Age-dependent poliovirus replication in the mouse central nervous system is determined by internal ribosome entry site-mediated translation

Abstract

Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.

FIG. 1.

IRES-mediated translation in cultured cells. (A) Schematic of bicistronic reporter DNA encoded by reporter plasmids and recombinant adenoviruses. The arrow indicates the transcription initiation site of the murine cytomegalovirus immediate early promoter. Firefly luciferase and Renilla luciferase have independent translation initiation and termination codons. SV40 An, simian virus 40 polyadenylation signal. The bicistronic reporter DNA is included in bicistronic plasmids used for DNA transformation of cultured cells and in recombinant adenoviruses used for assay of IRES-mediated initiation in mouse organs. (B) Translation mediated by the IRES of poliovirus (filled bars) or HRV2 (open bars) in the indicated cell lines transformed with a plasmid encoding the bicistronic mRNA. To control for transformation efficiency, Renilla luciferase expression was normalized to firefly luciferase expression. IRES activity (y axis) is the relative increase (n-fold) in Renilla luciferase translation compared with results obtained using a plasmid that does not have an IRES. Data points are the means of three transformations, and error bars indicate standard deviations. (C) Genome structure of poliovirus type 1 strain Mahoney (P1/M) and recombinant strain P1/HRV2. Poliovirus polyprotein (open box), predicted AUG initiation codons, and IRES are indicated. HRV2 sequence is shaded. (D) Single-cycle replication of poliovirus type 1 strain Mahoney (filled symbols) and P1/HRV2 (open symbols) in HeLa (square), Vero (circle), SH-SY5Y (triangle), and L20B (diamond) cell lines. Data points are the means of two infections.

FIG. 2.

Poliovirus replication in murine CNS. Virus titers in spinal cord (circle) and brain (triangle) in TgPVR (filled symbols) or nontransgenic (open symbols) mice were determined at the indicated days postinfection. (A) Virus titers in adult TgPVR mice infected with poliovirus type 1 strain Mahoney. (B) Virus titers in adult TgPVR mice infected with P1/HRV2. (C) Virus titers in neonatal TgPVR mice infected with poliovirus type 1 strain Mahoney. (D) Virus titers in neonatal TgPVR and nontransgenic mice infected with P1/HRV2. Data points are the geometric means of titers in organs of at least three mice.

FIG. 3.

IRES-mediated translation in brain and spinal cord in adult and neonatal mice. Animals were infected with recombinant adenoviruses that produce bicistronic mRNA with the IRES of poliovirus (PV) or HRV2 (Fig. 1A). To control for infection efficiency, Renilla luciferase expression was normalized to firefly luciferase expression. IRES activity (y axis) is the relative increase (n-fold) in Renilla luciferase translation compared with results obtained using an adenovirus vector that does not have an IRES. Data points are the means of results for five mice, and error bars indicate standard deviations.

FIG. 4.

Virulence and replication of P1/HRV2 recovered from serial infections of TgPVR mice. (A) P1/HRV2 virulence. Percentages of mice that are not paralyzed after infection with P1/HRV2 (square), second-passage infection (triangle), and third-passage infection (circle). (B) Titers of third-passage P1/HRV2 infection in murine brain and spinal cord. Virus titers in spinal cord (circle) and brain (triangle) in TgPVR (filled symbols) or nontransgenic (open symbols) mice were determined at the indicated days postinfection. Data points are the geometric means of titers in organs of at least three mice.

FIG. 5.

IRES-mediated translation in kidney and liver in adult and neonatal mice infected with recombinant adenoviruses. To control for infection efficiency, Renilla luciferase expression was normalized to firefly luciferase expression. IRES activity (y axis) is the relative increase (n-fold) in Renilla luciferase translation compared with results obtained using an adenovirus vector that does not have an IRES. Data points are the means of results for five mice, and error bars indicate standard deviations.