Multiple enzyme activities of flavivirus proteins.
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.
Padmanabhan
R
R
Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC, USA.
Mueller
N
N
Reichert
E
E
Yon
C
C
Teramoto
T
T
Kono
Y
Y
Takhampunya
R
R
Ubol
S
S
Pattabiraman
N
N
Falgout
B
B
Ganesh
V K
VK
Murthy
K
K
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*
D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases