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To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5.Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5.Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5.Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.