Suppression of Drug Resistance in Dengue Virus

Mateo, Roberto; Nagamine, Claude M.; Kirkegaard, Karla

Mutations in the NS5 methyltransferase domain confer resistance to the nucleoside inhibitor MK-0608. Nucleoside analog 2=-C-methyl-7-deaza-adenosine (also termed MK-0608, or 7-DMA) ( Fig. 3A) was originally developed as an inhibitor of hepatitis C virus polymerase (14) but also displays antiviral activity against dengue virus serotype 2 in cultured cells and in mice (15) . Inhibition of dengue virus serotype 2 in BHK-21 cells is shown in Fig. 3 . To select viruses resistant to MK-0608, dengue virus pools grown in C6/36 mosquito cells were serially passaged in BHK-21 cells in the presence or absence of increasing MK-0608 concentrations. For each passage, the multiplicity of infection was below 0.1 FFU per cell, which allowed any drug-resistant variants to propagate in the absence of coinfecting drug-susceptible virus. The amount of viral amplification at each passage is displayed as the ratio of total output virus to the initial inoculum (Fig. 3B ). Sequence analysis of passaged viruses revealed mutations in all populations, some of which were identical in the drug-treated and untreated populations (Table 1) . Such mutations likely conferred a growth advantage in BHK-21 cells; one of these, Q399H, has proven useful in adaptation to other mammalian cell lines (R. M. Deans et al., unpublished data). Notably, only the two viral pools passaged in the presence of MK-0608 acquired mutations in the NS5 coding region: A60T in pool 1 and Y201H in pool 2 (Table 1) . When tested individually, the NS5-A60T and NS5-Y201H mutations to select for resistance to the inhibitor (black bars). As a control for mammalian culture adaption of mosquito-grown virus, both initial populations were also passaged in the presence of 0.5% DMSO, the solvent for MK-0608 (white bars). After 17 passages, the two resulting virus pools and their DMSO-passaged counterparts were harvested and sequenced. Adaptation mutations were found in all populations (Table 1) . Drug-treated populations were passaged five more times with 50 M MK-0608. Unique mutations A60T (population 1) and Y201H (population 2) were found. Shown is the fold amplification for each passage, as the ratio of output over input virus. (C) Wild-type, NS5-A60T, and NS5-Y201H viruses were used to infect BHK cells in the absence or presence of MK-0608, as indicated. The yield after 24 h is shown. (D) Space-filling model of residues A60 and H200 in the three-dimensional structure of the dengue virus serotype 3 NS5 protein (26) . Residues A60 and Y201 of serotype 2, used in our studies, aligned with residues A60 and H200 of serotype 3, respectively. The methyltransferase domain of NS5 is shown in yellow, and GDD residues at the polymerase active site are shown in blue. each conferred significant resistance to MK-0608 compared to wild-type virus (Fig. 3C) . Interestingly, both mutations mapped to the methyltransferase domain of the NS5 protein (Fig. 3D) . Thus, it is likely either that MK-0608 inhibits methyltransferase activity or that allosteric interactions between the methyltransferase and the polymerase domains (16) relieve MK-0608's inhibition at the polymerase active site.