title: Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.
abstract: The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.
authors.affiliation: Research Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia. Electronic address: nicholas.eyre@adelaide.edu.au.
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Eyre
authors.affiliation: Research Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Aloia
authors.affiliation: Department of Medical Microbiology and Immunology and the La Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, University of Alberta, Edmonton, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Joyce
authors.affiliation: Research Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Monrat
authors.initials: M
authors.lastname: Chulanetra
authors.affiliation: Department of Medical Microbiology and Immunology and the La Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, University of Alberta, Edmonton, Canada.
authors.forename: D Lorne
authors.initials: DL
authors.lastname: Tyrrell
authors.affiliation: Research Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Beard
mesh_terms: D000818:Animals; D045604:Extracellular Fluid / Q000821:virology*; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D008156:Luciferases; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Exosome; Hepatitis C virus; Luciferase; NS5A; Virus assembly; Virus replication