Abstract
Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.
Highlights
Introduction ofnon-structural protein 5 (NS5) Mutations into a Genomic Length dengue virus (DENV) cDNA Clone—Mutations encoding changes in NS5 amino acids were introduced into the genomic length DENV serotype 2 strain New Guinea C (DENV-2) cDNA clone pDVWS601, which yields the virus v601 [43, 44]
The mutation K46A/R47A/E49A was engineered into a 1001-bp overlap extension PCR fragment (DENV-2 nucleotides 7165– 8165), which was HpaI7406/StuI7874 digested and introduced into the corresponding sites of pDVWS601 to produce pDVWS601NS5K46A,R47A,E49A
The NS5 mutation L512V was engineered into a 2077-bp fragment (DENV-2 nucleotides 8085–10161), which was AatII8570/MluI9732 digested and introduced into the corresponding sites of pDVWS601 and pDVWS601NS5K46A,R47A,E49A to produce pDVWS601-NS5L512V and pDVWS601-NS5K46A,R47A,E49A,L512V, respectively
Summary
Expression and Purification of the Polymerase Domains— Three WNV strain Kunjin ns gene constructs encoding a N-terminal His tag fused to pol (corresponding to aa 273– 905), pol (corresponding to aa 317–905), or pol (corresponding to aa 273 to 882) open reading frames were cloned into the pDEST14 vector, and expressed in Escherichia coli. The proteins were expressed and purified to homogeneity as follows: the E. coli strain C41pRos, transformed with either plasmid construct, was grown at 37 °C to an A600 of 0.6, induced with 0.5 mM isopropyl -D-thiogalactopyranoside, and further incubated 16 –18 h at 17 °C. The cell pellet was resuspended in 50 mM Tris buffer, pH 8.0, containing 150 mM NaCl, 10 mM imidazole, DNase I (2 g/ml), a protease inhibitor tablet (Sigma), and sonicated on ice. The sample was centrifuged, the supernatant collected and filtered through a 0.22-m filter. The protein was eluted with 50 mM Tris buffer, pH 8.0, containing 150 mM NaCl and 500 mM imidazole. Protein-containing fractions were applied onto a preparative Superdex 200 gel filtration column pre-equilibrated in 10 mM Tris buffer, pH 9.0, with 300 mM NaCl and 5% glycerol.
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