title: Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level.
abstract: Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
authors.forename: Hung-Lun
authors.initials: HL
authors.lastname: Hsu
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA.
authors.forename: Jean K
authors.initials: JK
authors.lastname: Millet
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
authors.forename: Deirdre A
authors.initials: DA
authors.lastname: Costello
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Whittaker
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Daniel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000472:pathogenicity / Q000502:physiology*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D016865:Reassortant Viruses / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014771:Virion / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009439:Neuraminidase