Identification of mutations conferring 5-azacytidine resistance in bacteriophage Qβ

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for ϕX174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 99%

Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance

Domingo‐Calap

Pereira-Gómez

Sanjuán

2012

“…Mutagenesis was carried out using the QuikChange II sitedirected mutagenesis kit (Stratagene). The primers used to build the sitedirected mutants and the procedures to isolate them have been described previously (40). A single plaque of each site-directed mutant was picked and the presence of the desired mutation verified.…”

Section: Methodsmentioning

confidence: 99%

“…We previously showed that experimental populations of Q␤ exposed to the nucleoside analogue 5-azacytidine (AZC) experience strong decreases in viral yield, which are concomitant with increases in mutation frequency (39). Serial transfers in the presence of AZC led to the emergence of two amino acid substitutions in the viral RNA polymerase, Thr210Ala and Tyr410His, which reduced sensitivity to AZC but had strong fitness costs in the absence of the drug (40). To investigate whether the mode of nucleoside analogue resistance in Q␤ is similar to those previously reported in animal viruses, we characterized the effects of the Thr210Ala and Tyr410His substitutions.…”

mentioning

confidence: 99%

Changes in Protein Domains outside the Catalytic Site of the Bacteriophage Qβ Replicase Reduce the Mutagenic Effect of 5-Azacytidine

Cabanillas

Sanjuán

Lázaro

2014

Self Cite

The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Q␤ replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Q␤ replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCEIn the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Q␤ replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Q␤. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Q␤ is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified. The high mutation rate shown by RNA viruses is one of the main factors determining their population structure and biological properties. RNA virus populations are extraordinarily diverse and contain a dynamic repertoire of mutants, often referred to as quasispecies (1, 2). Such low replication fidelity allows RNA viruses to be highly evolvable (3-5) but also situates them close to the maximum error rate compatible with the maintenance of genetic information (6-8). Since replication above this limit can result in the loss of infectivity (9, 10), the fidelity of the enzymes that copy RNA virus genomes must be tightly regulated. These considerations have motivated an antiviral therapy r...

“…In the case of RNA viruses, mutation rates are orders of magnitude higher than those of their DNA-based hosts (7). These high mutation rates have important practical implications; for instance, for the long-term durability of vaccination strategies (6) and antiviral drugs (2), for the stability of live attenuated vaccines (26), for the eventual success of antiviral therapies based on the concept of lethal mutagenesis (1), or to determine the risk of new emerging viruses (14). The spontaneous mutation rate of a virus can be evaluated in vivo using a variety of experimental approaches.…”

mentioning

confidence: 99%

Luria-Delbrück Estimation of Turnip Mosaic Virus Mutation Rate In Vivo

Iglesia

Martínez

Hillung

et al. 2012