To investigate whether individual amino acid sequence differences contributed to the neutralization resistant phenotype, PCR-based site-directed mutagenesis was used to introduce an amino acid change as described previously [24] . Briefly, a set of primers was used that each had either the wildtype or mutated sequence. The primer sequences and their HXB2 locations are shown below, where the substituted nucleotides are underlined. All mutants were confirmed by sequencing the entire env gene.