title: Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue.
abstract: NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4mM. The induced cultures were then grown for 20h at 18°C and cells were harvested by centrifugation at 6000xg for 15min at 4°C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H-12, Islamabad 44000, Pakistan. Electronic address: deebaamraiz@gmail.com.
authors.forename: Deeba
authors.initials: D
authors.lastname: Amraiz
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H-12, Islamabad 44000, Pakistan. Electronic address: zaidi.sahar@gmail.com.
authors.forename: Najam-Us-Sahar Sadaf
authors.initials: NU
authors.lastname: Zaidi
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H-12, Islamabad 44000, Pakistan. Electronic address: s.munazza.fatima@gmail.com.
authors.forename: Munazza
authors.initials: M
authors.lastname: Fatima
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D003716:Dengue Virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008826:Microbial Sensitivity Tests / Q000379:methods; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D000998:Antiviral Agents; C582223:NS5 protein, dengue virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase
keywords: Dengue virus; Expression; Polymerase activity; Purification; RNA-dependent RNA polymerase