Figure 6

Mutagenesis of the predicted HA segment domain 794–839 of human H3N2 viruses.

Plaque assays of recombinant viruses containing desired mutations were performed as described in Methods. The plots show the plaque radius for multiple individual plaques as a measure for virus replication. Dots represent individual plaques, red lines correspond to median plaque sizes. Point substitutions and their combinations in mutants are indicated in the domain structure. In each of the mutant series, structure-disrupting mutations with index 1 ([A1], [B1], etc.) were introduced to the left part of the structure, structure-disrupting mutations [A2], [B2], etc. were introduced to the right part, mutants containing the changes in both parts ([A3], [B3], etc.) were compensatory, restoring the structure. Mutant series A, B, C, E, G, and H affected one of the base pairs in the domain, mutant series D affected 2 pairs, and series F affected 4 pairs. Mutations [A1], [B1] and [B2] result in amino acid changes observed in natural strains, see Fig. 1, [C2] leads to N248K substitution (note that [C2] is silent in [D2] = [B2] + [C2], as observed in natural strains), other single nucleotide substitutions are silent. Despite three attempts to rescue the D1 and D3 mutants, no recombinant virus was produced. WT, A/PR/8/34 virus; 7xPR8 + HA_H3, recombinant virus with the A/Bilthoven/16190/68 (H3N2) HA segment.