title: A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I.
abstract: While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties.
  
  
  OBJECTIVE
  Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Zuiani
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Megan C
authors.initials: MC
authors.lastname: Schwarz
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: White
authors.affiliation: Departments of Molecular and Cellular Physiology and Structural Biology and the Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Luca
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.|Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.|Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Daved H
authors.initials: DH
authors.lastname: Fremont
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.|Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: David
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Evans
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA diamond@borcim.wustl.edu.|Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.|Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.|Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Diamond
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D019943:Amino Acid Substitution; D000914:Antibodies, Viral; D064113:CRISPR-Cas Systems; D002460:Cell Line; D015723:Gene Library; D005818:Genetic Engineering; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D006526:Hepatitis C / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000276:immunology / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051122:Scavenger Receptors, Class B / Q000502:physiology*; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; C554798:CD81 protein, human; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus