2A Protease Is Not a Prerequisite for Poliovirus Replication▿ †
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FIG. 1.PV genomic constructions. (A) Construction of PV and recombinant PVs. (B) Junctional amino acid sequences of pOM-EGFPΔP1, pOM-EGFPΔP1Δ2A, pOME, and pOMEΔ2A. The nucleic acid sequence (CCCGGG) recognized by SmaI, corresponding to the amino acid sequence Pro Gly, is shown by underlines. The inserted EMCV IRES are shown by the striped horizontal bars. Closed triangles indicate the sites of cleavage by 2Apro, and open triangles indicate the sites of cleavage by 3Cpro or 3CDpro. Nucleotide numbers of the deleted fragments are shown at the deletion positions.
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FIG. 2.Neutralization assay of PV1(M)OM, OME, OMEΔ2A, and Leon with anti-PV type 1 or type 2 monoclonal antibody. HeLa cells were mock infected (e, j, and o) or infected with PV1(M)OM (a, f, and k), OME (b, g, and l), OMEΔ2A (c, h, and m), or Leon (d, i, and n) in the absence (k to o) or presence of an anti-PV type 1 Mahoney monoclonal antibody (7m008, a to e) or anti-PV type 2 Leon monoclonal antibody (Thai p34-120, f to j). Eighteen hours after the infection, the CPE was observed under a microscope. Bar, 50 μm.
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FIG. 3.Plaque phenotypes of PV1(M)OM, OME, and OMEΔ2A. AGMK cells were infected with PV1(M)OM (a), OME (b), and OMEΔ2A (c). The cells were fixed 2 days after the infection with PV1(M)OM, and OME and 4 days after the infection with OMEΔ2A.
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FIG. 4.CPE in cells infected with or without PV1(M)OM, OME, and OMEΔ2A. (A) HeLa cells were mock infected (d and f) or infected with PV1(M)OM (a), OME (b), or OMEΔ2A (c and e) at an MOI of 10. Eight (a to d) or 24 h (e and f) after the infection (h.a.i.), morphological changes were observed under a microscope. Bar, 50 μm. (B) Percentages of CPE expression among all cells. (C) Rates of membrane blebbing among CPE-expressing cells. Average rates in two to three microscopic fields in one experiment were plotted. The rates were examined 0, 2, 4, 6, 8, 10, 12, and 24 h after the infection. Blue lines with squares indicate results for PV1(M)OM, pink lines with triangles indicate results for OME, and orange lines with circles indicate results for OMEΔ2A.
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FIG. 5.Apoptosis in cells infected with OMEΔ2A, detected by the TUNEL assay. HeLa cells were mock infected (d, h, and l), infected with PV1(M)OM (a, e, and i) or OMEΔ2A (b, f, and j), or treated without (d, h, and l) or with (c, g, and k) actinomycin D (Act D). Seven hours after the infection with PV1(M)OM, 8 h after the treatment with actinomycin D, and 21 h after the infection with OMEΔ2A, the cells were TUNEL stained. TUNEL-positive cells were fluorescent. Fluorescence (FL) images are shown at the top (a to d), bright-field (BF) images are shown in the middle (e to h), and merged (FL+BF) images are shown at the bottom (i to l). Bar, 50 μm.
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FIG. 6.Slot blot analysis of cells after transfection with or without synthesized viral RNA or after infection with or without viruses. (A) HeLa cells were transfected with or without (Mock) RNAs of pOM1, pOMΔ0.8, pOMΔ1.8, pOMΔP1, pOMΔP1Δ2A, or pOMΔP1Δ2AΔ2B, and cell lysates were collected 2 and 8 h later. The RNAs were detected with probes for the PV IRES sequence or GAPDH sequence. (B) A similar experiment was performed using the RNAs of pOM1, pOMΔP1, pOMΔP1Δ2A, pOMΔP1Δ2AΔ2B, pOM-EGFPΔP1, and pOM-EGFPΔP1Δ2A. (C) HeLa cells were infected with or without (Mock) PV1(M)OM, OMΔ0.8, OMΔ1.8, OMΔP1, or OMΔP1Δ2A, and cell lysates were collected 2 and 8 h later. The RNAs were detected with probes for the PV IRES sequence or GAPDH sequence. Relative amounts of PV RNA corrected to the amount of GAPDH RNA are shown.
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FIG. 7.CPE in the cells infected with PV1(M)OM, OMΔ0.8, OMΔ1.8, OMΔP1, and OMΔP1Δ2A. (A) HeLa cells were mock infected (f) or infected with PV1(M)OM (a), OMΔ0.8 (b), OMΔ1.8 (c), OMΔP1 (d), or OMΔP1Δ2A (e) at an MOI of 1,000, and cell morphology was observed 24 h later under a microscope. Bar, 50 μm. (B) Percentages of CPE expression among all cells. (C) Rates of membrane blebbing among CPE-expressing cells. Average rates in two to four microscopic fields in one experiment were plotted. The rates were examined at 2, 6, 10, and 24 h after the infection. Blue lines with filled squares indicate results for PV1(M)OM, green lines with triangles indicate results for OMΔ0.8, violet lines with inverted triangles indicate results for OMΔ1.8, orange lines with rhombuses indicate results for OMΔP1, pink lines with circles indicate results for OMΔP1Δ2A, and moss-green lines with open squares indicate results for mock-infected cells.
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FIG. 8.P1-null PV vector with or without the 2Apro coding region expresses EGFP. HeLa cells were mock infected (c, f, and i) or infected with OM-EGFPΔP1 (a, d, and g) or OM-EGFPΔP1Δ2A (b, e, and h) and observed 24 h later under a fluorescence microscope. Upper panels show fluorescence (FL) images (a to c), panels in the middle show bright-field (BF) images (d to f), and lower panels show merged (FL+BF) images (g to i). Bar, 100 μm.
- American Society for Microbiology
