Taken together, our results provide the first evidence that T-705 exerts mutagenic activity in influenza viruses and that lethal mutagenesis is either wholly or in part the anti-influenza A virus mechanism of T-705 in vitro. During serial passage, treatment with T-705 resulted in a disproportionate decrease in infectious virus without a corresponding decrease in RNA copy number, thereby reducing virus infectivity (the ratio of PFU to viral RNA copies). It also increased the mutation frequency and shifted the nucleotide profiles of individual NP gene clones. The greater increase in the virus mutant spectrum observed after passage in the presence versus the absence of T-705 suggests that T-705RTP is incorporated into the viral genome, although we did not address this possibility or the possibility that T-705 indirectly affects nuclear transcription or translation. We hypothesize that the small increase observed in the rate of viral mutation leads to a disproportionately larger reduction of viral infectivity as T-705 promotes accumulation of mutations in numerous genes, ultimately causing the generation of nonviable progeny. T-705-enhanced mutagenesis was required for virus extinction, which was not observed in parallel mock-treated samples. The stability of IC50 and EC50s over the course of multiple passages in MDCK cells suggests the absence of phenotypic changes in susceptibility to T-705; therefore, drug-resistant variants are unlikely to emerge in vitro.
Lethal mutagenesis is a mechanism by which drug treatment increases the viral mutation rate sufficiently to overwhelm the virus population's ability to retain fitness (35
). Our hypothesis that lethal mutagenesis is the antiviral mechanism of T-705 was based on available data about the antiviral mechanism of nucleoside analogs against poliovirus, vesicular stomatitis virus (36
), West Nile virus (34
), foot-and-mouth disease virus (38
), lymphatic choriomeningitis virus (39
), and human immunodeficiency virus type 1 (35
). Lethal mutagenesis is suggested when the percentage of noninfectious virus genomes is much higher after passaging with a nucleoside analog than after passaging in mock-treated cells; furthermore, incorporation of a nucleoside analog into the viral genome is known to induce hypermutation (34
We used two approaches to assess the anti-influenza mechanism of T-705: serial passage with a low virus infectious dose and serial passage with a high virus infectious dose. If the antiviral activity of T-705 is caused by acceleration of the viral mutation rate, then serial passage after the low infectious dose would allow accumulation of the mutations, resulting in lethal mutagenesis. Indeed, after low-dose inoculation we observed virus extinction after 24 serial passages in the presence of T-705. Quantification and infectivity testing of RNA genomes after low- and high-dose virus inoculation and serial passage showed strikingly lower specific infectivity in viruses passaged in the presence of T-705 than in mock-treated viruses (e.g., seasonal influenza A viruses showed a >60% decrease in specific infectivity after 23 passages with T-705), suggesting that T-705 cased lethal mutagenesis.
The lethal mutagenesis theory emphasizes the role of a class of defective interfering particles, which can also be produced during the natural course of influenza virus infection. Therefore, we substantiated the role of lethal mutagenesis by demonstrating that the interfering activity generated by T-705 treatment was greater than the interfering activity naturally associated with influenza virus replication. For that purpose, we inoculated cells with a high dose of infectious virus to allow the generation of defective interfering particles during the course of serial passage. In cells inoculated with the high dose, no viable viruses were detected after 3 serial passages with T-705, although virus remained detectable after passage 4 in mock-treated cells. After 3 passages, specific infectivity decreased by approximately 60% in the presence of T-705 but only by 20% in mock-treated viruses. Therefore, virus extinction required T-705-induced mutagenesis.
Our data suggesting the incorporation of T-705RTP into viral RNA are consistent with the lethal mutagenesis theory. Specifically, we demonstrated a 9-fold increase (A/Brisbane/59/2007) and a 5-fold increase (A/New Jersey/15/2007) in G→A transitions. In cells inoculated with a low virus dose, virus-specific increases in RNA C→T transition were observed after serial passage in treated, but not mock-treated, cells. G→A transitions were also increased at high infectious doses, but not significantly, likely reflecting dose-related rapid virus extinction. A→G mutations within the coding viral genome cause the production of functionally impaired mutant proteins.
We utilized Sanger sequencing for quasispecies substructuring; this method is much less sensitive than ultradeep sequencing, and therefore we cannot completely rule out the selection of a very small population of T-705-resistant influenza variants. However, if such mutants were selected, their prevalence was less than ~10% of the virus population and their fitness was severely impaired. Furthermore, the lethal mutagenesis theory suggests to us that lethal mutations in the viral genome would have likely prevented the survival of such mutants. Such a mechanism would add to the advantages of an antiviral strategy that targets the viral mutation rate rather than viral proteins.
We observed an increased number of G→A and C→T mutations, suggesting that T-705RTP base pairs with either cytosine or uracil. RNA viruses can also undergo an A→G/U→C mutation pattern caused by a large isoform of the RNA-specific adenosine deaminase (ADAR1-L); this pattern is usually induced by the innate immune response to viral infection (4
). ADAR1-L can deaminate adenosine (A) to inosine (I) in the cellular cytoplasm, as demonstrated in the case of hepatitis C viruses (40
). Inosine is then recognized as guanosine by decoding ribosomes and transcribing polymerases, leading to A→G transitions. However, we believe that the A→G mutation pattern observed in this study during passages with T-705 is attributable to viral polymerase errors caused by T-705, as no hypermutation was observed in mock-treated viruses. Moreover, the sites of the A→G mutations were not related to the presence of the A, C, or G nucleotides at positions −1, −2, +1, and +2 (data not shown), as would be observed in ADAR1-L-induced mutation (41
). Taken together, our results suggest that T-705 increases the influenza virus mutation rate beyond the biological tolerance threshold, causing lethal mutagenesis.
An early understanding of potential resistance to an agent is crucial to guide the development of novel antivirals and maximize their usefulness. It has not been established whether resistance to mutagenic agents such as T-705 emerges as readily as resistance to nonmutagenic inhibitors such as NA inhibitors and adamantanes. At least two other groups have attempted to generate T-705-resistant influenza viruses (7
). In these studies, the viruses were extinguished within 20 serial passages in the presence of T-705. Importantly, neither study characterized the passaged variants or sought to determine the exact conditions (cell line, drug concentrations, viral MOI, method of susceptibility testing) required for generation of a T-705-resistant strain.
Of the nucleoside analogs, only ribavirin is demonstrated to have anti-influenza activity (16
). Ribavirin and T-705 have analogous carboxamide groups. Multiple ribavirin-resistant mutants of many RNA viruses have been described (43
). These mutants showed amino acid substitutions in RdRP that increase the general template-copying fidelity of the enzyme, thereby restricting the incorporation of ribavirin triphosphate into RNA during viral RNA synthesis. However, to the best of our knowledge, no ribavirin-resistant influenza viruses have been reported.
It is postulated that an influenza virus is resistant to currently licensed antiviral agents if the virus is (i) infectious in the presence of a drug (e.g., forms plaques on MDCK cells), (ii) is phenotypically less susceptible, and (iii) possesses mutation(s) in the target protein. In our study, the T-705 EC50
s were 8 to 18 μM for both the seasonal and 2009 pandemic viruses. Notably, T-705 yielded comparable values for both oseltamivir-susceptible and oseltamivir-resistant viruses, suggesting that T-705 is effective against both. These data support previous reports of T-705 antiviral activity against oseltamivir-resistant and amantadine-resistant influenza viruses (6
). In addition, no specific amino acid substitutions in the polymerase gene were detected, and no viable viruses were detected after several serial passages. Thus, T-705-resistant mutants were not selected under our conditions.
Although a drug that increases the mutation rate of a virus may also increase the mutation rate of its host cells, the most recent generation of nucleoside analogs, including T-705, were designed to specifically target viral RdRP without significantly affecting human DNA and RNA synthesis (12
). Importantly, however, nucleoside analogs exert relatively high cytotoxicity. Ribavirin is the only known nucleoside analog with potent anti-influenza activity, and like all first-generation nucleoside analogs, it is cytotoxic (50
). We found that T-705 was not toxic to MDCK or human lung A549 cells at concentrations of >1,000 μM. This value is at least 10 times the toxicity threshold of ribavirin (CC50
of 94 μM in MDCK cells) (6
). The T-705 concentration sufficient to inhibit influenza virus replication in both human and canine cell lines was in the 10 μM range, resulting in a selective index (CC50
) of >1. Furthermore, we observed neither early (1 h posttreatment) nor late (24 h posttreatment) cytotoxic effects. Furuta and colleagues demonstrated that T-705RTP was not significantly incorporated into MDCK-cell DNA at T-705 concentrations of <637 μM (15
). Although the available evidence suggests that T-705 treatment would not severely affect cellular RNA and DNA synthesis in humans, further studies are warranted to investigate the long-term effect of T-705 on the human genome.
Overall, given the increased mutation frequency in the influenza virus genome during the T-705 treatment, the specific mutational bias, and the fact that this increase is dose dependent, this study demonstrated that T-705 is an influenza virus mutagen. This provides a novel and promising approach to influenza therapy. Further studies are required to clarify whether the increased mutation frequency in the influenza virus genome resulting from T-705 treatment is due to the direct incorporation of T-705 in the virus genome and/or indirectly due to the interaction with some unknown host factors that would lead to lowering the intracellular concentrations of nucleoside triphosphate pools.