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A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system
1Department of Microbiology, Institute of Medical Science, The University of Tokyo, Japan.
Abstract:
Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.
Insights
Poliovirus mutations in the internal ribosomal entry site (IRES) stem-loop II (SLII) reduced viral replication in mouse cells and neurovirulence. Revertants restored these functions, indicating nucleotide 107 is crucial for IRES activity in mouse cells.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- The internal ribosomal entry site (IRES) is critical for cap-independent translation initiation of poliovirus.
- The stem-loop II (SLII) structure within the IRES is essential for viral replication and pathogenesis.
Purpose of the Study:
- To investigate the role of SLII structure mutations in poliovirus replication and neurovirulence.
- To identify genetic determinants of IRES activity in different cell types.
Main Methods:
- Generation of poliovirus mutants with alterations in the SLII region.
- Assessment of viral replication in human (HeLa) and mouse (TgSVA) cells.
- Evaluation of neurovirulence in a mouse model.
- Isolation and characterization of naturally occurring revertants.
- Analysis of IRES activity using a cell-free translation system.
Main Results:
- SLII mutants replicated efficiently in HeLa cells but poorly in TgSVA cells, with attenuated neurovirulence.
- Revertants regained efficient replication in TgSVA cells and restored neurovirulence.
- Revertants exhibited enhanced IRES activity in TgSVA cell extracts.
- Mutations at nucleotide (nt) 107 and within nt 120-161 were identified as key revertant sites.
Conclusions:
- Nucleotide 107 of poliovirus RNA plays a critical role in IRES-mediated translation in mouse cells.
- Specific mutations within the SLII structure modulate IRES activity and affect poliovirus host range and virulence.
- The findings provide insights into the structural requirements for IRES function and poliovirus adaptation.