The A134V mutation affects cell surface expression level of HA Because influenza virus, as well as particles pseudotyped with HA, buds from the plasma membrane [11, 18] , we reasoned that changes in surface expression of H5-HA could have an impact on the production of H5pp. Thus, we have compared by flow cytometry plasma membrane expression levels of HA protein in cells transfected with H5Anh and H5Cam. Cells were labelled with an anti-H5N1 antibody, fixed and then stained with a PEconjugated secondary antibody. As assessed by measuring mean fluorescence intensity (MFI), cell surface expression of H5Anh was significantly less compared to H5Cam ( Fig. 4C ; p,0.01). Interestingly, introduction of the A134V mutation into H5Anh (AnhM8) increased its cell surface expression ( Fig. 4C ; p,0.02), and conversely, a V134A mutation in H5Cam (CamM2) reduced transport to the plasma membrane to a level that was not significantly different from that measured with H5Anh (Fig. 4C ). These data demonstrate that the Ala to Val substitution at position 134 enhances surface expression of H5-HA.