Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

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Ribavirin was evaluated as a potential treatment of yellow fever (YF) in a hamster model of the disease. Ribavirin treatment during the first five days after YF virus infection improved survival, reduced tissue damage in target organs (liver and spleen), prevented hepatocellular steatosis, and normalized alanine aminotransferase levels. The results of this study suggest that ribavirin may be effective in the early treatment of YF, and that its mechanism of action in reducing liver pathology in YF virus infection may be similar to that observed with ribavirin in the treatment of chronic hepatitis C virus infection.

The aim of this study was to investigate the role of luteolin in the mechanism of ultraviolet radiation B (UVB)-induced photoaging. An in vivo photoaging model was established using UVB irradiation of bare skin on the back of rats, and an in vitro photoaging model was established using UVB irradiation of human dermal fibroblasts (HDF). Skin damage was observed using hematoxylin-eosin (HE) and Masson staining, skin and cellular reactive oxygen species (ROS) levels were detected by DHE and DCF fluorescent probes, mitochondrial membrane potential was detected by JC-1 staining, and protein expressions were detected by immunofluorescence and Western Blot. Results from animal experiments showed that luteolin reduced UVB-induced erythema and wrinkle formation. Results from cellular assays showed that luteolin inhibited UVB-induced decrease in cell viability. In addition, in vitro and in vivo experiments showed that luteolin reduced oxidative stress levels, decreased activation of matrix metalloproteinases (MMPs) and increased collagen expression. Continued cellular experiments using 3-TYP, an inhibitor of Sirtuin 3 (SIRT3), revealed a loss of cellular protection by luteolin and a decrease in collagen, suggesting that luteolin acts by targeting and promoting SIRT3. luteolin is involved in the protection of skin cells against UVB radiation-induced ageing via the SIRT3/ROS/mitogen-activated protein kinases (MAPK) axis and it may be a promising therapeutic agent for the prevention of UVB photoaging.

Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and doublestranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ؎ 2 nucleotides. In the presence of Mg 2؉ , the site size increases to 29 ؎ 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex. The dengue virus (DENV)2 is a member of the Flaviviridae family and the etiological agent of dengue fever, a disease affecting worldwide ϳ100 million people each year (1-5). The Flaviviridae family includes such human pathogens as tick-borne encephalitis virus, hepatitis C virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus (1-5). The dengue virus exists in four distinct types, DENV1, -2, -3, and -4, which show ϳ60% genomic sequence identity (1). The dengue fever often develops into the dengue hemorrhagic fever, an acute form of the disease, possibly as a result of sequential infection with different types of the virus. It is characterized by a significant mortality, particularly for individuals with a weakened or undeveloped immune system. The small, 10.7-kbp positive strand RNA genome of the dengue virus encodes only 10 proteins: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) (3, 6 -8). The entire viral genome is translated as a single polyprotein, subsequently cleaved by viral and cellular proteases into functional entities.The NS5 protein is the viral replicative RNA-dependent RNA polymerase (1, 3, 6 -8). The primary structure of the fulllength polymerase encompasses 900 amino acids (i.e. it is the largest of the nonstructural proteins of the dengue virus) (6 -10). The enzyme is built of two functional domains (6 -10). The N-terminal domain includes the first 263 amino acids. It functions as a methyltransferase (MTase), whose activity is necessary for viral RNA recognition by the host cell translational apparatus. Correspondingly, amino acids ϳ273-906 form the polymerase domain, containing the active site of the RNA synthesis (Fig. 1a) (6 -8). The crystal structures of the isolated N-terminal and polymerase domains have been determined up t...