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 identify the mutations selected in the E1E2 HCV envelope protein genes during the culture in the presence of each lectin, we sequenced the genomic region encompassing the end of Core to the end of p7. Three mutations were identified after the three month culture with GNA: V284A in E1 and V392D / S449P in E2. Interestingly, the mutation S449P is expected to prevent the addition of the N-glycan on the E2N4 N-glycosylation site at position 448, by introducing a proline residue between the asparagine 448 and the serine 450. The mutation M338V in E2 was found after the culture of HCV in the presence of CV-N, ConA and GRFT. Interestingly, we also identified the mutation L755M in p7 in the three viruses isolated after culture with these lectins. Three other mutations, L612M, N417S and A400D, were also detected in the viruses selected with CV-N, ConA and GRFT, respectively. The N417S mutation has previously been shown to lead to a shift of the E2N1 N-glycan from N417 to N415 [26] . Importantly, when the virus was passaged in absence of lectin, we only detected the mutation I599V in E2. These mutations are summarized in bold characters in Table 1 . Using the European Hepatitis C Virus database (euHCVdb) [27] , we observed that mutations V284A, N417S, S449P and L755M occurred on residues which are highly conserved not only in genotype 2a HCV sequences but also in sequences of other genotypes (Table 2 ). In these sequences, only three synonymous mutations were identified (one in the end of Core, and two in p7, after GNA exposure). Altogether, these results suggest that a positive selection occurred on the HCV envelope protein genes in the presence of lectins. This was also supported by the cumulative behavior of the dS/dN ratio, obtained using the Synonymous / Non-synonymous Analysis Program (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html) [28] and comparing the four sequences obtained after lectin exposure and the control sequence obtained after culture without lectin (S2 Fig) .