Mutation in enterovirus 71 capsid protein VP1 confers resistance to the inhibitory effects of pyridyl imidazolidinone

Abstract

Enterovirus 71 is one of the most important pathogens in the family of Picornaviridae that can cause severe complications in the postpoliovirus era, such as encephalitis, pulmonary edema, and even death. Pyridyl imidazolidinone is a novel class of potent and selective human enterovirus 71 inhibitor. Pyridyl imidazolidinone was identified by using computer-assisted drug design. This virologic investigation demonstrates that BPR0Z-194, one of the pyridyl imidazolidinones, targets enterovirus 71 capsid protein VP1. Time course experiments revealed that BPR0Z-194 effectively inhibited virus replication in the early stages, implying that the compound can inhibit viral adsorption and/or viral RNA uncoating. BPR0Z-194 was used to select and characterize the drug-resistant viruses. Sequence analysis of the VP1 region showed that the resistant variants differed consistently by seven amino acids in VP1 region from their parental drug-sensitive strains. Site-directed mutagenesis of enterovirus 71 infectious cDNA revealed that a single amino acid alteration at the position 192 of VP1 can confer resistance to the inhibitory effects of BPR0Z-194.

FIG. 1.

Scheme for the generation and selection of BRV viruses. We used 1 μM BPR0Z-194 to select the potentially resistant viruses (passages 1 to 4) and 2.5 μM BPR0Z-194 to isolate truly resistant viruses from the viruses that survived at a lower BPR0Z-194 concentration (passages 5 and 6). After passage 6, the viruses were passaged again in the absence of 2.5 μM BPR0Z-194 and called BRV viruses.

FIG. 2.

Chemical structure of BPR0Z-194.

FIG. 3.

Time course study of the antiviral activity of BPR0Z-194. Vero cells were infected with EV71 at an MOI of 1. First, 10 μM BPR0Z-194 was added to the culture medium at the specified times. Then, for the 0-h time point, BPR0Z-194 was added at the time of adsorption. Virus adsorption was determined at 4°C.

FIG. 4.

One-step growth of BRV virus. Vero cells (2.5 × 10⁵/ml) were infected with BRV virus at an MOI of 5. The supernatant and debris from each well were collected separately at 1-h intervals from 1 to 12 h. The collected debris was freeze-thawed three times and then centrifuged at 1,000 rpm for 10 min. The virus titer was determined by plaque assay.

FIG. 5.

Sequence analysis of the BRV virus VP1 gene.