title: A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles.
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.
  
  
  RESULTS
  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.
authors.affiliation: HKU-Pasteur Research Centre, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China. tangd@hkucc.hku.hk
authors.forename: Dong-Jiang
authors.initials: DJ
authors.lastname: Tang
authors.forename: Yuen-Man
authors.initials: YM
authors.lastname: Lam
authors.forename: Yu-Lam
authors.initials: YL
authors.lastname: Siu
authors.forename: Chi-Hong
authors.initials: CH
authors.lastname: Lam
authors.forename: Shui-Ling
authors.initials: SL
authors.lastname: Chu
authors.forename: J S Malik
authors.initials: JS
authors.lastname: Peiris
authors.forename: Philippe
authors.initials: P
authors.lastname: Buchy
authors.forename: Béatrice
authors.initials: B
authors.lastname: Nal
authors.forename: Roberto
authors.initials: R
authors.lastname: Bruzzone
mesh_terms: D000818:Animals; D001717:Birds; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004285:Dogs; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000737:chemistry*; D007252:Influenza Vaccines / Q000737:chemistry*; D005585:Influenza in Birds / Q000821:virology; D016086:Lentivirus / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000737:chemistry
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D012794:Sialic Acids