title: Multiple enzyme activities of flavivirus proteins.
abstract: Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
authors.affiliation: Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC, USA.
authors.forename: R
authors.initials: R
authors.lastname: Padmanabhan
authors.forename: N
authors.initials: N
authors.lastname: Mueller
authors.forename: E
authors.initials: E
authors.lastname: Reichert
authors.forename: C
authors.initials: C
authors.lastname: Yon
authors.forename: T
authors.initials: T
authors.lastname: Teramoto
authors.forename: Y
authors.initials: Y
authors.lastname: Kono
authors.forename: R
authors.initials: R
authors.lastname: Takhampunya
authors.forename: S
authors.initials: S
authors.lastname: Ubol
authors.forename: N
authors.initials: N
authors.lastname: Pattabiraman
authors.forename: B
authors.initials: B
authors.lastname: Falgout
authors.forename: V K
authors.initials: VK
authors.lastname: Ganesh
authors.forename: K
authors.initials: K
authors.lastname: Murthy
mesh_terms: D000595:Amino Acid Sequence; D005416:Flavivirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases