Current Enzyme Inhibition, 2009, 5, 209-222 209 1573-4080/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Inhibition of Flaviviridae Replication Complex: Assays to Investigate the Modulating Effect of Potential Compounds Andrea Baier* Department of Molecular Biology, John Paul II Catholic University of Lublin, Lublin, Poland Abstract: Approximately up to 200 million new cases of infections caused by viruses of the Flaviviridae family, like hepatitis C virus (HCV), West-Nile virus (WNV), dengue virus (DENV) and Japanese encephalitis virus (JEV) are regis- tered annually. Up to date, there is no effective antiviral therapy directed against Flaviviridae viruses. For the replication of all viruses the intact function of nonstructural proteins is necessary. The blockade of their activities leads to inhibition of the virus propagation. Thus, the nucleotide triphosphatase (NTPase) as well as helicase activities of NS3 and the RNA-dependent RNA polymerase (RdRp) activity of NS5 (or NS5B) appear to be exceptionally attractive targets for antiviral compounds. In this context, a systematic screening of chemical libraries and computational analysis are necessary which will lead to the development of effective inhibitors of the Flaviviridae replication complex. In this review the most promissing methods based on different techniques to quantify the enzymatic activities, like radio- activity, fluorescence, colorimetry as well as a coupled second enzymatic reaction are summarized and discussed. These methods serve as a starting point to establish a high throughput system (HTS) that permits more efficient screenings. Keywords: Flaviviridae, in vitro assays, NTPase/helicase, RNA-dependent RNA polymerase. The family Flaviviridae includes human and animal pathogenic viruses of global importance e.g. the human flaviviruses West-Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV). This genus consists of more than 70 species that are, on the basis of phylogenetic analyses, divided into 14 classes which in turn are grouped into three clusters: the mosquito-borne cluster, the tick-borne cluster and the non-vector cluster [1]. All flaviviruses of human importance are mosquito- or tick- borne viruses which may cause fulminant, hemorrhagic dis- eases and viral encephalitis. The second human pathogenic genus containing hepatitis C virus (HCV) and GBV-B is called hepacivirus and causes chronic or acute hepatits which may lead to liver cirrhosis and hepatocellular carcinoma [2- 4]. Bovine viral diarrhea virus (BVDV), classical swine fe- ver virus (CSFV) and border disease virus (BDV) [5] are animal pathogenic viruses belonging to the genus pestivirus and lead to severe diseases of the respective host usually followed by death. Further viruses which are not classified in one of the mentioned genera are GBV-A and GBV-C. Possible routes of transmission differ between the genera. HCV carriers are often intravenous-drug users or blood product recipients. The latter could be reduced by blood screening for HCV. Contrary to blood and sexual transmitted HCV, flaviviruses are transmitted by vectors. Only rats and mice are infected with no-vector viruses. In case of pestivi- ruses transmission is directly from one animal to another by the oro-nasal and transplacenteral route. Flaviviridae viruses are small (40-50 nm) enveloped par- ticles [2, 3, 6] with positive-sense single-stranded RNA *Address correspondence to this author at the Department of Molecular Biology, John Paul II Catholic University of Lublin, Al. Krasnicka 102, 20- 718 Lublin, Poland; Tel: +48 81 445 4603; Fax: +48 81 445 4611; E-mail: baier@kul.lublin.pl genome of the length of 9100 to 11000 bases which possess a single open reading frame (ORF) flanked by 5‘- and 3‘- terminal located untranslated regions (5’UTR and 3’UTR). In the case of flaviviruses the 5‘UTR is relatively short (ap- proximately 120 bases) and contains a cap structure type I (m7GppN1mpN2). In hepaci- and pestiviruses the 5‘UTRs consist of approximately 340 bases and reveal features of an internal ribosomal entry site (IRES) mediating the binding to the ribosome [7, 8]. There are some indications that the adenosine- and/or uridine-rich stretches of the 3‘UTR are involved in the regulation of the nucleoside triphosphatase (NTPase) activity of the nonstructural protein 3 (NS3 pro- tein) associated NTPase/helicase [9, 10]. Furthermore, it may be important for the initiation of minus strand synthesis [11]. VIRAL REPLICATION Genomic replication proceeds very similarly within in the Flaviviridae family. After binding to the cell by specific re- ceptors and entry of the virus via receptor-mediated endocy- tosis the viral RNA is uncoated by acid-catalyzed membrane fusion and translation is initiated by IRES- or cap-mediated processes. The viral RNA is translated into one viral poly- protein which is co- and posttranslationally cleaved by viral and host proteases [12-14]. RNA synthesis occurs in cyto- plasmic replication complexes localized at the perinuclear membranes [15, 16]. All NS proteins appear to be involved in RNA replication. The genomic RNA is encapsulated into the core shell consisting of capsid proteins, enveloped by viral surface protein embedded cellular derived lipid mem- branes (Assembly). The virus particle is budded through intracellular mem- branes into cytoplasmic vesicles [17, 18]. By the cellular secretory pathway the virus particles are transported to the plasma membrane and undergo a maturation procedure. Vi- rus release into the extracellular compartment is connected