Figure 6.
Figure 6. Mutations in envelope region E2425–483 mediate enhanced viral entry and escape from neutralizing antibodies. To map envelope regions mediating enhanced entry and viral escape, we exchanged four regions spanning C-terminal E1 and N-terminal E2, E1, E2- HVR1, and E2-HVR2 of the envelope glycoproteins of selected variant VL and nonselected variant VC of patient P01 (see Figs. 2–4). These regions include aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively. (A) Deduced amino acid sequences of the exchanged region between P01VC (black) and P01VL (blue). Amino acid changes are indicated in red bold letters. (B) Construction of recombinant chimeric HCVpps P01VCVL221–483 and P01VLVC221–483, P01VCVL-E1 and P01VLVC-E1, P01VCVL-HVR1 and P01VLVC-HVR1, and P01VCVL-HVR2 and P01VLVC-HVR2 by exchanging E1E2 envelope domain aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively, of nonselected variant VC (patient 01) depicted in white and escape isolate VL (patient 01) depicted in blue (see Figs. 2–4). HVR1 and HVR2 are shown in orange, and CD81 binding domains (CD81 BD) are shown in green. Positions of E2 epitopes I and II are indicated (Zhang et al., 2007, 2009). The number of mutations within each region is shown. (C) Viral entry of HCVpps containing chimeric envelope proteins in Huh7 cells. HCVpps were incubated with Huh7 cells, and infection was analyzed as described in Fig. 3. Results are expressed in relative light units (RLU) plotted in a logarithmic scale. The threshold for a detectable infection is 3 × 103 RLU and was determined as described in Fig. 3. (D) Neutralization of HCVpps by autologous pretransplant serum was performed as described in Fig. 4. End point dilution titers are indicated for each variant. Dashed lines indicate the threshold for a positive neutralization titer corresponding to 1:40. Calculation of neutralization and determination of threshold titers are described in Materials and methods. Chimeric HCVpps are depicted in dashed blue. Statistically significant differences (repeated measures ANOVA) in HCVpp entry or neutralization between VC and VL wild-type and mutant variants are indicated by asterisks (**, P < 0.001). Ctrl, negative control; MT, mutation.

Mutations in envelope region E2425–483 mediate enhanced viral entry and escape from neutralizing antibodies. To map envelope regions mediating enhanced entry and viral escape, we exchanged four regions spanning C-terminal E1 and N-terminal E2, E1, E2- HVR1, and E2-HVR2 of the envelope glycoproteins of selected variant VL and nonselected variant VC of patient P01 (see Figs. 2–4,34). These regions include aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively. (A) Deduced amino acid sequences of the exchanged region between P01VC (black) and P01VL (blue). Amino acid changes are indicated in red bold letters. (B) Construction of recombinant chimeric HCVpps P01VCVL221–483 and P01VLVC221–483, P01VCVL-E1 and P01VLVC-E1, P01VCVL-HVR1 and P01VLVC-HVR1, and P01VCVL-HVR2 and P01VLVC-HVR2 by exchanging E1E2 envelope domain aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively, of nonselected variant VC (patient 01) depicted in white and escape isolate VL (patient 01) depicted in blue (see Figs. 2–4,34). HVR1 and HVR2 are shown in orange, and CD81 binding domains (CD81 BD) are shown in green. Positions of E2 epitopes I and II are indicated (Zhang et al., 2007, 2009). The number of mutations within each region is shown. (C) Viral entry of HCVpps containing chimeric envelope proteins in Huh7 cells. HCVpps were incubated with Huh7 cells, and infection was analyzed as described in Fig. 3. Results are expressed in relative light units (RLU) plotted in a logarithmic scale. The threshold for a detectable infection is 3 × 103 RLU and was determined as described in Fig. 3. (D) Neutralization of HCVpps by autologous pretransplant serum was performed as described in Fig. 4. End point dilution titers are indicated for each variant. Dashed lines indicate the threshold for a positive neutralization titer corresponding to 1:40. Calculation of neutralization and determination of threshold titers are described in Materials and methods. Chimeric HCVpps are depicted in dashed blue. Statistically significant differences (repeated measures ANOVA) in HCVpp entry or neutralization between VC and VL wild-type and mutant variants are indicated by asterisks (**, P < 0.001). Ctrl, negative control; MT, mutation.

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