate for human 66 norovirus. MNV is considered today the best surrogate since many 67 molecular features and fundamental mechanisms of replication are 68 conserved [13]. From the reference antiviral molecules screened 69 using this assay, 2 0 -C-methylcytidine (2CMC) was identified as an 70 inhibitor of MNV replication. We report on the particular charac- 71 teristics of the anti-norovirus activity of this nucleoside analogue. 72 2. Materials and methods 73 2.1. Compounds 74 Ribavirin, 1-(b-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxam- 75 ide (Virazole; RBV) was purchased from ICN Pharmaceuticals 76 (Costa Mesa, CA). Simvastatin and dextran sulfate 5000 and 77 10,000 were purchased from Sigma–Aldrich (Bornem, Belgium). 78 All other molecules were synthesized as described before [19–22]. 79 2.2. Cells and viruses 80 MNV (strain MNV-1.CW1) was propagated in RAW 264.7 cells 81 grown in DMEM (Life Technologies, Gent, Belgium) supplemented 0006-291X/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.bbrc.2012.10.003 ⇑ Corresponding author. E-mail address: Johan.Neyts@rega.kuleuven.be (J. Neyts). Biochemical and Biophysical Research Communications xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 29371 No. of Pages 5, Model 5G 13 October 2012 Please cite this article in press as: J. Rocha-Pereira et al., Inhibition of norovirus replication by the nucleoside analogue 2 0 -C-methylcytidine, Biochem. Bio- phys. Res. Commun. (2012), http://dx.doi.org/10.1016/j.bbrc.2012.10.003