To further characterize the relationship between these changes and symptoms severity, we introduced them by site-directed mutagenesis in the ancestral TEV clone. In addition, all three possible pairs of non-synonymous mutations and the triple non- synonymous mutant were also created. A. thaliana Ler plants were inoculated with these nine mutant clones and maintained in the same growth conditions for three weeks. The results of this experiment are summarized in Table 1 . All mutant genotypes were viable and replicated and accumulated in the plants, as confirmed by Western blot analysis (data not shown). Among the three single mutants, the only clone that produced visible symptoms was the one containing the L2013F allele in VPg. These symptoms were, nonetheless, qualitatively milder than those produced by TEV-At17 ( Figure S1 ). Concerning the three double mutants, only the combination of VPg and P3 substitutions induced symptoms that were qualitatively more severe than those produced by the single VPg L2013F mutant (Table 1 ) and almost as severe as those observed for TEV-At17. By contrast, mutation 6K1 T1210M does not have any effect on aggravating the symptoms associated with VPg L2013F. The combination of substitutions in P3 and 6K1 did not produce any symptom. Finally, the triple mutant recreated the strong symptoms characteristic of TEV-At17 (Table 1 and Figure S1 ). All together, these results suggest that the presence of substitution L2013F in the VPg protein is enough for triggering symptoms and that the severity of these symptoms is enhanced by the presence of substitution A1047V in P3, suggesting an epistatic interaction between these two mutations. The role of substitution T1210M in the 6K1 peptide remains unclear. It has been recently reported that the correct interaction between potyvirus' VPg and host's eIF4E is required to initiate a successful infection [27] . Recessive resistance of peppers to potyvirus depends on the substitution of relevant amino acid residues in eIF4E that disrupt the normal interaction between this translation factor and VPg. Resistance-breaking viral strains restore the normal interaction [27] . Therefore, we can hypothesize that TEV-AT17 has enhanced its ability to infect A. thaliana Ler by improving the interaction of its VPg with the host's translation initiation factor eIF4E.