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. 2010 Sep;54(9):3686-95.
doi: 10.1128/AAC.00561-10. Epub 2010 Jul 6.

Characterization of Dengue Virus Resistance to Brequinar in Cell Culture

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Free PMC article

Characterization of Dengue Virus Resistance to Brequinar in Cell Culture

Min Qing et al. Antimicrob Agents Chemother. .
Free PMC article

Abstract

Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.

Figures

FIG. 1.
FIG. 1.
Cytotoxicity and spectrum of BQR antiviral activity. (A) Structure of BQR. (B) CFI assay. A549 cells were infected with DENV-2 (MOI, 0.3) in the presence of 2-fold serial dilutions of BQR. After incubation at 37°C for 48 h, the expression of viral envelope protein was quantified by an immunodetection method (see Materials and Methods). Average results from three experiments are shown. (C) Spectrum of BQR antiviral activity. Vero cells were infected with the indicated viruses at an MOI of 0.1; the infected cells were immediately treated with BQR. For DENV-2, WNV, YFV, PWV, and WEEV, culture media were collected at 42 h p.i. and viral titers were measured using plaque assays. For VSV, culture medium was collected at 16 h p.i. and the viral titer was measured. Average results and standard deviations (n = 3) are presented. (D) Cytotoxicity of BQR in Vero and A549 cells. Cytotoxicity was examined by incubation of Vero and A549 cells with the indicated concentrations of BQR. Cell viability was measured by an MTS assay and is presented as a percentage of the colorimetric absorbance derived from the compound-treated cells compared with that derived from the mock-treated (0.9% DMSO) cells. Average results from three experiments are shown.
FIG. 2.
FIG. 2.
Mechanism of BQR-mediated inhibition of DENV-2. (A) Time-of-addition analysis of BQR in DENV-2 infection. Vero and A549 cells were infected with DENV-2 at an MOI of 2 at 4°C for 1 h. The infected cells were washed three times with PBS. BQR (5 μM) was then added to the cells at the indicated time points postinfection. The supernatants were assayed for determination of viral titers at 24 h postinfection. As controls, 0.9% DMSO was added to the infected cells at 0, 10, and 20 h p.i. for estimation of its effect on viral production. (B) Transient replicon assay. (Left panel) A luciferase-reporting replicon (10 μg) was electroporated into A549 cells. The transfected cells were immediately incubated with 5 μM BQR or 0.9% DMSO (as controls) and the luciferase activities were measured at the indicated time points. (Right panel) A549 cells were preincubated with 5 μM BQR for 24 h, after which the replicon (10 μg) was electroporated into these cells. The transfected cells were then incubated with medium containing 5 μM BQR or 0.9% DMSO, and the luciferase signals were measured at the indicated time points. For analyzing the effect of pyrimidine on the antiviral activity of BQR, 5 μM BQR and 20 μM uridine were added to the transfected cells; luciferase activities were measured at the indicated time points. Error bars indicate the standard deviations from three independent experiments.
FIG. 3.
FIG. 3.
Pyrimidine reverses the antiviral effect of BQR. (A) Uridine reverses the antiviral effect of BQR in the CFI assay. A549 cells were infected with DENV-2 and treated with different doses of BQR and uridine. At 48 h p.i., viral envelope protein was quantified by the CFI assay. (B) Specificities of pyrimidines to reverse the antiviral effect of BQR. Vero cells (4 × 104 cells/well in a 96-well plate) were infected with DENV-2 VLPs (5 × 105 focus-forming units). The cells were then treated with 1 μM BQR in the presence or absence of 25 μM uridine (U), cytidine (C), adenosine (A), or guanosine (G). Luciferase activities were assayed at 48 h postinfection. Error bars indicate the standard deviations from three independent experiments.
FIG. 4.
FIG. 4.
Selection and sequencing of BQR-resistant DENV-2. (A) Scheme for selection of BQR-resistant DENV-2. P1 through P6 were selected at 0.5 μM BQR, P7 through P12 were selected at 1.0 μM, and P13 through P21 were selected at 2.0 μM. (B) Resistance analysis. Vero cells were infected with wild-type or P21 viruses (MOI, 0.1) in the presence of BQR or 0.9% DMSO (as a negative control). At 48 h p.i., the viral titers in culture fluids were quantified by plaque assays. Resistance is quantified by comparison of the viral titers from the BQR-treated infections with the viral titers from the DMSO-treated infections. Error bars indicate the standard deviations from three independent experiments. (C) Mutations recovered from the P21 resistant virus. The locations of the nucleotide and amino acid changes are indicated. (D) Amino acid sequence alignment. (Left panel) Sequence alignment of a flavivirus envelope protein region; (right panel) sequence alignment of a flavivirus RdRp region. The sequences of DENV-1, DENV-2, DENV-3, DENV-4, WNV, KUNV, JEV, and YFV are derived from the sequences with GenBank accession numbers U88535, M29095, M93130, AY947539, AF404756, D00246, AF315119, and X03700, respectively. (E) Locations of resistance mutations on crystal structures of DENV-2 envelope protein and DENV-3 RdRp. (Left panel) DENV-2 envelope protein structure (Protein Data Bank code 1OKE [20]). The two envelope protein subunits are in yellow and green; the mutated residue M260 is labeled in red. (Right panel) DENV-3 RdRp structure (Protein Data Bank code 2HFZ [36]). The fingers, palm, and thumb subdomains are indicated; the GDD active site of RdRp is shown in yellow; the priming loop is colored in cyan; the mutated residue Q802 is shown in red. The images were prepared using the PyMol program.
FIG. 5.
FIG. 5.
Analysis of resistance mutations. (A) Plaque morphologies of WT, Env M260V, NS5 E802Q, and Env M260V-NS5 E802Q viruses. Plaques were developed in the absence of BQR. (B) Growth kinetics of WT and mutant viruses. Vero and C6/36 cells were infected with the WT and mutant viruses at an MOI of 0.1. After 1 h of incubation, the cells were washed three times with PBS; the medium was then replenished. Viral titers in culture fluids were quantified at the indicated time points using plaque assays. (C) Resistance analyses of WT mutant viruses. The resistance assays were performed as described in the legend to Fig. 4B. (D) Resistance analyses of WT DENV-1 and DENV-2. Vero cells were infected with WT DENV-1 and DENV-2 at an MOI of 0.1 in the presence of BQR. Viral titers in culture fluids were quantified by plaque assays at 48 h postinfection. (Top panel) Viral titers; (bottom panel) relative viral titers (relative viral titers are the viral titers from the BQR-treated infections/viral titers from the mock-treated infections). Error bars indicate the standard deviations from three independent experiments.
FIG. 6.
FIG. 6.
Analysis of NS5 E802Q mutation. (A) Luciferase-reporting replicon analysis. A549 cells were preincubated with 5 μM BQR for 24 h, after which an equal amount of WT or NS5 E802Q replicon RNA (10 μg) was electroporated into the cells. The transfected cells were incubated with medium containing BQR (5 μM) or 0.9% DMSO (as a control). Luciferase activities were measured at the indicated time points posttransfection. Average results from three independent experiments are presented. (B) Recombinant proteins of full-length NS5. Full-length WT and mutant E802Q NS5 of DENV-2 (2.5 μg each) were analyzed by SDS-PAGE and stained with Instant Blue (Expedeon Ltd., United Kingdom). The proteins were expressed and purified as reported elsewhere (23). The molecular masses of protein markers are labeled. (C) Inhibition of de novo RNA synthesis by BQR. Subgenomic RNA of DENV-2 (containing the 5′-terminal 169 nucleotides directly connected to the 3′-terminal 462 nucleotides of the genome) was incubated with equal amounts of recombinant NS5 (0.25 μg of WT or E802Q) of DENV-2, as described previously (23). The reaction mixtures were incubated with the indicated concentrations of BQR. The RdRp products were analyzed on a 10% denaturing polyacrylamide gel with 7 M urea and quantified using a PhosphorImager. For both WT and E208Q NS5, the RdRp activity in the absence of BQR was set equal to 100%, and the remaining RdRp activities in the presence of BRQ are indicated. A representative result from three experiments is shown. (D) Enhancement of de novo RNA synthesis by the E802Q mutation. The RdRp products, derived from the WT and mutant NS5 in the absence of BQR, as shown in panel C, were quantified; the RdRp product from the WT NS5 is set equal to 100%. The average results of three experiments are presented, with error bars representing standard deviations.
FIG. 7.
FIG. 7.
Effects of Env M260V on RNA synthesis, virion assembly/release, and BQR resistance. BHK-21 cells were transfected with WT or Env M260V genome-length RNA. The transfected cells were incubated with different concentrations of BQR. At 24 h p.t., the cells were washed three times with PBS and the intracellular viral RNA was quantified (using the host GAPDH RNA level for normalization). (A) Relative amounts of intracellular viral RNA, using the amount of viral RNA derived from the DMSO-treated sample set equal to 100%. At 24 h p.t., the supernatants were filtered through 0.2-μm-pores-size filters and the extracellular viral RNA was quantified. (B) Relative amounts of extracellular WT RNA (set equal to 100%) and Env protein mutant (MT) RNA without BQR treatment. (C) Relative amounts of extracellular viral RNA for BQR-treated samples; for either WT or Env protein mutant RNA, the amount of RNA derived from the mock-treated sample is set equal to 100%. Average results from three independent experiments are presented.
FIG. 8.
FIG. 8.
VLP analysis of NS5 and envelope protein mutations. (A) Scheme for DENV-2 VLP production. VLPs were prepared by sequential transfection of BHK-21 cells with a luciferase-reporting replicon and a vector expressing viral CprMEnv proteins. See Materials and Methods for details. (B) Western blotting of WT and mutant (MT) envelope proteins. The doubly transfected cells were lysed at 30 h after the first electroporation. The lysates were blotted for envelope proteins using mouse monoclonal 4G2 IgG and horseradish peroxidase-labeled anti-mouse IgG as primary and secondary antibodies, respectively; γ-tubulin was also blotted as a loading control. The combinations of WT and NS5 mutant replicons and WT and Env mutant structural proteins are indicated above the blotting image. (C) Kinetics of replicon replication in VLP packaging cells. Luciferase activities were measured from the doubly transfected VLP packaging cells at the indicated time points after the first replicon transfection. Average results from three experiments are shown. (D) Effects of envelope protein and NS5 mutations on VLP assembly/release. VLP production in culture fluid was measured by determination of the amount of extracellular viral RNA at 48 and 72 h after the first transfection. The relative amounts of RNA are shown; the amount of viral RNA derived from the VLP (collected at 72 h p.t.) containing the WT replicon and WT structural proteins was set equal to 100%. Average results from three independent experiments are presented.

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