Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor

Abstract

Arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The pyrimidine biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative- and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and pyrimidine biosynthesis inhibitors for the treatment of arenavirus infections.

FIG 1

Chemical structure of A3 and its molecular mass.

FIG 2

A3 inhibits arenavirus multiplication in Vero cells. Subconfluent monolayers of Vero cells were infected (MOI, 0.01) with LCMV (A) or Candid#1 (B). After 90 min of infection, viral inocula were removed and cells were treated with the indicated concentrations of A3 (i) or ribavirin (Rib) (ii). Aliquots of TCS were collected at the indicated times (hours postinfection), and virus titers (reported in FFU/ml) were determined via an immunofocus assay. Dotted lines indicate the limits of detection for the assay. Viral infections were performed in triplicates. Mean values and standard deviations are shown. Statistical analyses were conducted using a paired, two-tailed Student's t test within Microsoft Excel. *, P < 0.05.

FIG 3

A3 inhibits LCMV multiplication in A549 and BHK-21 cells. Subconfluent monolayers of A549 (A) and BHK-21 (B) cells were infected with LCMV (MOI, 0.01) and treated with the indicated concentrations of A3 (i) or ribavirin (Rib) (ii). Aliquots of TCS were obtained at the indicated times (hours postinfection) and titrated (resulting in data reported in FFU/ml) by using immunofluorescence. Dotted lines indicate the limits of detection for the assay. Viral infections were performed in triplicates. Mean values and standard deviations are shown. Statistical analyses were conducted using Student's t test within Microsoft Excel. *, P < 0.05.

FIG 4

Effects of A3 on cell viability. Vero (A), A549 (B), and BHK-21 (C) cells were seeded in 96-well plates and cultured for 16 h prior to starting treatment with the indicated concentrations of A3 for 24 h or 48 h, followed by addition of 15 μl/well of the CellTiter 96 Aqueous One solution reagent. After incubation at 37°C for 20 min, the quantity of formazan product generated in each well, which is directly proportional to the numbers of viable cells, was determined in an ELISA. Viability of A3-treated cells was calculated as the percentage of values obtained with DMSO-treated cells (set at 100%). Mean values and standard deviations of three independent assays are shown.

FIG 5

Evaluation of IC₅₀ and CC₅₀ levels for A3 in A549 cells. (i) To determine the IC₅₀, subconfluent monolayers of A549 cells were infected with r3LCMV GFP/Gluc (MOI, 0.01) in the presence of increasing concentrations of A3 (A) or ribavirin (B). Viral titers (bars) and Gluc expression (lines) were determined at 48 h.p.i. Dotted lines indicate both the viral limits of detection and background levels of Gaussia luciferase expression for the assay. Viral infections were performed in triplicates. Mean values and standard deviations are shown. (ii) To determine CC₅₀ values, A549 cells were treated as described in the legend for Fig. 4, with the indicated concentrations of A3 (A) or ribavirin (B) for 24 h, followed by cell viability analysis. Mean values of three independent assays are shown. Nonlinear regression curves were determined, and the respective IC₅₀ and CC₅₀ values were calculated.

FIG 6

A3 exhibits a dose-dependent inhibitory effect on the activities of both the LCMV and Candid#1 minigenomes. BHK-21 cells in 12-well plates were transfected with LCMV (A) or Candid#1 (B) MG systems. At 5 h posttransfection, transfection medium was replaced with fresh medium containing the indicated concentrations of A3 (A and B, i graphs) or ribavirin (A and B, ii graphs). At 72 h posttransfection, cell lysates were prepared to determine CAT expression levels in an ELISA. Values correspond to mean values and standard deviations of two independent experiments. For each experiment, each sample was examined in duplicate.

FIG 7

A3 exhibits a dose-dependent inhibitory effect on viral RNA replication and transcription in LCMV-infected BHK-21 cells. BHK-21 cells were infected (MOI, 0.1) with LCMV. After 90 min of adsorption, the virus inoculum was removed, cells were washed, and fresh medium was added that contained the indicated concentration of A3. At the indicated time postinfection, total cellular RNA was isolated and analyzed by Northern blotting hybridization using a ³²P-labeled DNA NP probe. Methylene blue (MB) staining of the membrane was used to assess RNA quality and equal loading and transfer of RNA samples, determined by the amounts of 28 S rRNA.

FIG 8

Dose-dependent inhibitory activity of A3 on recombinant trisegmented arenavirus. Vero cells were infected (MOI, 0.1) with r3LCMV/CAT-GFP (A) or r3Candid#1/CAT-GFP (B). After 90 min of infection, the virus inoculum was removed, cells were washed, and fresh medium was added that contained the indicated concentration of A3. At 48 h.p.i. GFP expression was directly visualized by fluorescence microscopy (A and B, images i). After imaging, cell lysates were prepare to determine CAT expression levels (A and B, graphs ii). Virus titers in TCS (A and B, graphs iii) were determined on Vero cells, based on GFP expression, which was directly visualized by fluorescence microscopy at 48 h.p.i. Mean values and standard deviations were calculated using Microsoft Excel software. Viral titers and representative images of at least three independent infections are shown.

FIG 9

A3 exhibits a dose-dependent inhibitory effect on the activity of the LASV minigenome. 293T cells in 12-well plates were transfected with the components of the LCMV or LASV T7 MG systems. At 5 h posttransfection, transfection medium was replaced with fresh medium containing the indicated concentrations of A3. At 60 h posttransfection, cell lysates were prepared to determine CAT expression levels in an ELISA.

FIG 10

Uracil and orotate addition reverses the antiviral effect of A3. Subconfluent monolayers of Vero (A) and A549 (B) cells were infected with LCMV (MOI, 0.01). After viral infection, cells were washed and treated with A3 (5 μM) and with the indicated increasing concentrations of uracil (Ura [A and B, graphs i]) and orotic acid (Oro [A and B, graphs ii]). Vehicle treatment (DMSO) was used as an internal control. Virus in TCS at the indicated hours postinfection were determined by immunofluorescence (results are reported as FFU/ml). Dotted lines indicate the limits of detection for the assay. Viral infections were performed in triplicates. Mean values and standard deviations are shown. Statistical analysis were conducted using Student's t test from Microsoft Excel. *, P < 0.05.

FIG 11

A3 reduces metabolites of de novo pyrimidine biosynthesis during LCMV infection. (A) Schematic representation of de novo pyrimidine biosynthesis pathway: stars indicate compounds analyzed by mass spectrometry. PALA and A3 inhibitors work to block their enzymatic target. Solid squares represent enzymatic complexes. In gray, enzymatic reactions (arrows) and metabolites (italic) are shown from the salvage pathway that allow the incorporation of uracil into the de novo pathway. Modified from reference . (B) Metabolite changes upon A3, LCMV, or A3 plus LCMV treatment. A549 cells were treated with A3 compound (5 μM) and/or infected with LCMV (MOI, 1). At 24 h.p.i., metabolite changes were analyzed by LC/MS-MS. Experiments were performed in quadruplicates. Mean values and standard deviations are displayed as log₂-converted values.

FIG 12

Inhibition of LCMV infection by PALA. Subconfluent monolayers of A549 (A) and BHK-21 (B) cells were infected with LCMV (MOI, 0.01) and treated with the indicated concentrations of PALA. Aliquots of TCS were obtained at the indicated times postinfection and titrated (results in FFU/ml) by immunofluorescence. Viral infections were performed in triplicates. Dotted lines indicate the limits of detection for the assay. Mean values and standard deviations are shown. Statistical analyses were conducted using Student's t test within Microsoft Excel. *, P < 0.05. PALA cytotoxicity in A549 (C) and BHK-21 (D) cells was evaluated as described for Fig. 3. Cell viability was normalized to DMSO-treated cells (set as 100%). Mean values and standard deviations of three independent assays are shown.

FIG 13

A3 inhibits human DHODH in vitro. (A) DHODH catalyzes the conversion of l-dihydroorotic acid to the oxidized orotate, thereby reducing dichloroindophenol (DCIP, blue) into DCIPH₂ (colorless). (B) In vitro DHODH inhibition was measured by adding serial dilutions of A3 and the known DHODH inhibitors brequinar, teriflunomide, and leflunomide. DMSO was included as a negative control. IC₅₀s were calculated based on four independent experiments.

FIG 14

Additive antiviral effects of A3 and ribavirin. Subconfluent A549 (A) and BHK-21 (B) cells were infected with LCMV (MOI, 0.01) and treated with the indicated concentrations of ribavirin (Rib) (i) in the presence or absence of 0.5 μM A3. Similarly, LCMV-infected cells were treated with the indicated concentrations of A3 (ii) in the presence or absence of ribavirin (Rib; 20 μM in A549 cells [A, graph ii] and 1 μM in BHK-21 cells [B, graph ii]). Viral titers in TCS at the indicated times were determined based on immunofluorescence (reported as FFU/ml). Viral infections were performed in triplicates. Dotted lines indicate the limits of detection for the assay. Mean values and standard deviations are shown. Statistical analysis were conducted using Student's t test within Microsoft Excel. *, P < 0.05.