Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice

Abstract

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3'-5' exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

FIG. 1.

Drug susceptibility profiles of HPMPC^R VAC (strain Lederle) clones. HEL cells were grown to confluence in 96-well dishes, infected with 50 PFU of virus (seven HPMPC^R or five WT clones, each in duplicate), and cultured for 2 or 3 days. The media contained serial dilutions of the test compound, and the concentration of drug required to inhibit CPE by 50% (IC₅₀) is shown for each virus type. Mean IC₅₀ values ± standard deviations (error bars) for each group of clones are indicated (averaged over at least two independent experiments).

FIG. 2.

Vaccinia virus E9L gene and mutational map. The VAC E9L gene encodes 1,006 amino acids and comprises DNA polymerase B exonuclease [DNA pol B exo] and DNA polymerase B [DNA pol B] domains plus six highly conserved sequence elements common to B-family DNA polymerases (I to VI) (32). The gene encoded by VAC strain Lederle bears three preexisting sequence polymorphisms, which differentiate the gene from that encoded by VAC strain WR. All of the viruses isolated by passage in HPMPC-containing media also encoded new A314T and A684V mutations (boxed). The PCR amplicons used for marker rescue studies are shown at the bottom of the top panel. Also shown are the known map sites for mutations conferring resistance to cytosine arabinoside (AraC), phosphonoacetic acid (PAA), and aphidicolin (Aph) (36). The two bottom panels show the sequence context of A314T and A684V mutations and alignments to other B-family DNA polymerases. RB69, phage RB69 gp49; HCMV, human cytomegalovirus; HSV, herpes simplex virus (type 1); EBV, Epstein-Barr virus; Sc, Saccharomyces cerevisiae Pol1; Hs, Homo sapiens polymerase α; T4, phage T4 gp49; AdV, human adenovirus type 5.

FIG. 3.

Drug resistance properties of HPMPC^R recombinant viruses. The figure compares the activities of different compounds against recombinant viruses encoding the indicated mutations. The data are presented as a ratio of the IC₅₀ for the recombinant virus versus the IC₅₀ for the parent VAC strain WR and are plotted on a log scale to facilitate comparison of a great range of differences in drug resistance (ratios of >1) or hypersensitivity (ratios of <1). Mean IC₅₀ values were determined using a CPE assay and HEL cells and derive from at least two independent experiments. Additional details are available from the authors upon request.

FIG. 4.

Effects of HPMPC and PAA on vaccinia virus growth. (A) Effects of HPMPC on the growth of different recombinant VAC strains as judged by a plaque reduction assay. About 200 PFU of each virus was plated on BSC40 cells in the presence of various concentrations of drug, incubated for 2 days, and stained to visualize plaques. Each data point was determined in triplicate, and the mean ± standard error (error bar) plotted as a percentage of the number of plaques at zero drug concentration. A nonlinear regression analysis was used to determine EC₅₀ values. The calculated EC₅₀ values were 53 ± 3 (•), 140 ± 20 (□), 240 ± 20 (▪), 790 ± 40 (┌), 890 ± 60 (▵), and 1,340 ± 50 (×) μM. (B and C) Effects of HPMPC and PAA on the growth of different recombinant VAC strains as judged by virus yield reduction assays. HEL cells were infected with ∼200 PFU of each virus in the presence of the indicated drug concentrations and cultured for 3 days, and the yield was determined using plaque assays on HEL cells. The results show the means ± standard deviations (error bars) of two independent experiments.

FIG. 5.

Growth properties of HPMPC^R viruses. BSC40 cells were infected with the indicated viruses at an MOI of 0.03 and cultured at 37°C, and the yield was determined by plaque assay on BSC40 cells for virus harvested at each of the indicated time points. Each measurement was determined in triplicate; the error bars are approximately the size of the data points.

FIG. 6.

Effects of HPMPC^R alleles on disease in mice following intranasal infection. Groups of five NMRI mice were infected with 4,000 (□), 400 (▵), or 40 (▿) PFU of the indicated virus or mock infected with saline (○). For each trial with a recombinant virus, a parallel study was performed using mice infected with the parental VAC strain WR. The mice were then monitored for weight loss over the next 30 days. The percentage of change in total weight for each group of mice [or the surviving member(s)] is plotted. A filled symbol (e.g., ▪) denotes a time point where one or more mice in a cohort were euthanized because of weight loss in excess of 25%.

FIG. 7.

Virus encoding the A684V substitution mutation exhibits a mutator phenotype. Six different stocks of each virus were prepared and expanded by two rounds of passage without drug (each starting from a single plaque), virus titers were determined, and ∼1,000 PFU was plated on BSC40 cells in the presence of 60 μM isatin-β-thiosemicarbazone (IBT). The proportion of IBT^R virus was determined after staining and counting the resulting plaques 2 days later, relative to the number of PFU determined in the absence of drug. (A) Variation in the proportion of IBT^R virus for different viral stocks. (B) “Box and whisker” plots showing medians, quartiles, and ranges for each virus group. Statistically significant differences between the median numbers of IBT^R plaques between mutants and WT are indicated (*, P < 0.01, Mann-Whitney U test).

FIG. 8.

HPMPC treatment of infections caused by HPMPC^R virus in vivo. Groups of five NMRI mice were infected with 4,000 PFU of recombinant virus encoding the A314T and A684V mutations (□, ▵, and ▿) or mock infected with saline (○). Animals were treated subcutaneously with saline (□, ○) or HPMPC at a dose of 50 (▿) or 10 (▵) mg/kg/day for 5 days. The change in total weight (expressed as a percentage) for each group of mice is plotted. A filled symbol (e.g., ▪) denotes a time point where one mouse was euthanized.

FIG. 9.

HPMPC^R alleles mapped onto the structure of RB69 DNA polymerase. (A) Structure of the RB69 DNA polymerase with DNA bound in the polymerase active site (13). Space-filling models and color codes are used to mark the locations of residues that are homologs of the VAC E9L residues A684 and T688 and the associated α-helix (yellow), VAC E9L Y688 (orange), and the incoming deoxynucleoside triphosphates (turquoise). Residue A684 is the uppermost of the two yellow-colored amino acids most clearly seen in panels A2 and A3. (B) Structure of the RB69 DNA polymerase with DNA bound in the exonuclease site (25). An NMY element (mauve) marks the tip of a β-hairpin structure that interacts with the 3′ terminus of the primer strand of the bound DNA and approximates the proposed location of the VAC A314T mutation (Fig. 2). These images were generated using CN3D v4.0 and crystallographic coordinates deposited as mmdbId 16732 and 11301.