Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni 2؉ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.Dengue viruses, members of the family Flaviviridae, are transmitted by mosquitos. There are four serotypes (types 1 to 4) that cause widespread human diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. About 40% of the world population living in tropical and subtropical regions of the world is at risk for infection. Of the 1 million cases of dengue hemorrhagic fever cases per year, about 5% are fatal. There is currently no effective vaccine or antiviral drug to protect against dengue diseases (1, 2). The virus has a single strand RNA genome of positive polarity. The viral RNA has a type 1 cap at the 5Ј-end and is devoid of poly(A) at the 3Ј-end. The RNA genome codes for a single polyprotein precursor (3,391 amino acid residues for DEN2-New Guinea C strain) (3) arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5.1 Processing of the N-terminal region by the host signal peptidase associated with the endoplasmic reticulum yields three structural proteins that are assembled into the virion (C, prM, and E) (4 -6). Processing of C-prM by signal peptidase occurs via a post-translational mechanism in which cleavage of the cytoplasmic domain of C in the C-prM precursor by the viral protease precedes the signal peptidase cleavage (7-9). prM undergoes further cleavage to M at a late step during virion morphogenesis mediated by a cellular protease in the post-Golgi acidic compartment (10). In addition, an endoplasmic reticulum membrane-bound host protease with the characteristics of the signalase mediates cleavages at the NS1-NS2A (11) and NS4A-NS4B junctions (12)(13)(14).A trypsin-like serine protease domain within the N-terminal 180 amino acid residues of NS3 was identified by sequence comparisons (15,16). Analysis of polyprotein proces...

Replication of positive strand flaviviruses is mediated by the viral RNA-dependent RNA polymerases (RdRP). To study replication of dengue virus (DEN), a flavivirus family member, an in vitro RdRP assay was established using cytoplasmic extracts of DEN-infected mosquito cells and viral subgenomic RNA templates containing 5-and 3-terminal regions (TRs). Evidence supported that an interaction between the TRs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements is required for RNA synthesis. Two RNA products, a template size and a hairpin, twice that of the template, were formed. To isolate the function of the viral RdRP (NS5) from that of other host or viral factors present in the cytoplasmic extracts, the NS5 protein was expressed and purified from Escherichia coli. In this study, we show that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo initiation. Furthermore, the incubation temperature during initiation, but not elongation phase of RNA synthesis modulates the relative amounts of the hairpin and de novo RNA products. A model is proposed that a specific conformation of the viral polymerase and/or structure at the 3 end of the template RNA is required for de novo initiation.The dengue virus, which is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is estimated to infect 100 million people per year worldwide (1-3). The virus is spread by the mosquito, Aedes agypti, which puts ϳ40% of the world at risk for dengue infection (1). Approximately 5% of infected individuals worldwide develop hemorrhagic or shock manifestations, which can commonly result in death (1). The dengue virus type 2 (DEN2) 1 is the most prevalent of the four dengue serotypes.The virus contains a positive strand, 5Ј-capped RNA, 10,723 nucleotides in length (for New Guinea-C strain; Ref. 4), which encodes a single polyprotein precursor, arranged in the order, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (for a review, see Ref. 5). This precursor is processed in the endoplasmic reticulum by a combination of the signal peptidase and the viral serine protease to generate three structural proteins of the virion, C, prM, and E (6 -8) and at least seven nonstructural (NS) proteins.NS3, the second largest protein encoded by the virus, contains a serine catalytic triad within the N-terminal 180 amino acids, and it requires NS2B for protease activity (9 -18). The crystal structures of the protease domain alone and in complex with an inhibitor have been reported (19,20). However, the function of other nonstructural proteins in viral replication is poorly understood.NS3 also contains conserved motifs found in several NTPase/ RNA helicases (21-23). According to the current model, replication is initiated by the viral RNA-dependent RNA polymerase (RdRP) by synthesis of minus (Ϫ) strand to form a doublestranded RNA intermediate, which then serves as a template for genomic positive strand (24 -27). The vira...

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.

Dengue viruses (DEN), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. The effects of mycophenolic acid (MPA) and ribavirin (RBV) on DEN replication in monkey kidney (LLC-MK2) cells were examined. MPA (IC 50 =0?4±0?3 mM) and RBV (IC 50 =50?9±18 mM) inhibited DEN2 replication. Quantitative real-time RT-PCR of viral RNA and plaque assays of virions from DEN2-infected and MPA (10 mM)-and RBV (¢200 mM)-treated cells showed a fivefold increase in defective viral RNA production by cells treated with each drug. Moreover, a dramatic reduction of intracellular viral replicase activity was seen by in vitro replicase assays. Guanosine reversed the inhibition of these compounds, suggesting that one mode of antiviral action of MPA and RBV is by inhibition of inosine monophosphate dehydrogenase and thereby depletion of the intracellular GTP pool. In addition, RBV may act by competing with guanine-nucleotide precursors in viral RNA translation, replication and 59 capping.Dengue viruses (DEN1-4), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. Of the 1 million annual cases of dengue haemorrhagic fever/dengue shock syndrome, about 2-5 % are fatal. Currently, there is no vaccine or antiviral drug to treat DEN infections (Barrett, 2001;Gubler, 1998;Halstead & Deen, 2002). DEN2 New Guinea C strain, used in this study, has a single-stranded RNA genome (10 723 nt) of positive polarity (Irie et al., 1989). The aim of this study was to examine the antiviral action of mycophenolic acid (MPA) and ribavirin (RBV) on DEN2-infected LLC-MK2 cells by determining the number of infectious particles, levels of virion-associated RNAs and intracellular viral replicase activity by plaque assays, quantitative real-time RT-PCR (qRT-PCR) and in vitro replicase assays, respectively. MPA, a non-nucleoside analogue, is a potent, non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme required for biosynthesis of guanine nucleotides (reviewed by Allison & Eugui, 2000). GTP is required for translation, transcription and replication processes. Therefore, inhibition of IMPDH is expected to inhibit not only proliferation of eukaryotic cells, but also replication of DNA and RNA viruses (Markland et al., 2000). However, RBV, a nucleoside analogue, is a competitive inhibitor of IMPDH.It is approved as an inhaled drug for treatment of respiratory syncytial virus infection, as well as orally, together with alpha interferon, for treatment of hepatitis C virus (HCV) infections.DEN2 was propagated in mosquito (C6/36) cells as described previously (Charnsilpa et al., 2005). LLC-MK2 cells were infected with DEN2 under single-step growth conditions (Dulbecco & Vogt, 1954) at an m.o.i. of 10 and incubated for 72 h with 1 % fetal bovine serum. The plaque assay was performed essentially as described previously (Charnsilpa et al., 2005).To quantify the virus-associated RNA, qRT-PCR was used as described previously (Houng et al., 2000). The detection of...

Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5-and 3-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is reduced by ϳ60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation. The dengue virus (DENV)3 is a mosquito-borne flavivirus (MBFV) in the Flaviviridae family that consists of over 70 members, many of which are significant human pathogens (1). The MBFV members are classified into three subgroups: DENV, yellow fever virus, and Japanese encephalitis virus (JEV) (2, 3). The four serotypes of DENV (DENV1 to -4) cause an estimated 50 million cases of infections, with ϳ10% of those leading to severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome (4 -6).The viral genome is a single-stranded RNA of positive (ϩ) polarity containing ϳ11 kilobases (10,723 nt for DENV2 New Guinea C strain, GenBank TM accession number M29095 (7)). The 3Ј-end is non-polyadenylated, and the 5Ј-end has a type I cap structure (for a review, see Ref. 8). Flanking the single long open reading frame are the 5Ј-and 3Ј-UTRs, which contain conserved cis-acting RNA secondary structure elements required for translation and replication (9 -18). The viral RNA is translated to form a polyprotein precursor, which is processed by host and viral proteases in the endoplasmic reticulum membrane. Protein processing gives rise to three structural (C, prM, and E) and seven nonstructural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 in that order (for reviews, see Refs. 8,19,and 20) and references therein). According to the current model for replication, assembly of the viral replicase complex occurs in a cytoplasmic membrane organelle, followed by negative (Ϫ)-strand RNA synthesis starting at the 3Ј-end of the viral genome, resulting in a double-stranded replicative form. NS3 and NS5 are multifunctional pr...