Article Figures & Data
Figures
- Figure 1.
Genomic organization of poliovirus and one-step growth curves for mono-crePV and dual-crePV. A, structure of the PV1(M), dual-crePV, mono-crePV, and A133Gmono-crePV genome. The single-stranded RNA is covalently linked to the viral-encoded protein VPg at the 5′-NTR. The 5′-NTR consists of two cis-acting domains, the cloverleaf and the IRES, which are separated by a spacer region. The IRES controls translation of the polyprotein (open box), consisting of a structural region (P1) and nonstructural regions (P2 and P3) specifying the replication proteins. Within the 2CATPase coding region, the cis replication element (cre) is indicated. The 3′-NTR contains a heteropolymeric region and is polyadenylated. RNA replication requires all three structural elements: cloverleaf, cre, and the 3′-NTR. The duplicated cre was inserted into the spacer between cloverleaf and IRES (dual-crePV). The native cre in 2CATPase was inactivated by mutation as indicated by an X (mono-crePV). A point mutation (A133G) was engineered into domain II of the 5′-NTR in mono-crePV (A133Gmono-crePV). B, one-step growth curves for mono-crePV and dual-crePV in HeLa cells (top) and SK-N-MC cells (bottom). Cells were infected at a MOI of 10 and incubated at 37°C or 39.5°C. The virus titer was determined by plaque assay on monolayers of HeLa cells as described in Materials and Methods.
- Figure 2.
One-step growth curves of polioviruses in different human and mouse neuroblastoma cells. Cells were infected as described in Fig. 1B with PV1(M) (▴), mono-crePV (•), and A133Gmono-crePV (▪). A, human SK-N-SH at 37°C and 39.5°C. B, human SH-SY5Y at 37°C and 39.5°C. C, human SK-N-MC at 37°C and 39.5°C. D, mouse Neuro-2aCD155 at 37°C and 39.5°C.
- Figure 3.
Schematic presentation of A133Gmono-crePV therapy on Neuro-2aCD155 tumors in CD155 tgA/J mice with established immunity against poliovirus. Stage I, CD155 tgA/J mice were immunized i.p. with live mono-crePV (1 × 108 pfu) thrice with an interval of 1 wk. Stage II, 21 d after the last immunization, 1 × 107 Neuro-2aCD155 cells were transplanted s.c. in the animals. Stage III, intratumoral treatment of the s.c. tumor with A133Gmono-crePV (1 × 108 pfu) or PBS at days 0, 2, 4, and 6. Stage IV, mice that survived without signs of tumors for 6 mo were rechallenged with Neuro-2aCD155 cells (1 × 107 cells) in the contralateral flank.
- Figure 4.
Abolition of established neuroblastoma implants in CD155 tgA/J mice with A133Gmono-crePV. Neuro-2aCD155 was introduced as a tumor implant s.c. in CD155 tgA/J mice, and multiple intratumoral injections of 1 × 108 pfu of A133Gmono-crePV (solid arrows) were given when the tumor volume reached ∼170 mm3 (day 0). ○, control animals were given PBS; •, virus-treated animals showed regression of the tumors. One of the 12 virus-treated animals was sacrificed at day 8 (dotted arrow) for tumor analysis. Two of the 11 mice observed long term developed tumors as indicated.
- Figure 5.
Expression of CD155 in tumor cells. Whole-cell lysates of tumors from mice untreated with A133Gmono-crePV (lanes 1–4), and tumor from the mouse that was treated with A133Gmono-crePV and sacrificed at day 8 ( Fig. 4, dotted arrow; lane 5) and tumors from two mice with recurrent tumors (lanes 6 and 7) were resolved on a 10% SDS-PAGE gel followed by Western blotting with anti-CD155 antibody NAEZ-8 (top) or anti-actin antibody (bottom).
Tables
- Table 1.
Neuropathogenicity of wt poliovirus PV(M), dual-crePV, mono-crePV, and A133Gmono-crePV
Virus PLD50 (pfu) * in PVR transgenic mice PLD50 (pfu) * in PVR transgenic A/J mice wt PV1(M) 101.8 102.0 Dual-crePV >107.0 >107.0 Mono-crePV >107.0 >107.0 A133Gmono-crePV 104.5 104.8 -
↵* Defined as the amount of virus that causes paralysis or death in 50% of PVR transgenic mice or PVR transgenic A/J mice after i.c. inoculation.
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Volume 67, Issue 6
