Mechanism of action at the molecular level of the antiviral drug 3(2H)-isoflavene against type 2 poliovirus

Abstract

The mechanism of action of the antiviral compound 3(2H)-isoflavene against Sabin type 2 poliovirus has been studied, and interference with virus uncoating was demonstrated. Isolation and sequencing of drug-resistant variants revealed single amino acid substitutions (I194M or D131V) in the VP1 capsid protein. While M194 is located in a hydrophobic pocket and should partially fill the space occupied by the isoflavene ring, V131 is exposed on the VP1 surface, forming a contact with VP4. The D131V mutation most likely induces local conformational changes in VP1 and/or VP4 that affect viral flexibility. Two dependent variants, N53S of VP1 and K58E of VP4, both located on the inner surface of the capsid, near the threefold axis of symmetry, were also selected. Both mutations affected viral stability, allowing the transition to 135S particles in the absence of drug, without the involvement of the viral receptor.

FIG. 1.

Localization of drug-resistant and -dependent mutations and modeling of drug binding. (A) Close-up of the 3(2H)-isoflavene binding site near VP1 Ile194. The 3(2H)-isoflavene ligand (yellow) is modeled into the bottom of the hydrophobic pocket formed by VP1 Ile110, Phe134, Phe136, Tyr159, Pro181, Ile194, Val196, and Leu240. Mutation of Ile194 into a longer Met side chain (indicated by an orange arrow) partially fills the bottom of the pocket, abolishing efficient binding of the isoflavene. Also shown is VP1 Asp131 (red), a second mutation (Asp→Val) that confers drug resistance. Asp131 forms contacts with VP4 (through His265) and the backbone (NH of Tyr198) of one of the beta strands that is part of the pocket wall. In particular, loss of the latter contact could induce a local conformational change of the Val196-Pro197-Tyr198 backbone segment (highlighted in red), leading to a change in shape and, possibly, also leading to a change in the accessibility of the pocket. (B) The locations of the two mutations resulting in a drug-dependent phenotype, VP1 Asn53 to Ser and VP4 Lys58 to Glu, are shown in the context of a VP1 to VP4 pentamer seen from inside the capsid (VP1, blue; VP2, green; VP3, red; VP4, white). Both residues lie on the inner surface of the capsid and are far from the isoflavene-binding pocket (yellow).

FIG. 2.

Effect of 3(2H)-isoflavene on RNA synthesis of PV2 Sabin, R2/01, and D6/01 variants. (A) HeLa cells monolayers at 60% confluency were transfected with purified viral RNA from PV2 Sabin and viral variants in the presence of 0.5 mg of DEAE-dextran per ml. Following incubation for 6 h in the presence or absence of the drug (20 μM), RNAs were extracted, adsorbed on filter paper (10 μg), and hybridized with a probe (specific for PV2 nucleotides 1500 to 1940). After autoradiography, RNAs were quantified as described in Materials and Methods. (B) HeLa cell monolayers were infected with viral variants and the reference Sabin PV2 strain at MOIs of 10 and incubated at 37°C in the absence or presence of the antiviral compound (20 μM). After a single step of viral replication (6 h postinfection), total RNAs were extracted and adsorbed (5 μg) on filter paper. Hybridization was performed as described for panel A. Solid bars, without antiviral compound; shaded bars, with antiviral compound.

FIG. 3.

Effect of 3(2H)-isoflavene on thermal inactivation of PV2 Sabin and R2/01. Each virus (5 × 10⁸ PFU) was incubated overnight at 4°C in the presence or absence of antiviral compounds. Following 30 min of incubation at the indicated temperatures, the viruses were chilled on ice and titers were determined by plaque assay (the data represent the means of three separate experiments). Solid bars, without antiviral compounds; shaded bars, with WIN51711; open bars, with 3(2H)-isoflavene.

FIG. 4.

Effect of 3(2H)-isoflavene on binding of PV2 Sabin, R2/01, and D6/01 variants. HeLa cell monolayers were infected with 200,000 cpm of [³⁵S]methionine-labeled purified viruses in the presence or absence of the drug (20 μM). After 1 h of incubation at 4°C, the cells were washed with PBS and lysed with 0.2% Nonidet P-40, and the cell-bound radioactivity was measured in a beta-scintillation counter. A polyclonal antibody against PV2 Sabin was used as the virus binding inhibitor. The experiment was repeated three times. Solid bars, without 3(2H)-isoflavene; shaded bars, with 3(2H)-isoflavene; open bars, with anti-PV2 antibody.

FIG. 5.

Effect of 3(2H)-isoflavene on sucrose gradient sedimentation profiles of PV2 Sabin (Sab), R2/01, and D6/01 variants. (A) Binding of purified [³⁵S]methionine-labeled viruses to HeLa cells was synchronized by 1 h of incubation at 4°C in the presence or absence of the compound (20 μM). After removal of unbound virus, cells were incubated in the presence or absence of the compound at 37°C for the indicated times and then lysed. Cell-associated radiolabeled viral particles were centrifuged through a 15 to 30% sucrose gradient. Fractions were collected, and radioactivity was measured in a beta-scintillation counter. The percentages of mature 160S and 135S viral particles are indicated. WIN 51711 was used in the assay as an inhibitor of uncoating. (B) [³⁵S]methionine-labeled variant D6/01 virions were incubated with or without compound at 37°C without cells for the indicated times. Virus-associated radioactivity was analyzed on a 15 to 30% sucrose gradient, as specified above. The percentages of the 160S, 135S, and 80S viral particles are indicated.

FIG. 5.

Effect of 3(2H)-isoflavene on sucrose gradient sedimentation profiles of PV2 Sabin (Sab), R2/01, and D6/01 variants. (A) Binding of purified [³⁵S]methionine-labeled viruses to HeLa cells was synchronized by 1 h of incubation at 4°C in the presence or absence of the compound (20 μM). After removal of unbound virus, cells were incubated in the presence or absence of the compound at 37°C for the indicated times and then lysed. Cell-associated radiolabeled viral particles were centrifuged through a 15 to 30% sucrose gradient. Fractions were collected, and radioactivity was measured in a beta-scintillation counter. The percentages of mature 160S and 135S viral particles are indicated. WIN 51711 was used in the assay as an inhibitor of uncoating. (B) [³⁵S]methionine-labeled variant D6/01 virions were incubated with or without compound at 37°C without cells for the indicated times. Virus-associated radioactivity was analyzed on a 15 to 30% sucrose gradient, as specified above. The percentages of the 160S, 135S, and 80S viral particles are indicated.

FIG. 6.

Thermostability of the dependent variant. HeLa cells were incubated with the D6/01 variant in the absence of the drug for 1 h at 4°C. After infection for 6 h in the presence or absence of the drug at the indicated temperatures, the cells were lysed and viral titers were determined by plaque assay in the presence of the compound. The drug-plating index is expressed as the ratio between the plaque titer in the presence of the drug and that in the absence of the drug.