title: A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication.
abstract: Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.
authors.affiliation: Institute of Virology and Cell Biology, University of Lübeck, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Isken
authors.affiliation: Institute of Virology and Cell Biology, University of Lübeck, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Langerwisch
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Jirasko
authors.affiliation: Joint Laboratory for Structural Biology of Infection and Inflammation of the University of Hamburg and the University of Lübeck, DESY, Hamburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Rehders
authors.affiliation: Joint Laboratory for Structural Biology of Infection and Inflammation of the University of Hamburg and the University of Lübeck, DESY, Hamburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Redecke
authors.affiliation: Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Harish
authors.initials: H
authors.lastname: Ramanathan
authors.affiliation: Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Brett D
authors.initials: BD
authors.lastname: Lindenbach
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.affiliation: Institute of Virology and Cell Biology, University of Lübeck, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Tautz
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D017124:Conserved Sequence; D018274:Electroporation; D004789:Enzyme Activation / Q000502:physiology; D016679:Genome, Viral; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase