title: New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.
abstract: BACKGROUND
  The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target.
  
  
  RESULTS
  Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle.
  
  
  CONCLUSIONS
  Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.
authors.affiliation: Inflammatory and Infectious Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Shiryaev
authors.forename: Elliot R
authors.initials: ER
authors.lastname: Thomsen
authors.forename: Piotr
authors.initials: P
authors.lastname: Cieplak
authors.forename: Eugene
authors.initials: E
authors.lastname: Chudin
authors.forename: Anton V
authors.initials: AV
authors.lastname: Cheltsov
authors.forename: Mark S
authors.initials: MS
authors.lastname: Chee
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kozlov
authors.forename: Alex Y
authors.initials: AY
authors.lastname: Strongin
mesh_terms: D000595:Amino Acid Sequence; D057166:High-Throughput Screening Assays; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis; D020815:Polyproteins / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010455:Peptides; D020815:Polyproteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases