Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives - PubMed (original) (raw)

Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives

H Shimizu et al. J Virol. 2000 May.

Abstract

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.

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Figures

FIG. 1

FIG. 1

Structure of MRL-1237, enviroxime, HBB, and guanidine hydrochloride.

FIG. 2

FIG. 2

Drug susceptibility of cDNA-derived MRL-1237- and guanidine-resistant mutants. The effect of 2C mutations on drug susceptibility was examined with MRL-1237 and guanidine hydrochloride by plaque-reduction assay. A wild-type [PV1(M)] and drug-resistant [PV1(M)2C-F164Y, PV1(M)2C-I120V, PV1(M)2C-N179A, or PV1(M)2C-N179G] mutants derived from cDNA were inoculated to HeLa cells. After adsorption, HeLa cell monolayers were incubated for 72 h without drug (solid bars), with 2 mM guanidine (striped bars), or with 50 μM of MRL-1237 (open bars).

FIG. 3

FIG. 3

Effect of guanidine hydrochloride and benzimidazoles on the replication of cDNA-derived guanidine-dependent mutants. Drug-dependent phenotypes of PV1(M)2C-M187L/A233S (solid bars) and PV1(M)2C-M187L/A233T (open bars) were examined in the plaque assay. After viral adsorption, the cell monolayers were incubated for 72 h in the presence or absence of the drug. Final concentrations of MRL-1237, guanidine hydrochloride, HBB, and enviroxime were 50, 2,000, 200, and 30 μM, respectively.

FIG. 4

FIG. 4

Effect of MRL-1237 and guanidine on poliovirus-specific RNA synthesis. Poliovirus-specific RNA synthesis in virus-infected HeLa cells was determined by the Northern ELISA assay described in Materials and Methods. (A) The HeLa cell monolayers were inoculated with a wild-type PV1(M) virus, with an MRL-1237-resistant PV1(M)2C-F164Y virus, or with a guanidine-dependent PV1(M)2C-M187L/A233S virus. The infected cells were incubated in MEM without drug (solid bar), with 2 mM guanidine (striped bar), or with 50 μM MRL-1237 (open bar) for 5 h. The extracted RNAs were biotinylated, and 100 ng of biotinylated RNA per 130 μl was hybridized with 100 ng of a DIG-labeled poliovirus-specific DNA probe. Finally, the biotinylated poliovirus-specific RNA was visualized by the peroxidase-tetramethylbenzidine system. The viral RNA production is presented as the mean OD450–620 (the absorbance at 450 nm with a reference wavelength of 620 nm) values of three individual RNA samples. Total RNA from mock-infected cells was also prepared as a negative control. (B) Poliovirus RNA was transcribed from a pVMT7(1)pDS306(T) clone and biotinylated as a positive control. The OD450–620 values were determined with dilutions of the biotinylated poliovirus RNA.

FIG. 5

FIG. 5

Amino acid alignment of the NTP-binding motif of the 2C proteins of picornaviruses. The sequence data were obtained from the GenBank sequence database. The names of virus species are abbreviated as follows and the GenBank accession number is shown in parentheses: polio1, Mahoney strain of poliovirus type 1 (J02281); EV70, J670/71 strain of enterovirus 70 (D00820); coxB3, Nancy strain of coxsackievirus B3 (M16560); coxB4, JVB strain of coxsackievirus B4 (X05690); coxA16, G-10 strain of coxsackievirus A16 (V05876); Rhino14, 1059 strain of human rhinovirus type 14 (K02121); Rhino2, HGP strain of human rhinovirus type 14 (X00429); FMDV, Argentine/61 strain of foot-and-mouth disease virus A10 (K02990); EMCV, R strain of encephalomyocarditis virus (M81861); HAV, LA strain of human hepatitis A virus (K02990). Amino acids are numbered from the amino terminus of the 2C protein of poliovirus type 1. The consensus A and B motifs of the NTP-binding proteins are indicated. Solid circles indicate amino acids critical for the viral replication of poliovirus, as previously demonstrated by site-directed mutagenesis (47, 70). Highly conserved amino acid residues among 10 picornavirus strains are shaded in gray. Major phenotypic determinants for drug resistance or dependence (Ile-120, Phe-164, Asn-179, and Met-187) are indicated as I, F, N, and M (boxed) and by large arrows. The minor determinants responsible for guanidine phenotypes are indicated by small arrows (72).

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