Poliovirus 3Dpol polymerase region is essential for cleavage of poliovirus 3AB in vivo

Figures

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  Fig 1. Schematic view of PV replicon mutants used in this study. Introduced amino acid substitutions or deletions are shown. These replicons encode firefly luciferase as a reporter for the replication and can replicate only in the presence of the 3CDpro provided in trans. For the analysis of cis 3AB cleavage, mutations examined in (A) and (D) were introduced in a PV polyprotein (AG-PV-2B2CP3) expression vector. The positions of the nucleotides are of PV1(Mahoney) genome (GenBank accession number V01149.1). (A) The 3C-R13N, 3C-K82N, and 3C-R84S amino acid substitutions, which abolish the binding to viral RNA and phospholipids, were introduced to the PV(3C/D[A/G]) replicon. The PV(3C/D[A/G]) replicon has a disrupted cleavage site between the 3Cpro and 3Dpol. (B) Deletions were introduced in the 3Dpol-coding region of the PV(3C-TGA) replicon. The PV(3C-TGA) replicon has a termination codon (TGA) immediately after the 3Cpro-coding region. (C) In-frame deletions were introduced in the 3Dpol coding region of the PV(3C/D[A/G]) replicon. For the mutants with deletions immediately after the 3Cpro-coding region, the cleavage sites between 3Cpro and 3Dpol retain the WT sequence (Q/G) to minimize…

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  Fig 2. Experimental design for trans rescue of replication of PV replicons and infection of PVpv. (A) Schematic view of the trans-rescue experiment. For the replication assay, PV 3CDpro was expressed in the presence of DOX (1 mg/mL) and GC376 (100 μM) at 37°C for 17 h. The RNA transcripts of each PV replicon mutant with a firefly luciferase reporter were transfected into the cells (4 × 104 cells) in the absence of DOX and GC376 and in the presence or absence of GuHCl (a 2C inhibitor, 2 mM). The luciferase signals in the cells were analyzed at 7 h p.t. For the infectivity assay, PV 3CDpro was expressed in the presence of DOX (1 mg/mL) and in the presence or absence of GuHCl (2 mM) at 37°C for 5 h. PVpv mutants with a firefly luciferase reporter were then added to the cells (8 × 103 cells). The luciferase signals in the cells were analyzed at 17 h p.i. (B) Replication and infectivity of PV mutants with amino acid substitutions that abolish the binding to viral RNA and phospholipids. The luciferase signals measured at 7 h p.t. of RNA or 17 h p.i. with PVpv are shown. Replication levels are the ratio of the luciferase activities observed in the absence of GuHCl to those observed in the…

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  Fig 3. Replication and infectivity of PV mutants with deletions in the 3Dpol-coding region. The luciferase signals measured at 7 h p.t. of RNA or 17 h p.i. with PVpv are shown. Replication levels are the ratio of the luciferase activities observed in the absence of GuHCl to those observed in the presence of GuHCl. Identified essential genomic regions are highlighted in a schematic view of the 3Dpol-coding region. The data represent the mean and standard deviation of three independent experiments with two biological replicates.

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  Fig 4. Replication and infectivity of PV mutants with in-frame deletions in the 3Dpol-coding region. The luciferase signals measured at 7 h p.t. of RNA or 17 h p.i. with PVpv are shown. Replication levels are the ratio of the luciferase activities observed in the absence of GuHCl to those observed in the presence of GuHCl. Identified essential genomic regions are highlighted in a schematic view of the 3Dpol-coding region. The data represent the mean and standard deviation of three independent experiments with two biological replicates.

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  Fig 5. Replication and infectivity of PV mutants with termination codons (TGATAA) in the 3Dpol-coding region. The luciferase signals measured at 7 h p.t. of RNA or 17 h p.i. with PVpv are shown. Replication levels are the ratio of the luciferase activities observed in the absence of GuHCl to those observed in the presence of GuHCl. Identified essential coding regions are highlighted in a schematic view of the 3Dpol-coding region. The data represent the mean and standard deviation of three independent experiments with two biological replicates.

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  Fig 6. Effect of host factors and mutations on cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells expressing AG-PV-2B2CP3 proteins with the indicated amino acid substitutions or without the substitutions (i.e., wild type, WT). The cells were treated with DOX (1 mg/L) for 17 h in the presence or absence of a 3C protease inhibitor (GC376, 100 μM), a PI4KB inhibitor (T-00127-HEV1, 10 μM), or OSBP inhibitors (T-00127-HEV2, -HEV3, and -HEV4, 10 μM). Viral proteins were detected by anti-3A or -2C antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of three independent experiments with two to three biological replicates. NS, not significant.

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  Fig 7. Effect of insertion of premature termination codons (TGATAA) in the 3Dpol-coding region on cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells expressing AG-PV-2B2CP3 proteins with the indicated length of the 3Dpol peptides or without the insertion (i.e., WT). The cells were treated with DOX (1 mg/L) for 17 h. The PV-2B2CP3(WT)-expressing cells were treated with DOX in the presence or absence of a 3C protease inhibitor (GC376, 100 μM) or a PI4KB inhibitor (T-00127-HEV1, 10 μM). Viral proteins were detected by anti-3A or -2C antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of two independent experiments with two to three biological replicates. NS, not significant.

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  Fig 8. The effect of 3CDpro and 3Dpol provided in trans on the cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells expressing AG-PV-2B2CP3 proteins that have premature termination codons (TGATAA) in the 3Dpol-coding region, resulting in partial 3Dpol peptides as indicated (70 or 202 aa of the N-terminal peptides), along with AG-3CD(WT) or AG-3D(WT) proteins. The cells were treated with DOX (1 mg/L) for 17 h. Viral proteins were detected by anti-3A, -2C, or -3D antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of three independent experiments with two biological replicates. NS, not significant.

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  Fig 9. Effect of 3CDpro activity provided in trans on cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells co-expressing AG-PV-2B2CP3(3D-0 aa) and AG-3CD variants (WT or C147A) or AG-3D(WT). The cells were treated with DOX (1 mg/L) for 17 h. Viral proteins were detected by anti-2C, -3A, or -3D antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of three independent experiments with two to three biological replicates. NS, not significant.

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  Fig 10. Effect of aa substitution in the 3B region of a PV polyprotein on the cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells expressing AG-PV-2B2CP3 variants. The cells were treated with DOX (1 mg/L) for 17 h. Viral proteins were detected by anti-2C, -3A, or -3D antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of three independent experiments with two biological replicates. NS, not significant.

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  Fig 11. Effect of 3Dpol variants provided in trans on cleavage of 3AB in vivo. Western blot analysis of viral proteins in the cells co-expressing AG-PV-2B2CP3(3D-0 aa) and AG-3D variants. The cells were treated with DOX (1 mg/L) for 17 h. Viral proteins were detected by anti-2C, -3A, or -3D antibodies. The percentage of cleaved 3AB is highlighted in color. The data are representative of two independent experiments with two biological replicates. NS, not significant.