title: Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.
abstract: Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
authors.affiliation: Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10021, USA.
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Evans
authors.forename: Rainer
authors.initials: R
authors.lastname: Gosert
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Yuan
authors.forename: Hubert E
authors.initials: HE
authors.lastname: Blum
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Goff
authors.forename: Brett D
authors.initials: BD
authors.lastname: Lindenbach
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012093:Replicon / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase