Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity.
The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus, the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent Kd value of 53nM for binding to the 5'-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development.
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd., Nakhon Pathom 73170, Thailand.
Maliwan
M
Kamkaew
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd., Nakhon Pathom 73170, Thailand. Electronic address: sarin.chi@mahidol.ac.th.
Sarin
S
Chimnaronk
D020121:5' Untranslated Regions; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008345:Manganese / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D020121:5' Untranslated Regions; D002413:Cations, Divalent; D012367:RNA, Viral; D011994:Recombinant Proteins; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase
Dengue virus; In vitro polymerase assay; Polyhistidine tag; RNA-dependent RNA polymerase (RdRp); Surface plasmon resonance