Two amino acid substitutions in the 130-loop of the receptor binding domain (RBD) are sufficient to confer H5Anh pseudotyping ability Sequence alignment of H5-HA proteins revealed a striking amino acid divergence at 9 positions over a short stretch of only 33-amino-acid-long region around the 130-loop of RBD (Table 1) , which accounted for over 30% of the amino acid differences found in the entire HA molecule (576 amino acids in length). Therefore this region was chosen for site-directed mutagenesis to generate a series of H5Anh mutants that were subsequently tested for their ability to pseudotype lentiviral vectors. The level of protein expression for all H5Anh mutants in producer cells was comparable, albeit slightly lower for AnhM1 and AnhM6 (Fig. 3A) . Interestingly, all H5Anh mutants that harbored residues alanine-valine at positions 133-134 (AnhM1-5, Table 1 ) displayed a largely restored ability of H5Anh to produce pseudotyped particles, despite other sequence differences at the 130-loop flanking region (Fig. 3B ). By contrast, AnhM6, which contains H5Cam-like 130-loop flanking sequences but serine-alanine at positions 133-134, did not generate efficiently H5Anh-pp in culture supernatant (Fig. 3B) . These data clearly demonstrate that amino acid residues at positions 133-134 are crucial for efficient H5pp production. More specifically, substitution of the two amino acids S133-A134 of H5Anh with A133-V134, which are unique to H5Cam sequence, confers H5Anh the ability to be incorporated into the lipid envelope of lentiviral particles and is essential for efficient production of transduction-competent H5Anh-pp.