As residue 134 is in the 130-loop of the receptor binding site, we next investigated the impact of A134V mutation on receptor binding properties. We employed a cell-based assay using soluble H5-HA proteins that were engineered by removing TMD and Ctail of HA (Fig. 5A ) as described in Material and Methods. Stable cell lines were generated to express sH5Anh, sH5Cam and their reciprocal single amino acid mutant forms (sH5AnhM8 with A134V and sH5CamM2 bearing V134A; see also Table 1) , and soluble HA proteins were affinity-purified as described under Material and Methods. When analyzed on native gels, purified soluble H5-HA proteins contained mostly the homotrimeric form ( Fig. 5A ) that can bind to the sialic acid-containing cellular receptors. We observed that sH5Anh bound strongly to MDCK cells, whereas the A134V mutation reduced the binding to a much lower level (Fig. 5B ). By contrast, sH5Cam bound weakly to MDCK cells and, as predicted, the single V134A change induced a major increase in the binding of sH5Cam to MDCK cells (Fig. 5B) .