Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.Flaviviridae are enveloped viruses with positive-strand RNA genomes that have been grouped into three genera, Hepacivirus, Pestivirus, and Flavivirus (11,59). Several members of the Flavivirus genus, e.g., dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, and West Nile virus (WNV), are medically important arthropod-borne pathogens afflicting humans. DENV infects 50 to 100 million people each year, with ϳ500,000 patients developing the more severe disease dengue hemorrhagic fever, leading to hospitalizations and resulting in approximately 20,000 deaths, mainly in children (24,26,27,29). Based on serological studies, DENVs are further classified into four distinct serotypes, DENV 1 to 4, whose respective genomes share ϳ60% sequence identity, with ϳ90% sequence identity within a serotype (7, 26). The DENV RNA genome spans about 10.7 kb and contains a type I methyl guanosine cap structure at its 5Ј end but is devoid of a polyadenylate tail. The genomic RNA is translated into a single polyprotein (58), which is cleaved into three structural (C-prM-E) and seven nonstructural (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) proteins by both the viral and cellular proteases (28). The viral serine protease is within the N-terminal region of NS3, and recent structural studies show that part of its catalytic site is formed by the viral cofactor NS2B upon substrate binding (18). The C-terminal region of NS3 forms the RNA helicase domain, which is thought to either separate a double-stranded RNA template into individual strands or disrupt secon...

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

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A delicate balance of signals regulates cell survival. One set of these signals is derived from integrin-mediated cell adhesion to the extracellular matrix (ECM). Loss of cell attachment to the ECM causes apoptosis, a process known as anoikis. In searching for proteins involved in cell adhesion-dependent regulation of anoikis, we identified Bit1, a mitochondrial protein that is released into the cytoplasm during apoptosis. Cytoplasmic Bit1 forms a complex with AES, a small Groucho/transducin-like enhancer of split (TLE) protein, and induces cell death with characteristics of caspase-independent apoptosis. Cell attachment to fibronectin counteracts the apoptotic effect of Bit1 and AES. Increasing Bit1 expression enhances anoikis, while suppressing the expression reduces it. Thus, we have elucidated an integrin-controlled pathway that is, at least in part, responsible for the cell survival effects of cell-ECM interactions.

Flavivirus NS5 protein encodes methyltransferase and RNAdependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.The genus Flavivirus from the family Flaviviridae contains more than 70 viruses, many of which are important human pathogens causing major public health threats worldwide. Dengue virus (DENV) 2 is a mosquito-borne flavivirus responsible for 50 -100 million human infections and ϳ20,000 deaths each year (1). Besides DENV, West Nile virus (WNV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus also cause significant human diseases (1). No antiviral therapy is currently available for treatment of flavivirus infections. Therefore, development of antiviral therapy is urgently needed for flaviviruses.The flavivirus genome is a single strand, plus-sense RNA of about 11 kb in length. The genomic RNA contains a 5Ј-untranslated region (UTR), a single open reading frame, and a 3Ј-UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins (2). Three structural proteins (capsid, pre-membrane, and envelope) are primarily involved in virus particle formation, and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are mainly responsible for viral replication (2). Among these, NS3 and NS5 possess enzymatic activities and have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5Ј-RNA triphosphatase, and nucleoside triphosphatase (3-5). NS5 is the largest and the most conserved viral protein. The N-terminal part of NS5 is a methyltransferase that methylates the N-7 and 2Ј-O positions of the viral RNA cap structure (6 -9);...

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Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G) 20 ]. Chemical modification of the initial "hit" improved the compound potency to an IC 50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 M. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC 50 of 5 M). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC 50 s of >100 M against hepatitis C virus RdRp and human DNA polymerase ␣ and ␤. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.