Article Figures & Data
Figures
- FIG 1
(A) Crystal structure of DENV3 NS5 is displayed as a cartoon (PDB code 4V0R). The MTase domain is colored in yellow, the RdRp domain is in cyan, and linker residues 263 to 266 are in red. The inset shows a closeup view of the linker region. The KDKE catalytic tetrad of MTase is shown as magenta sticks and labeled; the GDD active site is in green, and the priming loop is in blue and labeled. (B) Sequence alignment of the linker regions between the MTase and RdRp domains of NS5 from DENV1 to -4 and various flaviviruses showing the secondary structures observed in DENV3 NS5. WNV, West Nile virus; YFV, yellow fever virus; TBEV, tick-borne encephalitis virus. (C) Superposition of the MTase domain of the flaviviral NS5 structures with structural information for the linker region. The distance between Cα's of the first and fourth linker residues was measured. DENV3 NS5, PDB code 4V0R; DENV2 MTase, PDB code 1L9K; JEV NS5, PDB code 4K6M.
- FIG 2
Protein expression and in vitro enzymatic activities of DENV3 NS5 and its mutants. (A) SDS-12% PAGE of purified WT and mutant NS5 proteins purified as described previously (19); melting temperature values measured by Thermofluor assay are indicated in parentheses. Numbers at left are molecular masses in kilodaltons. (B) A de novo initiation/elongation assay was performed using viral untranslated region (UTR) sequence as the template as described previously (19, 20). (C) Elongation assays of DENV3 WT and mutant NS5 proteins were performed with a heteropolymeric RNA template annealed with four primers as described previously (10, 13). (D) N7 MTase activities of the WT and NS5 mutant were measured with the in vitro-transcribed first 110 nucleotides (nt) of the DENV genome with unmethylated cap G (12, 21). (E) 2′-O-MTase activities of DENV3 WT and mutant NS5 proteins were measured with a 7-mer single-stranded RNA with cap 0 structure (12, 21). Two independent experiments were performed for each assay in triplicate (RdRp assays) or duplicate (MTase assays). Percent activities relative to DENV3 WT NS5 are shown above each bar.
- FIG 3
Replication profiles of NS5 linker mutants. (A) NS5 linker mutations were introduced into an infectious DENV2 cDNA clone. Ten micrograms of in vitro-transcribed infectious clone RNA was electroporated into BHK-21 cells, and viral replication was monitored over a course of 5 days as described previously (18). Intracellular viral RNA replication was detected by reverse transcription-qPCR. The gray dashed line represents the background detection of uninfected cells. The IGG protein possesses a G insertion after I265, and the IPG protein has a P insertion after I265. (B) Extracellular viral RNA in the supernatants detected by reverse transcription-qPCR. The gray dashed line denotes background signal of uninfected supernatant. (C) Infectious virus titer measured by standard BHK-21 plaque assay. (D) IFA images showing dsRNA and NS5 costaining and percent infection of cells at 72 h postelectroporation (22).
- FIG 4
All atom root mean square deviations (RMSD) from molecular dynamics simulations of DENV NS5 structure generated using 4V0R (15) and 4HDG (23) and a schematic model depicting the dynamics of NS5 regulated by the interdomain linker. (A) Molecular dynamics simulations carried out with Gromacs-5.0.5 (www.gromacs.org) show that the V264P mutation exhibits a destabilizing effect on NS5 structure, and large conformational changes are observed in two independent simulations, while the V264G and V264I mutations have a minor effect on the structure and dynamics of NS5 compared to the wild-type protein. V264 is conserved and forms van der Waals interactions with side chains of residues R262 and E267. Molecular dynamics simulations suggest that I265 in DENV2 may adopt a similar interaction. (B) Schematic model showing that NS5 multiple enzymatic functions may require a flexible linker that permits the protein to adopt a greater variety of conformations (and thus yields viable viruses), as opposed to the V264P mutation, which abolishes 310-helix formation for the compact conformation of NS5 (suggested by molecular dynamics simulations), resulting in loss of some of the functions and abolished replication.
