Mutagen resistance and mutation restriction of St. Louis encephalitis virus

Abstract

The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus, is a mosquito-borne flavivirus that has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modelling of the E416K polymerase mutation indicated its location in the pinky finger domain of the RdRp, distant from the active site. Deep sequencing of the E416K mutant revealed lower genetic diversity than wild-type SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed that E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with West Nile virus high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference response, suggesting an impaired capacity to escape RNA interference could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and give further insight into the consequences of altered fidelity of flaviviruses.

Fig. 1.

Initial characterization and experimental design. (a) Susceptibility of SLEV-IC to ribavirin on BHK cells. BHK cells were pretreated with maintenance media containing 0, 250, 500, 750 or 1000 µM ribavirin for 1 h. Infections were done at a m.o.i. of 0.01, and viral titres were assessed in triplicate assays at 72 h p.i. and depicted as mean±sd (b) Schematic of SLEV serial passage in BHK cells to create ribavirin resistance. Duplicate wells were infected with SLEV-IC and deemed lineages A and B throughout passaging. Infectious titres were determined with and without ribavirin treatment following 72 h growth for each passage and compared to the unpassed SLEV-IC control. Treated viruses were passaged again in BHK cells with multiple concentrations of ribavirin.

Fig. 2.

Selection of SLEV with reduced ribavirin susceptibility. (a) Serial passage of SLEV in BHK cells in the presence of ribavirin. Log₁₀ reduction in infectious titre refers to the log difference between infectious titres with and without treatment. At each passage, viruses were subjected to 250 and 500 µM ribavirin. The response to 500 µM ribavirin throughout the passage experiment is shown. Inocula for passages 2–4 were taken following treatment with 250 µM and passages 5–8 following treatment with 500 µM ribavirin. The pretreatment period was increased from 1 to 4 h for passages 7 and 8. (b). Ribavirin susceptibility of selected clones. Graph represents the mean overall difference in infectious titre of each clone (mean reduction ±sd), after treatment with two concentrations (250 and 500 µM) of ribavirin and growth on BHK cell culture, as compared to infectious titre obtained after growth of same clone without ribavirin. Significance is determined by Student’s t-test as compared to mean overall reduction obtained with the SLEV-IC control. *P≤0.05, **P<0.01.

Fig. 3.

Resistance is not host cell or mutagen dependent. (a) Ribavirin susceptibility of selected clones on HeLa cells. Cells were pretreated and held with or without 100 µM ribavirin, in triplicate, following infection at a m.o.i. of 0.01, and harvested 3 days p.i. Reduction in titre was determined by subtracting titre obtained in the presence of ribavirin from the titre obtained without drug. (Mean viral reduction±sd is shown; *P≤0.05, Student’s t-test). (b) T-1106 susceptibility of selected clones on BHK cells. Cells were pretreated and held with or without 100 µM T-1106, in triplicate, following infection at a m.o.i. of 0.01, and harvested 3 days p.i. Reduction in titre was determined by subtracting titre obtained in the presence of ribavirin from the titre obtained without drug. (Mean viral reduction±sd is shown; *P≤0.05, Student’s t-test).

Fig. 4.

Structure of the NS5 protein with SLEV-E416K. The MTase domain (cyan) and polymerase domain (blue) are shown in ribbon form looking into the active site. Residue E416 is displayed as space-filling spheres (red), and the catalytic GDD sequence (motif C) is in stick representation (magenta).

Fig. 5.

In vitro growth kinetics of ribavirin-resistant SLEV clones in vertebrate and invertebrate cell lines. Cells were inoculated in triplicate at a m.o.i. of 0.01. Supernatant titres were determined by plaque assay on Vero cells. Mean titres±sd are shown. *P<0.05, paired t-test, as compared to the SLEV-IC. ^aPeak titre, P<0.05, t-test as compared to the SLEV-IC.

Fig. 6.

Lack of attenuation of ribavirin-resistant SLEV clones in mosquito cells lacking a functional RNAi response. Cells were inoculated in triplicate at a m.o.i. of 0.01 and harvested at 96 h p.i. Supernatant titres were determined by plaque assay on Vero cells. Mean titres±sd are shown. Significantly lower viral loads relative to SLEV-IC (*P<0.05, **P<0.01, paired t-test) were identified in three of four SLEV variants in CxT but not C6/36 cells.

Fig. 7.

Decreased mutation frequency of 8B-9 relative to SLEV-IC. Mean alternate allele frequency of SLEV-IC and 8B-9 in BHK and C6/36 cells±sem across the sequenced genome (~3000 bp). Alternate allele refers to any base call other than that of the majority consensus. Significantly higher frequencies were measured for SLEV-IC in both cell lines relative to SLEV 8B-9 (**Mann–Whitney U test, P<0.001).