Effect of mutational increase on the recognition of RNA structural motifs in 5’ genomic region of the hepatitis C virus by biochemical factors.
Samuel Prieto-Vega (1), Celia Perales (2,3), Esteban Domingo (2,3), Sunnie Thompson (4) and Jordi Gómez (1,3).
1: IPBLN Granada, 2:CBMSO Madrid, 3: CIBERehd, 4: UAB Alabama.
The 5' region of the genome of the hepatitis C virus (HCV) RNA is very rich in structural elements that can be identified in vitro by biochemical and biophysical factors, such as for example, by interaction with the hepatic micro RNA miR-122 and the ribosomal 40S subunit, or by sensitivity to RNases III and P, and to UV light (254nm). These motifs involve regions of primary, secondary and tertiary structure of the RNA.
The primary objective was to assess the effect of the mutational increase in populations of RNA molecules in the region 1-570 of the HCV genome, in the identification of structural motifs by the factors outlined above. A protocol of mutagenic PCR and in vitro transcription was used to generate mutated RNA populations. The second objective was to evaluate the number of mutations that accumulate in this region of the viral RNA before reaching error catastrophe threshold in a viral culture in the presence of ribavirin.
The effect of the incorporation of mutations in populations differs depending on the structural levels and on the individual structural motifs. They are especially detrimental in recognition sequences of miR 122 (primary structure), the tRNA-like structure (tertiary structure) for RNase P, and the secondary structure that determines a conformational change in 1-570 RNA for RNase III. The mutational effect on the structure of 1-570 RNA was also analysed in native gels and with single and double chain RNases, RNases T1 and V1, respectively. The conformational dispersion gradually increases with the number of mutations; however, resistance to RNases shows a more complex behaviour for which we as yet have no explanation. Overall, the experimental analysis of the sensitivity of different structural motifs in the context of a long RNA modifies the concept that was established theoretically with isolated structural motifs, for example, the robustness of the secondary structure to mutation being clearly depending on the RNA context.
The mutational load at which 50% loss of identification of each of the structures is reached is lower than the value that is achieved with treatment of HCV with ribavirin in cell culture, but overall, the effect of the increase of mutation on all structural motifs makes plausible the hypothesis that the functions carrying these structures could be sensitive to therapeutic strategies based on error catastrophe.
1: IPBLN Granada, 2:CBMSO Madrid, 3: CIBERehd, 4: UAB Alabama.
The 5' region of the genome of the hepatitis C virus (HCV) RNA is very rich in structural elements that can be identified in vitro by biochemical and biophysical factors, such as for example, by interaction with the hepatic micro RNA miR-122 and the ribosomal 40S subunit, or by sensitivity to RNases III and P, and to UV light (254nm). These motifs involve regions of primary, secondary and tertiary structure of the RNA.
The primary objective was to assess the effect of the mutational increase in populations of RNA molecules in the region 1-570 of the HCV genome, in the identification of structural motifs by the factors outlined above. A protocol of mutagenic PCR and in vitro transcription was used to generate mutated RNA populations. The second objective was to evaluate the number of mutations that accumulate in this region of the viral RNA before reaching error catastrophe threshold in a viral culture in the presence of ribavirin.
The effect of the incorporation of mutations in populations differs depending on the structural levels and on the individual structural motifs. They are especially detrimental in recognition sequences of miR 122 (primary structure), the tRNA-like structure (tertiary structure) for RNase P, and the secondary structure that determines a conformational change in 1-570 RNA for RNase III. The mutational effect on the structure of 1-570 RNA was also analysed in native gels and with single and double chain RNases, RNases T1 and V1, respectively. The conformational dispersion gradually increases with the number of mutations; however, resistance to RNases shows a more complex behaviour for which we as yet have no explanation. Overall, the experimental analysis of the sensitivity of different structural motifs in the context of a long RNA modifies the concept that was established theoretically with isolated structural motifs, for example, the robustness of the secondary structure to mutation being clearly depending on the RNA context.
The mutational load at which 50% loss of identification of each of the structures is reached is lower than the value that is achieved with treatment of HCV with ribavirin in cell culture, but overall, the effect of the increase of mutation on all structural motifs makes plausible the hypothesis that the functions carrying these structures could be sensitive to therapeutic strategies based on error catastrophe.