Construction of plasmid, bacterial expression, purification, and assay of dengue virus type 2 NS5 methyltransferase.
Dengue virus (DENV), a member of mosquito-borne flavivirus, causes self-limiting dengue fever as well as life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its positive sense RNA genome has a cap at the 5'-end and no poly(A) tail at the 3'-end. The viral RNA encodes a single polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is processed into 3 structural proteins (C, prM, and E) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional enzymes performing various tasks in viral life cycle. The N-terminal domain of NS5 has distinct GTP and S-adenosylmethionine (SAM) binding sites. The role of GTP binding site is implicated in guanylyltransferase (GTase) activity of NS5. The SAM binding site is involved in both N-7 and 2'-O-methyltransferase (MTase) activities involved in formation of type I cap. The C-terminal domain of NS5 catalyzes RNA-dependent RNA polymerase (RdRp) activity involved in RNA synthesis. We describe the construction of the MTase domain of NS5 in an E. coli expression vector, purification of the enzyme, and conditions for enzymatic assays of N7- and 2'O-methyltransferase activities that yield the final type I 5'-capped RNA ((7Me)GpppA2'OMe-RNA).
Department of Microbiology and Immunology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Siwaporn
S
Boonyasuppayakorn
Radhakrishnan
R
Padmanabhan
D002855:Chromatography, Thin Layer; D003716:Dengue Virus / Q000201:enzymology*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism*; D012314:RNA Cap Analogs / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification*
C582223:NS5 protein, dengue virus; D012314:RNA Cap Analogs; D012315:RNA Caps; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C509442:m7GpppA