The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein

Abstract

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.

FIG. 1.

BFA specifically inhibits poliovirus RNA replication. (A) Poliovirus growth curve with or without BFA. HeLa cells were infected with wild-type poliovirus at an MOI of 5 PFU/cell in the absence (▪) or presence (•) of 2.0 μg of BFA/ml. Virus was harvested from parallel wells at the time points indicated. Titers of <10⁵ PFU/ml are shown at 10⁵ PFU/ml. (B) Poliovirus replicon translation and replication with or without BFA. Poliovirus replicon (FLuc) RNA has the capsid-coding sequence replaced by the luciferase gene. In the absence of replication, luciferase levels (in relative light units [RLU]) in HeLa cells transfected with replicon RNA are a measure of input poliovirus RNA translation. Translation alone was measured by transfecting cells with a mutant replicon, FLuc-3D^pol238A, possessing an inactive viral polymerase (▴). Normal poliovirus replication was measured by transfecting cells with wild-type FLuc (▪). Luciferase levels in the presence of 2.0 μg of BFA per ml are shown in cells transfected with wild-type FLuc (•) or mutant FLuc-3D^pol238A (♦). (C) Titration of BFA inhibition of poliovirus. HeLa cells were infected at an MOI of 0.001 and incubated with the indicated concentration of BFA (μg/ml). Virus was harvested, and titers were determined at 48 h postinfection.

FIG. 2.

Identification of BFA-resistant viruses. (A) Phenotype of BFA-resistant viruses. HeLa cells were infected at a low MOI (0.001 PFU/cell) and then incubated for 2 days in the absence or presence of 0.5 μg of BFA/ml. WT, wild-type poliovirus; −, control uninfected cells; bfa1, bfa2, bfa3, and bfa4, potential drug-resistant mutants tested. All viruses replicated and spread throughout the plate under normal growth conditions (no BFA), causing 100% cytopathic effect (CPE). Wild-type poliovirus did not cause CPE in the presence of 0.5 μg of BFA/ml. Candidate mutant bfa1 caused partial CPE in the presence of 0.5 μg of BFA/ml, since pinpoint plaques are visible. Candidate mutant bfa1 was later confirmed to have a single point mutation in the 2C gene at position 4361. Candidate mutants bfa3 and bfa4 caused 100% CPE in the presence of 0.5 μg of BFA/ml (also in 1.0 μg and 2.0 μg of BFA/ml [data not shown]). Candidate mutants bfa3 and bfa4 were later confirmed to both contain the double point mutations G4361A (in gene 2C) and C5190T (in gene 3A). Mutant bfa2 was not identified in the original screen but is instead the molecular-clone-derived Mo5190T. Mutant bfa2 exhibited significant drug resistance and caused >90% CPE in the presence of 0.5 μg of BFA/ml. (B) Location of BFA resistance (BFA^r) mutations in the poliovirus genome. Poliovirus is a positive-strand RNA virus with an ∼7,500-nucleotide genome. The full-length poliovirus genome is diagrammed on the bottom, with expanded versions of the 2C and 3A genes shown above it. 2C is 329 aa long and 3A is 87 aa long. One BFA^r mutation was found in replication protein 2C, a valine-to-isoleucine change at aa 80. A second BFA^r mutation was found in replication protein 3A, an alanine-to-valine change at aa 27. Known domains of 2C and 3A are indicated. 2C contains an NTP^ase motif and highly conserved motifs B and C (24), as well as a zinc finger (indicated by “Zn”) (36). 2C also contains at least one membrane-binding domain (mem) (19) and an RNA-binding domain (6, 39). 3A contains a 22-aa hydrophobic stretch (hydro) in its C terminus that is thought to play a role in membrane binding (48).

FIG. 3.

Kinetic analysis of BFA-resistant virus growth. HeLa cells were infected at an MOI of 5 with the virus strains indicated below in the absence (▪) or presence (•) of 2.0 μg of BFA/ml. Virus was harvested at the indicated time points, and then titers were determined by plaque assay. (A) Wild type (WT); (B) MoBFA^r-DB; (C) Mo4361A; (D) Mo5190T. Titers off the bottom of the scale are indicated as 10⁵ PFU.

FIG. 4.

Kinetic analysis of BFA-resistant replicons. (A) HeLa cells or (B) 293 cells were transfected with Fluc replicon RNAs corresponding to wild type (WT) or those carrying mutations that confer resistance to BFA (Mo4361A, Mo5190T, or MoBFA^r-DB). Transfections were performed in the absence (solid symbols) or presence (open symbols) of 2.0 μg of BFA/ml. Cells were harvested at the indicated time points, and the luciferase activity was determined as described in Materials and Methods. (C) Transfections were carried out in the presence of guanidinium HCl to determined luciferase production in the absence of RNA replication. Luciferase was determined at 1 and 6 h posttransfection.

FIG. 5.

Golgi phenotype of BFA-resistant virus. HeLa cells were infected at an MOI of 1 with either wild type (WT) or MoBFA^r-DB. Cells were fixed at 7 h postinfection and costained for poliovirus protein 2C (green) and a Golgi marker (GM130, red). The diffuse stain corresponds to Golgi complex dispersion as BFA disrupts the complex, as do both wild-type (WT) and MoBFA^r-DB poliovirus infections. Control stains demonstrating the specificity of the anti-poliovirus 2C antibody are also shown.

FIG. 6.

Subcellular localization of virus replication. HeLa cells were infected with wild-type poliovirus (WT) or MoBFA^r-DB for 7 h in the presence or absence of 2.0 μg of BFA/ml and then fixed and stained for poliovirus protein 2C (green) and ER-resident protein calnexin (red). Cells were imaged by using confocal deconvolution microscopy.