Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

Adele Boccuto,Mattia Mori,Carla Veo,Federica Giammarino,Francesca Picarazzi,Francesco Saladini,Ilaria Vicenti,Filippo Dragoni,Maurizio Zazzi,Alessia Lai,Gianguglielmo Zehender,Carla Della Ventura

doi:10.3390/ijms22052670

Abstract

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Highlights

Zika virus (ZIKV) is an emerging human pathogen belonging to the Flaviviridae family, a group of arthropod-borne positive-sense single-stranded RNA viruses [1]
While this study supports the analogy between ZIKV and hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) in their interaction with sofosbuvir, it does not provide any clue to sofosbuvir resistance selection in ZIKV RdRp, a key component of drug profiling
In vitro systems are not directly comparable, the time to emergence of sofosbuvir-resistant mutants appears to be similar with ZIKV (95 ± 20 days in this study) and HCV (90 ± 14 days in the work showing selection of the S282T substitution) [47]

Summary

Introduction

Zika virus (ZIKV) is an emerging human pathogen belonging to the Flaviviridae family, a group of arthropod-borne positive-sense single-stranded RNA viruses [1]. While ZIKV has been long known to be transmitted by the bite of Aedes spp. mosquitoes, additional transmission routes have been demonstrated in the last few years, including sex, blood transfusion, and vertical transmission [2,3,4,5]. During the recent epidemic, ZIKV infection has been associated with severe diseases, including multiorgan failure [10]; neurological complications, such as Guillain-Barré syndrome (GBS) in adults; and congenital ZIKV syndrome in newborns [11,12,13], possibly associated with increased virulence and neurotropism of the Asiatic lineage. The size of the epidemic and severity of the disease have renewed interest in the ZIKV infection, which can no longer be considered a benign disease [9,14]

Methods

Results

Conclusion