Quasispecies Changes with Distinctive Point Mutations in the Hepatitis C Virus Internal Ribosome Entry Site (IRES) Derived from PBMCs and Plasma

Figures

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  Figure 1. Nucleotide differences detected in 5'UTR sequences derived from patient PBMCs and plasma. Nucleotide changes in PBMC-derived IRES sequences (n=225) are indicated in blue while nucleotide changes in plasma-derived IRES sequences (n=225) are indicated in red. Similarities in nucleotide frequency variation between PBMC and plasma-derived samples are represented in green dots while differences are represented in yellow dots. Structural domains are indicated in Roman letters (I-IV). The start codon AUG is highlighted in stem-loop domain IV. Substitution sites are indicated as squares and arrows indicate mutations described in the text. Nucleotide sequence and putative secondary RNA structure of the 5'UTR [nt. 1-383 genotype 1b (GenBank, AJ238799.1)] was adapted from Honda et al. [24].

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  Figure 2. Entropy differences between the PBMC and plasma. Entropy differences between sequences (nt 100 to 300 of the HCV IRES) derived from PBMCs (n=225) and plasma (n=225). Each bar represents the entropy difference at a single nucleotide position. Significant differences between the entropy of PBMC and plasma are indicated by orange bars while no significant differences are indicated by blue bars. The results were generated using a Perl script and the figure was produced in R software programming language.

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  Figure 3. Distribution of nucleotide combinations at positions 107, 204, and 243 in PBMC- and plasma-derived IRES samples in different patients. Data represent the frequencies in percent of all possible nucleotide combinations at positions 107, 204, and 243 detected in PBMC- and plasma-derived HCV IRES samples from different patients.

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  Figure 4. Phylogenetic tree analysis of all PBMC- and plasma-derived HCV IRES sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (-1129.7196) is shown. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 450 nucleotide sequences. PBMC-derived HCV IRES nucleotide sequences (n=225; coloured in blue) and plasma-derived HCV IRES nucleotide sequences (n=225; coloured in red) (a). HCV IRESes with the triple adenine variant (A-A-A) in PBMC (n=36; blue triangles) and in plasma (n=26; red triangles) (b). All positions containing gaps and missing data were eliminated. There were a total of 200 positions (nt 100 to 300) in the final dataset. The bootstrap values along the branches indicate percent confidence of branches. Bootstrap values greater than 70% are shown. The scale bar corresponds to 0.01 substitutions per site.…

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  Figure 5. The effect of single point mutations on the thermodynamic stability of the relevant IRES domains. The thermodynamic stability of the relevant domains was calculated using the RNAeval program with RNA free energy parameters and ΔG set at 37°C. Blue cells indicate mutations detected only in PBMC-derived IRESes while red cells indicate mutations detected only in plasma-derived IRESes.