T84 cells and electrophysiology. Measurements of short-circuit current (I sc ) on monolayers (0.33-cm 2 inserts) of the polarized human intestinal cell line T84 were performed as previously described (41) . A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles BACKGROUND: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. METHODOLOGY/FINDINGS: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. CONCLUSIONS: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses. H5N1 influenza virus is highly pathogenic in poultry, certain bird populations, and has occasionally infected human causing severe clinical outcomes [1] [2] [3] . Since the first human outbreak in 1997, there have been more than 600 confirmed human cases of H5N1 infection with a mortality rate of approximately 60% [4] . To initiate an infection, like all other subtypes of influenza viruses, H5N1 virus first binds to cell surface glycan receptors via its surface glycoprotein hemagglutinin (HA) and is subsequently internalized via endocytic pathways [5] [6] [7] . HA is a homotrimeric type I transmembrane glycoprotein, which can be cleaved into HA1 and HA2 subunits [8] . A furin-dependent polybasic cleavage site has been shown to be characteristic of highly pathogenic avian influenza viruses [9, 10] , although not all H5-HAs contain the polybasic cleavage site. In cells infected by influenza virus, HA protein is first synthesized as a precursor (HA0), which is then oligomerized, glycosylated and ultimately transported to the plasma membrane where assembly and budding of progeny virions takes place [11] . Then, during the final stage of the virus life cycle, neuraminidase (NA), the second major envelope glycoprotein of influenza, cleaves the terminal sialic acids from the cell surface glycans to allow release of the virus from the host cell [12] .