title: Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.
abstract: The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada; Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Aviad
authors.initials: A
authors.lastname: Levin
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Neufeldt
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pang
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Kristen
authors.initials: K
authors.lastname: Wilson
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Darci
authors.initials: D
authors.lastname: Loewen-Dobler
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Joyce
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Wozniak
authors.affiliation: Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: D Lorne J
authors.initials: DL
authors.lastname: Tyrrell
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D018936:Hepatitis C Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals / Q000502:physiology*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018936:Hepatitis C Antigens; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D014758:Viral Core Proteins