Amiloride derivatives inhibit coxsackievirus B3 RNA replication

Abstract

Amiloride derivatives are known blockers of the cellular Na(+)/H(+) exchanger and the epithelial Na(+) channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.

FIG. 1.

Antiviral activity and cytotoxicity of HMA, EIPA, benzamil, and amiloride. HeLa cells infected with CVB3 (MOI of 0.01) or mock infected were treated with various concentrations of HMA (A), EIPA (B), benzamil (C), and amiloride (D) for 48 h or left untreated. Infected cells were then lysed into culture medium by freeze-thawing, and virus yields were quantified by plaque assay. Metabolism of mock-infected cells was measured using the fluorescent indicator Alamar blue. •, CVB3 yield; ○, cell metabolism. The data points are averages ± standard errors of the mean from three independent experiments.

FIG. 2.

(A) Time courses of CVB3 production and release. HeLa cells were infected with CVB3 at an MOI of 1. At the indicated times culture supernatants were collected, cells were lysed in equal volumes of fresh medium, and intra- and extracellular virus titers were measured by plaque assay. Total virus yield was quantitated as a sum of intra- and extracellular titers. •, total virus yield; ○, extracellular virus yield. (B and C) Time-of-addition assay. The cells were infected as above, and 25 μM EIPA or 400 μM amiloride was added to the culture medium at the indicated times. At 10 h postinfection virus yields were measured as above. White bar, untreated virus. (D) Time-of-removal assay. The cells were infected in the presence of 25 μM EIPA or 400 μM amiloride. At various times postinfection culture supernatants were replaced with compound-free medium, and the infection was allowed to continue until 10 h postinfection when total virus yields were measured. Effects of EIPA (▴) and amiloride (▪) on CVB3 production are shown in panels B and D. The effects of EIPA (striped bars) and amiloride (solid bars) on CVB3 release are shown in panel C. The data points are averages ± standard errors of the mean from three independent experiments with duplicate samples.

FIG. 3.

Effects of amiloride, EIPA, and guanidine on viral RNA yield in a single replication cycle. HeLa cells were infected with CVB3 (A) or HRV2 (B) at an MOI of 10. After virus adsorption, the cells were labeled with [5,6-³H]uridine and treated with 400 μM amiloride, 25 μM EIPA, or GHCl (500 μM and 2 mM) or left untreated until 9 h (CVB3) or 10 h (HRV2) postinfection. Cytoplasmic RNA was then extracted and analyzed by formaldehyde-agarose gel electrophoresis and autoradiography. Virus titers in cell lysates were determined in parallel. The data are from one representative experiment of three. Contrast and brightness of the images were increased slightly by using Adobe Photoshop CS.

FIG. 4.

CVB3 RNA and protein syntheses in the presence of amiloride and EIPA. (A) Time course of CVB3 RNA synthesis. HeLa cells were infected with CVB3 at an MOI of 10 or mock infected. At the indicated times the cells were labeled with [5,6-³H]uridine in the presence of actinomycin D for 30 min. RNA was then extracted and ³H incorporation was quantitated using a scintillation counter. •, infected cells; ▴, mock-infected cells. The values are averages ± standard errors of the mean. (B and C) Effects of compounds on viral RNA and protein syntheses. The cells were infected as described above. The indicated compounds were added to the cultures at 4 h postinfection, and 30 min later the cells were labeled with [5,6-³H]uridine (B) or [³⁵S]methionine-cysteine (C) for 30 min in the same medium. Cytoplasmic RNA was then extracted and analyzed by formaldehyde-agarose gel electrophoresis and autoradiography (B). Alternatively, the cells were lysed, and the proteins were analyzed by SDS-polyacrylamide gel electrophoresis, followed by visualization on a phosphorimager. Positions of select CVB3 proteins are marked on the right in panel C. Contrast and brightness of images in panels B and C were increased slightly by using Adobe Photoshop CS.

FIG. 5.

Effects of mutations upon the kinetics of CVB3 replication. HeLa cells were infected with wild-type (•), 3D-S299T (▴), 3D-A372V (▵), 2A-D48G (○), or 2A-D48G/3D-A372V (□) viruses at an MOI of 1. At the indicated times culture supernatants were collected, cells were lysed in equal volumes of fresh medium, and intra- and extracellular virus titers were measured by plaque assay. Total virus yield was quantitated as a sum of intra- and extracellular titers. (A) Total virus yield. (B) Extracellular virus yield. The data are averages ± standard errors of the mean from three independent experiments.

FIG. 6.

Effect of mutations upon CVB3 susceptibility to amiloride and EIPA. HeLa cells were infected with the indicated viruses at an MOI of 0.01 (A) or 1 (B) and treated with 400 μM amiloride (black bars) or 25 μM EIPA (gray bars) or left untreated. Total virus yields were measured at 48 h (A) or 10 h (B) postinfection. (A) Effect of amiloride on virus yields over multiple replication cycles. (B) Effect of amiloride or EIPA on virus yields in a single replication cycle. WT, wild-type plasmid-derived CVB3; A3 isolate, amiloride-resistant isolate of CVB3 generated by passaging in the presence of 400 μM amiloride. The data are averages ± standard errors of the mean from three independent experiments.