Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3' nontranslated region

Abstract

We have proposed a cancer treatment modality based on poliovirus chimeras replicating under the translational control of an internal ribosomal entry site (IRES) derived from human rhinovirus type 2. Insertion of the heterologous IRES causes a neuron-specific propagation deficit and eliminates neurovirulence inherent in poliovirus without affecting viral growth in cells derived from malignant gliomas. We now report the elucidation of a molecular mechanism responsible for the cell type-specific defect mediated by the rhinovirus IRES. Rhinovirus IRES function in neuronal cell types depends on specific structural elements within the 3' non-translated region of the viral genome. Our observations suggest long-range interactions between the IRES and the 3' terminus that control IRES-mediated gene expression and virus propagation.

Fig. 1.

Genetic structure (Left), growth properties (Center), and viral gene expression in Sk-N-Mc neuroblastoma cells (Right) of PV (A), CBV3 (C), and their derivatives PV-RIPO (B) and CBV-RICO (D), containing the HRV2 IRES (indicated by a gray box; B and D). One-step growth curves were established in HeLa (solid diamonds) and Sk-N-Mc neuroblastoma (open squares) cells. The kinetics of viral gene expression in Sk-N-Mc cells were assayed through Western blot detection of polioviral gene products 2BC and 2C (A and B) and coxsackieviral proteins 3AB and 3A (C and D) at the indicated intervals postinfection (p.i.).

Fig. 2.

Sequence and predicted secondary structure of the 3′ NTRs of CBV3 (A), PV1(S) (B), HRV2 (E), CBV3 SLD Z (F), and engineered variants (C, D, and G). The structure and position of the three distinct SLDs in the CBV3 3′ NTR are indicated (34). The PV1(S) 3′ NTR was altered by insertion of the CBV3 Z domain (boxed in gray) [PV1(S) + Z; C]. The CBV3 3′ NTR was modified by deletion of the Z domain (CBV3 ΔZ; D) or exchange thereof with the entire 3′ NTR of HRV2 (CBV3 ΔZ + HRV2; G).

Fig. 3.

Replication profiles of CBV-RICO (shown on top) containing chimeric 3′ NTRs in HeLa (solid diamonds) and Sk-N-Mc neuroblastoma (open squares) cells. We analyzed constructs containing the cognate CBV3 3′ NTR (A), the PV1(S) 3′ NTR (B), the PV1(S) 3′ NTR containing the SLD Z (C), the CBV3 3′ NTR with the SLD Z deleted (D), the HRV2 3′ NTR (E), or the HRV2 3′ NTR replacing the CBV3 SLD Z (F). (Right) Western blot analyses of the kinetics of viral gene expression in Sk-N-Mc neuroblastoma cells for the corresponding constructs.

Fig. 4.

Effect of 3′ NTR manipulation on IRES activity of WT CBV3 (on top). Particle propagation and viral gene expression of WT CBV3 (A) and CBV3 ΔZ (B) in HeLa (solid diamonds) and Sk-N-Mc neuroblastoma (open squares) cells.

Fig. 5.

(A) Genetic structure of two HRV2 IRES (boxed in gray) driven rLuc reporter constructs with a full-length CBV3 (Upper) or CBV3 ΔZ3′ NTR (Lower), respectively. (B) Stability of reporter RNAs containing the complete CBV3 (Upper) or a CBV3 ΔZ 3′ NTR (Lower) in Sk-N-MC cells at different intervals posttransfection. Negative control (-) represents “mock” transfection without transfection reagent. (C) The kinetics of rLuc expression in Sk-N-Mc (gray) and HeLa (black) cells transfected with in vitro transcript RNA containing the CBV3 ΔZ3′ NTR (hatched bars) or the full-length CBV3 3′ NTR (solid bars). The data represent the averages of three independent experiments.