title: Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools.
abstract: Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses.
  
  
  OBJECTIVE
  Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. For example, drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses.
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Landowski
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Dabundo
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA.
authors.forename: Anthony V
authors.initials: AV
authors.lastname: Nicola
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA haguilar@vetmed.wsu.edu.
authors.forename: Hector C
authors.initials: HC
authors.lastname: Aguilar
mesh_terms: D002460:Cell Line; D006801:Humans; D045405:Nipah Virus / Q000502:physiology*; D014771:Virion / Q000502:physiology*; D014773:Virology / Q000379:methods*; D022701:Virosomes / Q000378:metabolism; D053586:Virus Internalization
chemical_list: D022701:Virosomes