FIG 8.

Epitope mapping on the NA of N1-5-VHHm escape NIBRG-14ma mutants. MDCK cells were infected with NIBRG-14ma in the presence of 3.7 μM N1-5-VHHm. Evidence of resistant viruses was based on a cytopathic effect on infected cell cultures and was monitored by plaque purification of candidate escape viruses. The NA coding sequence of the resultant escape viruses was amplified by RT-PCR and sequenced. One selected escape virus carried an Ile-to-Thr change at position 437. The deduced amino acid sequence in NA was modeled with PyMol (Delano Scientific, http://www.pymol.org) with the H5N1 NA structure derived from A/Vietnam/1194/2004 (PDB code 2HTY). (A) Top (left) and lateral (right) views of the surface-exposed amino acid residues of the NA dimer. The I437T amino acid substitution of the NA of the N1-5-VHHm escape mutant is surface exposed and marked in yellow. Residues that form the catalytic-site framework are depicted in red. The N1 “150 loop” is shown in green. For comparison, the residues involved in MAb HCA-2 binding are shown in dark blue (residues 222 to 230) and those involved in MAb 3H10 binding are shown in orange (N272, V339, and S340). (B) Sequence alignment of relevant N1 NAs. The I437T change in one of the N1-5-VHHm escape viruses is highlighted in yellow. The residues highlighted in red are important for MAb 3H10 binding. (C) Fluorogram showing the T-to-C substitution in the cloned cDNA of the N1-5-VHHm escape virus (left). Mono- and bivalent NA-specific VHHs bind to NIBRG-14 virus but not to I437T escape virus (right). ELISA plates were coated with 8 HA units of NIBRG-14 or I437T virus and, after blocking, incubated with different concentrations of N1-3-VHHm, N1-3-VHHb, N1-5-VHHm, or N1-5-VHHb. N1-VHH binding was revealed with an anti-His tag MAb.