A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function

Abstract

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Ribavirin Treatment Failure-Associated Mutation, Y1320H, in the RNA-Dependent RNA Polymerase of Genotype 3 Hepatitis E Virus (HEV) Enhances Virus Replication in a Rabbit HEV Infection Model

“…A major advantage of rabbit HEV-3ra over human HEV-3 is that the mutations in HEV-3ra can be readily tested with a homologous animal model since HEV-3ra naturally infects rabbits and causes persistent infection. Nevertheless, it should be noted that the rabbit HEV-3ra LR grows less efficiently in cultured cells than human HEV-3 Kernow-C1/p6 ( 21 , 22 , 50 , 60 – 62 ).…”

“…Of note, the Y1320H/K1383N/G1634R/triple mutant is associated with RBV resistance in a clinical case, and in vitro studies showed that Y1320H and G1634R enhance HEV replication, whereas K1383N decreases HEV replication but increases RBV susceptibility, thereby contradicting the clinical observation ( 21 ). More recently, additional amino acid substitutions (P25S, G38S, A64T, G71R, P79S, S95P, V245I, and T324S) in HEV-3 ORF2 protein are reportedly associated with a sustained viral response despite RBV therapy, and the P79S mutant impaired antibody-mediated neutralization of HEV-3, thus potentially acting as an immune decoy ( 20 , 22 ). Nonetheless, due to the lack of a tractable and relevant animal model for chronic HEV-3 infection, the mechanisms of action or clinical relevance of these RBV treatment failure-associated mutations in HEV-3 replication and pathogenesis are largely unknown.…”

Expanding the Hepatitis E Virus Toolbox: Selectable Replicons and Recombinant Reporter Genomes

“…In addition, participation of the ORF2 and ORF3 proteins in RNA replication cannot be evaluated. The recent description of an ORF2 single nucleotide variant which may affect viral RNA replication [57] is supporting this concern. Hence, results obtained in a subgenomic replicon system should be validated in a full-length HEV RNA replication and/or infection system.…”

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes

“…The number of FFU were counted using the analyzing software of the Immunospot analyzer or FIJI in combination with CellProfiler [85, 86]. For visualization of full 96-well fluorescence images a maximum filter (r=10 pixels) was applied as previously described [87].…”