Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor

Abstract

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.

FIG. 1.

Construction procedure of a cosmid carrying the fusion gene of MPH upstream region and hPVR structural region.

FIG. 2.

Detection of transgene and hPVR in Tg mice. (A) Southern blot analysis of BamHI digests of the genomic DNA prepared from the livers of MPVRTg25-61 (lane 1), C57BL/6 (lane 2), and PVRTg21 (lane 3). Similar analysis was performed on the DNA of HeLa cells (lane 4) and chimera cosmid shown in Fig. 1 (lane 5). Experimental conditions are described in Materials and Methods. (B) Western blot analysis of hPVR in MPVRTg25-61 (lanes 1 to 4), PVRTg21 (lanes 5 to 8), and HeLa cells (lane 9). Membrane fractions of various tissues of MPVRTg25-61 and PVRTg21 were prepared, and hPVR in these fractions was detected as described in Materials and Methods.

FIG. 3.

Structures of viral genomes. Genome organizations of PV1 Mahoney (A), HCV genotype 1b (B), and the chimeric virus 2A-369 (C) are shown. Numbers in parentheses represent nucleotide numbers from the 5′ end of PV or HCV genome. The HCV IRES region, the 2A^pro cleavage site coding sequence, and restriction cleavage sites for SalI and MluI in the genome of the 2A-369 virus are shown at the bottom of the figure. The cloverleaf-like RNA structure of PV is located at the 5′ end of the 2A-369 genome.

FIG. 4.

Plaque size of 2A-369 virus. Plaques of Mahoney virus and 2A-369 virus on HeLa S3 cells are shown. HeLa cells were infected with viruses and incubated in medium containing 1% NCS and 1% agar at 36°C for 72 h. Infected cells were stained with 1% crystal violet in ethanol to visualize plaques.

FIG. 5.

Time course of virus titers in the liver after virus inoculation. The livers of MPVRTg25-61 mice (right panel) and C57BL/6 (non-Tg) mice (left panel) were inoculated with 10⁴ PFU of Mahoney virus (□) or 2A-369 virus (⧫). The virus titers in the liver at indicated times were measured as described in Materials and Methods and plotted. At each experimental condition, at least five mice were used.

FIG. 6.

Time course of virus titers in the brain after virus inoculation. MPVRTg25-61 mice (right panel) and C57BL/6 (non-Tg) mice (left panel) were intracerebrally inoculated with Mahoney virus (□) or 2A-369 virus (⧫). Virus titers in the brain at the indicated times were measured as described in Materials and Methods and plotted. At each experimental condition, at least three mice were used. “P” in the figure indicates a paralyzed mouse.

FIG. 7.

Mouse neurovirulence test. MPVRTg25-61 mice were intracerebrally inoculated with 10⁴ PFU of Mahoney virus (□) or 2A-369 virus (⧫). The infected mice were observed for paralysis and mortality every 24 h for up to 14 days.

FIG. 8.

PV antigens in the inoculated tissues. Into the brain (A, B, E, and F) or liver (C, D, G, and H) of MPVRTg25-61 (E to H) or C57BL/6 (non-Tg) (A to D), 10⁴ PFU of Mahoney virus (A, C, E, and G) or 2A-369 virus (B, D, F, and H) were inoculated. The tissue sections were prepared 96 hpi, and the frozen sections were immunostained as described in Materials and Methods. Red represents nucleic acids, and green represents PV antigens. A scale bar (10 μm) is indicated in panel H.