title: Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.
abstract: Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.
authors.affiliation: School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Urbanowicz
authors.affiliation: School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: C Patrick
authors.initials: CP
authors.lastname: McClure
authors.affiliation: School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Barnabas
authors.initials: B
authors.lastname: King
authors.affiliation: School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Mason
authors.affiliation: School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Ball
authors.affiliation: NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.|School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tarr
mesh_terms: D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012190:Retroviridae / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014773:Virology / Q000379:methods*; D053586:Virus Internalization
chemical_list: D014759:Viral Envelope Proteins