Characterization of Hepatitis C Virus IRES Quasispecies – From the Individual to the Pool

Figures

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  FIGURE 1. Graphical abstract summarizing the experimental workflow.

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  FIGURE 2. Distribution of mutations across the HCV IRES from patient samples with respect to sustained response and no antiviral response to HCV treatment. (A) Sustained responders (SRs), localization of 53 individual mutations found in the literature. (B) Non-responders (NRs), localization of 102 individual mutations found in the literature (Yamamoto et al., 1997; Lu et al., 1999; Saiz et al., 1999; Soler et al., 2002; Thelu et al., 2004; Yasmeen et al., 2006; Ogata et al., 2008; El Awady et al., 2009). Total numbers of mutations (muts.) found within the corresponding domains in the SR and NR groups are depicted.

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  FIGURE 3. Results documenting a workflow of the analysis of individual HCV IRES clones. Different stages of analysis of some clones derived from Patient 4 (P4) are depicted as follows. (A) Denaturing gradient gel electrophoresis of selected samples obtained from P4. Clones marked with asterisks (∗) showed identical sequences and similar migration pattern in DGGE with a majority of the clones analyzed from the P4 (wild type). Clones shown in italics were not sequenced. The remaining clones were sequenced, and some were analyzed by flow cytometry. (B) Flow cytometry analyses of IRES activity of selected HCV IRES clones in a bicistronic pRG system transfected into CCL-13 cells; Y and X axes represent red (DsRed2) and green (EGFP) fluorescence, respectively. Lines inside the dot plots indicate gating of the corresponding EGFP and DsRed2 cell populations. B/A represents non-transfected cells. B/B represents a flow cytometry analysis of cells expressing only DsRed2 from empty pRG plasmid and serving as a negative control to set up a baseline of the experiment. B/C represents a positive control cells expressing pRG-refIRES bicistronic plasmid containing standard HCV IRES (subtype 1a, from nt 1 to…

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  FIGURE 4. Analysis of HCV IRES activity of selected pRG-IRES clones in human CCL-13 cells using flow cytometry. Columns represent mean EGFP fluorescence intensity per cell, which was calculated from all CCL-13 cells transfected with the respective pRG-IRES, in other words, from all cells expressing DsRed2 above the threshold level in the sample. All the values were normalized to refIRES activity, which was set to 100%. refIRES is the standard HCV IRES (subtype 1a, from nt 1 to nt 385 of the original sequence; GI:329737) cloned into the pRG plasmid (Vopalensky et al., 2008). Empty pRG plasmid serves as a negative control and represents the baseline for the analysis. HCV IRES samples from Patient 4 (P4) are the same as those depicted in Figure 3 and Table 3. At least seven independent biological replicates (independent pRG-IRES transfections) for each of the pRG-IRES plasmids were measured. Significant differences are marked with double asterisks (p ≤ 0.01) or an asterisk (p ≤ 0.05).

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  FIGURE 5. Flow cytometry analyses of the collective activities of HCV IRES libraries transfected into CCL-13 cells. (A) Untransfected CCL-13 cells only. (B) Cells transfected with the empty pRG plasmid. (A,B) These experiments were used to set up gates for the subsequent analysis as depicted by vertical and horizontal lines. (C) Cells transfected with the pRG plasmid containing the reference HCV IRES (subtype 1a, from nt 1 to nt 385 of the original sequence; GI:329737). (D) Patient 4 – cells transfected with pRG-HCV IRES sc library. (E) Patient 7 – cells transfected with pRG-HCV IRES sc library. (F) Patient 9 – cells transfected with pRG-HCV IRES sc library. (G) Patient 4 – cells transfected with pRG-HCV IRES wash library. (H) Patient 7 – cells transfected with pRG-HCV IRES wash library. (I) Patient 9 – cells transfected with pRG-HCV-IRES wash library. Y and X axes represent red and green fluorescence, respectively. Lines inside the dot plots indicate gating of the corresponding EGFP and DsRed2 cell populations. (D–F) Each sample consists of approximately 150 positive colonies. (J) Seven independent biological replicates (independent transfections) of the samples depicted in A–I. All…