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Title: A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles
Authors: Tang, DJ
Lam, YM
Siu, YL
Lam, CH
Chu, SL
Peiris, JS
Buchy, P
Nal, B
Bruzzone, R
Keywords: Animals;Birds;CHO Cells;Cell Membrane;Cricetinae;DNA Mutational Analysis;Dogs;HEK293 Cells;Hemagglutinin Glycoproteins, Influenza Virus;Humans;Influenza A Virus, H5N1 Subtype;Influenza Vaccines;Influenza in Birds;Lentivirus;Madin Darby Canine Kidney Cells;Mutation;Protein Binding;Protein Structure, Tertiary;Sialic Acids
Issue Date: 2012
Publisher: Public library of science
Citation: PLoS One, 7(11), e43596, 2012
Abstract: 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
Description: © 2012 Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ISSN: 1932-6203
Appears in Collections:Biological Sciences
Dept of Life Sciences Research Papers

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