28th International Conference on Antiviral Research location:Rome, Italy date:11-15 May 2015
The nucleobase analogue T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide; favipiravir) is a unique antiviral compound with broad and selective anti-RNA virus activity and a high barrier for resistance. Two hypotheses have been proposed for its mechanism of action (MOA) as anti-influenza virus agent: lethal virus mutagenesis and non-obligate chain termination.
When we passaged influenza virus in suboptimal concentrations of T-705 (3.2-20 µM), we observed that viral infectivity rapidly declined. After 14 passages, deep-sequencing of the entire viral genome revealed that T-705 increased the mutation rate by 4- to 16-fold. The majority (67%) of the mutations were G-to-A or C-to-U transitions, which is consistent with T-705 being a pseudobase mimicking both guanine and adenine due to its rotating carboxamide. These observations concur with a recent report (Baranovich et al., J. Virol., 2013) that T-705 acts as a lethal virus mutagen. Importantly, while these authors detected only a few missense mutations in the viral neuraminidase gene, we identified on average 27 mutations, of which one-third were missense, in the viral ribonucleoprotein (vRNP) components, i.e. PB1, PB2, PA and NP.
The rotating carboxamide and dual MOA of T-705 are reminiscent of ribavirin, another antiviral to which different MOAs have been attributed. Similar passaging experiments with ribavirin resulted in a 2- to 15-fold increased mutation rate, and 86% were G-to-A or C-to-U transitions. On average, 16 mutations were located in the vRNP proteins, of which half were missense.
The accumulation of mutations under T-705 or ribavirin resulted in a significant reduction in viral replication fitness, i.e. by a factor 1800 (T-705) and 120 (ribavirin) in the 14th virus passage, as determined by titration in cell culture and RT-qPCR.