Giuseppe
Emeritus
[Source: PLoS ONE, full text: (LINK). Abstract, edited.]
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A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles
Dong-Jiang Tang<SUP>1</SUP><SUP>?a</SUP><SUP>*</SUP>, Yuen-Man Lam<SUP>1</SUP>, Yu-Lam Siu<SUP>1</SUP>, Chi-Hong Lam<SUP>1</SUP>, Shui-Ling Chu<SUP>1</SUP>, J. S. Malik Peiris<SUP>1</SUP><SUP>,</SUP><SUP>2</SUP>, Philippe Buchy<SUP>4</SUP>, B?atrice Nal<SUP>1</SUP><SUP>,</SUP><SUP>3</SUP><SUP>?b</SUP>, Roberto Bruzzone<SUP>1</SUP><SUP>,</SUP><SUP>5</SUP>
<SUP></SUP>
1 HKU-Pasteur Research Centre, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 2 Center for Influenza Research ? School of Public Health, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 3 Department of Anatomy, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 4 Virology Unit, Institut Pasteur in Cambodia, Phnom Penh, Kingdom of Cambodia, 5 Department of Cell Biology and Infection, Institut Pasteur, Paris, France
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.
Citation: Tang D-J, Lam Y-M, Siu Y-L, Lam C-H, Chu S-L, et al. (2012) A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles. PLoS ONE 7(11): e43596. doi:10.1371/journal.pone.0043596
Editor: Stefan P?hlmann, German Primate Center, Germany
Received: March 23, 2012; Accepted: July 23, 2012; Published: November 2, 2012
Copyright: ? 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.
Funding: This work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: tangd@hkucc.hku.hk
?aCurrent address: School of Basic Science, Guangzhou Medical University, Guangzhou, People's Republic of China
?bCurrent address: Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, United Kingdom
-Dong-Jiang Tang<SUP>1</SUP><SUP>?a</SUP><SUP>*</SUP>, Yuen-Man Lam<SUP>1</SUP>, Yu-Lam Siu<SUP>1</SUP>, Chi-Hong Lam<SUP>1</SUP>, Shui-Ling Chu<SUP>1</SUP>, J. S. Malik Peiris<SUP>1</SUP><SUP>,</SUP><SUP>2</SUP>, Philippe Buchy<SUP>4</SUP>, B?atrice Nal<SUP>1</SUP><SUP>,</SUP><SUP>3</SUP><SUP>?b</SUP>, Roberto Bruzzone<SUP>1</SUP><SUP>,</SUP><SUP>5</SUP>
<SUP></SUP>
1 HKU-Pasteur Research Centre, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 2 Center for Influenza Research ? School of Public Health, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 3 Department of Anatomy, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China, 4 Virology Unit, Institut Pasteur in Cambodia, Phnom Penh, Kingdom of Cambodia, 5 Department of Cell Biology and Infection, Institut Pasteur, Paris, France
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.
Citation: Tang D-J, Lam Y-M, Siu Y-L, Lam C-H, Chu S-L, et al. (2012) A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles. PLoS ONE 7(11): e43596. doi:10.1371/journal.pone.0043596
Editor: Stefan P?hlmann, German Primate Center, Germany
Received: March 23, 2012; Accepted: July 23, 2012; Published: November 2, 2012
Copyright: ? 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.
Funding: This work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: tangd@hkucc.hku.hk
?aCurrent address: School of Basic Science, Guangzhou Medical University, Guangzhou, People's Republic of China
?bCurrent address: Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, United Kingdom
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