A single A134V mutation in the 130-loop of the RBD of H5-HA is the critical determinant for H5pp production To further delineate the respective roles of A133 and V134 for efficient pseudotyping, two additional H5Anh mutants were generated with a single amino acid substitution either at position 133 (S133A, AnhM7) or 134 (A134V, AnhM8) ( Table 1) . These experiments revealed that the A134V mutation was sufficient to confer H5Anh the ability to be incorporated into transductioncompetent pseudo-particles; whereas the S133A mutation was not (Fig. 4A) . We also generated reciprocal mutants of H5Cam that contained either the two residues found at positions 133-134 of H5Anh (viz., S133-A134; CamM1), or only one single V134A change (CamM2), or A133S substitution (CamM3) ( Table 1) . Again, the presence of valine at position 134 was found to be crucial for efficient H5pp production, whereas the A133S substitution had only a marginal effect, consistent with the results obtained with H5Anh mutants (Fig. 4A ). To confirm that the effect of valine at position 134 was indeed on the production of H5pp, we analyzed by Western blot both cellular lysates and culture supernatants containing H5pp that were concentrated by ultracentrifugation (Fig. 4B ). This series of experiments showed that incorporation of the ''Anhui-like'' single mutant CamM2 into the pseudotyped lentiviral particles was below the antibody detection limit, as also seen for wild-type H5Anh; whereas the ''Cambodialike'' single mutant AnhM8 induced H5pp production with an efficiency similar to H5Cam ( Fig. 4A-B) . Altogether, these experiments demonstrate that valine at position 134 (V134) is a critical residue for efficient H5pp production.