It is widely believed that HA is targeted to lipid rafts at the plasma membrane and the transmembrane domain has been described to be important for lipid rafts association of HA [25] . Therefore, we first swapped the transmembrane regions between H5Anh and H5Cam. We also noticed that the cleavage of H5Cam appears to be more efficient (Fig. 1A, 2B) . Thus, mutants with or without sequence variations found at the poly-basic cleavage site (AnhCam1, AnhCam2 and AnhCam3) were generated and analysed. However, none of these H5Anh mutants showed appreciable improvement in their ability to generate H5pp, when compared with wild type H5Anh. In fact, the production of H1 and trypsin-dependent H5 pseudo-particles has been reported [31, 32] , hence indicating that HA cleavage is not a determining factor for pseudotyping efficiency. Then by multiple sequence alignment, we identified a small region around the 130-loop of the receptor binding site of HA which appeared to be a ''hot-spot'', harboring several sequence variations among different H5N1 clades. Through a series of mutagenesis studies, we have found that one single residue at position 134 is a critical switch to dictate the ability of H5 HA to pseudotype lentiviral vectors for the production of H5pp. Similar to influenza virus, H5pp generated with an HIVbackbone bud at the plasma membrane [18] ; therefore the simplest explanation is that the mutation at position 134 may result in a change in cell surface expression of HA. Indeed we have observed a small but consistent change in cell surface HA expression due to mutations at position 134 (Fig. 4C) . To exclude the possibility that this finding merely reflected a differential binding to the two HA of the rabbit anti-H5 polyclonal serum (described in Material and Methods), we used another polyclonal serum from a different source (a duck anti-H5 serum described in Ref. 18 ) and found that the results of cell surface HA staining were similar (data not shown). The fact that the A134V mutation increased cell surface expression of H5Anh, may partially explain the effect of this amino acid substitution on H5pp production. Considering that the variation between H5Cam-pp and H5Anhpp production resulted in a 3 to 4 log difference in luciferase activity, it is likely that A134V mutation may have an impact on other properties of H5-HA, including binding to sialic acid receptors, which contribute to the observed phenotype. It has been reported in the case of H3-HA pseudotyping that lentiviral particles which incorporate sialic acid binding-incompetent H3-HA (derived from A/Aichi/2/68) can be efficiently generated and released into culture supernatant in the absence of exogenous bacterial NA; whereas the wild-type Aichi-HA fails to do so [33] . Although the difference in pseudotyping observed with wild-type Aichi-HA and its receptor binding-incompetent mutant is diminished when bacterial NA is added, the study by Bosch et al. [33] implies that changes in receptor binding properties can affect pseudotyping efficiency of lentiviral vectors by influenza HA.