Synergistic Lethal Mutagenesis of Hepatitis C Virus

Abstract

Lethal mutagenesis is an antiviral approach that consists of extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad-spectrum nature, which renders the strategy potentially effective against emergent RNA viral infections. Here we describe the synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method implemented in CompuSyn graphics software, with the average dose reduction index (DRI) being above 1 (68.02 ± 101.6 for favipiravir and 5.83 ± 6.07 for ribavirin) and the average combination indices (CI) being below 1 (0.52 ± 0.28). Furthermore, analogue concentrations that individually did not extinguish high-fitness HCV in 10 serial infections extinguished high-fitness HCV in 1 to 2 passages when used in combination. Although both analogues displayed a preference for G → A and C → U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. The prospects for synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Broad and Dynamic Diversification of Infectious Hepatitis C Virus in a Cell Culture Environment

“…For Illumina deep sequencing, PCR products were purified (QIAquick gel extraction kit; Qiagen), quantified (Qubit dsDNA assay kit; Thermo Fisher Scientific), and tested for quality (Bioanalyzer DNA 1000 LabChip; Agilent). Three amplicons of NS5B spanning genomic residues 7626 to 7962, 7941 to 8257, and 8229 to 8653 were analyzed from intracellular viral RNA using the Illumina MiSeq platform, with the 2 ϫ 300-bp mode with v3 chemistry; the fastq files were processed as previously described (74)(75)(76) to obtain the forward and reverse consensus haplotypes with abundances at or above 0.1% and median coverage of 99,694 reads per amplicon. Procedures for read cleaning, as well as controls for reliable mutant detection, and to ensure that two different amplifications of the same sample yield comparable read compositions, have been described (74,(76)(77)(78).…”

A review on favipiravir: the properties, function, and usefulness to treat COVID-19

“…The exact antiviral mechanism of favipiravir-RTP has not been yet known. However, there are three hypotheses for the mechanism of action: a) misincorporation of one or two consecutive favipiravir-RTP into the viral RNA and inhibiting the further RNA extension (chain termination) [21,22], b) the binding of the favipiravir-RTP to the active site of RdRp and blocking the enzyme activity [18] and c) lethal mutagenesis ( Figure 2) [23][24][25][26][27].…”

“…In the lethal mutagenesis process, favipiravir-RTP is misincorporated into a nascent RNA without termination of the RNA replication [23] (Figure 2). In the next cycle of RNA synthesis, the regions of the viral genome that has incorporated favipiravir-RTP will be prone to mutagenesis [23][24][25][26][27][28][29][30]. Favipiravir-RTP acts as a nucleotide and may promiscuously pairs to natural nucleotides cytosine (C) and uracil (U) [23][24][25][26][27][28][29][30].…”

“…It may be responsible for a large mutation frequency observed in the treated viral populations. An excess of mutations finally destroys the viruses [23][24][25][26][27][28][29][30].…”

“…In vitro and in vivo studies suggested the lethal mutagenesis as the most probable favipiravir mechanism of action [24][25][26][27]. However, two distinct studies support the chain termination as the responsible mechanism of action [31,32].…”