Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon

Abstract

Hepatitis E virus (HEV) is a common cause of acute hepatitis that results in high mortality in pregnant women and may establish chronic infections in immunocompromised patients. We demonstrate for the first time that alpha interferon (IFN-α) and ribavirin inhibit in vitro HEV replication in both a subgenomic replicon and an infectious culture system based on a genotype 3 strain. IFN-α showed a moderate but significant synergism with ribavirin. These findings corroborate the reported clinical effectiveness of both drugs. In addition, the antiviral activity of ribavirin against wild-type genotype 1, 2, and 3 strains was confirmed by immunofluorescence staining. Furthermore, the in vitro activity of ribavirin depends on depletion of intracellular GTP pools, which is evident from the facts that (i) other GTP-depleting agents (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide [EICAR] and mycophenolic acid) inhibit viral replication, (ii) exogenously added guanosine reverses the antiviral effects, and (iii) a strong correlation (R(2) = 0.9998) exists between the antiviral activity and GTP depletion of ribavirin and other GTP-depleting agents.

FIG 1

Inhibitory activities of IFN-α and RBV against HEV replication. Huh7 cells were transfected with capped p6/luc RNA and treated with RBV (A) or IFN-α (B) for 3 days. (C) Antiviral activities below or above those expected for RBV–IFN-α combinations. (D) Antiviral activities were also assessed in an infectious virus yield assay with RT-qPCR detection of viral RNA. *, P < 0.05; **, P < 0.01. GE, genome equivalents. (E) RBV (200 μM) inhibited formation of foci in HepG2/C3A cells infected with strains Sar 55, Akluj, Mex 14, LBPR-0379, Kernow-C1, and Kernow-C1 p6. (F) Cell viability of Huh7 cells used in the infectious virus yield assay after 20 days in the presence of IFN-α or ribavirin, as assessed by the MTS/PMS method. (G) Cell viability of HepG2/C3A cells treated with RBV at 200 μM for 3 days was assessed by the same method. Values represent means ± standard deviations (SD) for at least 3 independent experiments (A to D, F, and G) or 3 replicates (E).

FIG 2

Dependence of the antiviral activity of RBV on the depletion of intracellular GTP pools. MPA (A) and EICAR (B) are known inhibitors of IMPDH and are potent inhibitors of HEV replication in the replicon assay. Values represent means ± SD for at least 3 independent experiments.

FIG 3

Replenishing GTP pools salvages HEV replication. Addition of exogenous guanosine to the culture medium (40 μM) salvages HEV replication and abolishes the antiviral effects of RBV (A), MPA (B), and EICAR (C) in the replicon assay. (D) Relationship between the IC₅₀s for GTP depletion and EC₅₀s for inhibition of viral replication. Values represent means ± SD for at least 3 independent experiments.