Hepatitis C Virus Resistance to Carbohydrate-Binding Agents

Izquierdo, Laure; Oliveira, Catarina; Fournier, Carole; Descamps, Véronique; Morel, Virginie; Dubuisson, Jean; Brochot, Etienne; Francois, Catherine; Castelain, Sandrine; Duverlie, Gilles; Helle, Francois

To study the effect of the E1E2 mutations on the virus fitness, we introduced them into a modified JFH1 genome expressing a Renilla Luciferase reporter gene by reverse genetics [17] . Since the mutation L755M in p7 was fixed in three independent lectin exposures, we also decided to characterize it. Mutations were introduced alone or by combination of 2 or 3. A replicationdefective virus carrying a mutation in the NS5B GDD motif (GND) and a variant genome carrying a large in-frame deletion in the E1E2 coding region known to inactivate viral particle release (ΔE1E2) were used as negative controls for replication and assembly, respectively [16] . First, we assessed the impact of the mutations on HCV RNA replication. As shown in Fig 3A, some individual mutations or combinations of two mutations led to slight variations of the replication measured as Luciferase activities 24, 48 and 72 h after electroporation. An increased replication was observed at 72 h for the N417S, L612M and V284A-S449P mutants (1.6-, 1.6and 2.0-fold WT respectively, p < 0.05). In contrast, the mutant V284A-V392D showed a slight decrease of replication (0.6-fold WT, p < 0.05). However, the combinations of three mutations did not have any significant effect on replication as compared to WT (1. than the WT E2 which confirmed that this mutation leads to an impediment of the asparagine 448 N-glycosylation (Fig 4) . In addition, the detection of E2 for the mutants containing the N417S substitution was often weaker than the WT E2 because this residue is involved in the epitope of the 3/11 MAb which was used for the western blot. To further characterize our mutants, infectious virus production was measured at 48 h post-electroporation for each mutant and compared to that of the WT. Supernatant of cells electroporated with the ΔE1E2 mutants was used as negative control to determine the background level of Luciferase activity. As shown in Fig 3B, the production of infectious virus for A400D and A400D-L755M mutants was significantly decreased as compared to that of the WT (3.0-and 2.5-fold WT, p < 0.05 and p < 0.001, respectively). On the contrary, results suggest that other combinations of two mutations positively modulate infectivity. The production of infectious viral particles was markedly improved for the V392D-S449P and L612M-L755M mutants (3.5-and 3.0-fold WT, p < 0.001 and p < 0.05, respectively). However, the different combinations of three mutations that were found at the end of each selection did not have any significant effect on infectious viral particle production.