title: Relation between viral fitness and immune escape within the hepatitis C virus protease.
abstract: BACKGROUND
  The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally.
  
  
  OBJECTIVE
  We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness.
  
  
  METHODS
  Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection.
  
  
  METHODS
  NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication.
  
  
  RESULTS
  Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication.
  
  
  CONCLUSIONS
  Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.
authors.affiliation: Division of Clinical Virology, Karolinska Institutet at Karolinska University Hospital, S-141 86 Stockholm, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Söderholm
authors.forename: G
authors.initials: G
authors.lastname: Ahlén
authors.forename: A
authors.initials: A
authors.lastname: Kaul
authors.forename: L
authors.initials: L
authors.lastname: Frelin
authors.forename: M
authors.initials: M
authors.lastname: Alheim
authors.forename: C
authors.initials: C
authors.lastname: Barnfield
authors.forename: P
authors.initials: P
authors.lastname: Liljeström
authors.forename: O
authors.initials: O
authors.lastname: Weiland
authors.forename: D R
authors.initials: DR
authors.lastname: Milich
authors.forename: R
authors.initials: R
authors.lastname: Bartenschlager
authors.forename: M
authors.initials: M
authors.lastname: Sällberg
mesh_terms: D000208:Acute Disease; D000328:Adult; D000818:Animals; D003602:Cytotoxicity, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D005814:Genes, Viral; D014644:Genetic Variation / Q000276:immunology; D015789:HLA-A2 Antigen / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology; D006526:Hepatitis C / Q000276:immunology* / Q000821:virology; D019698:Hepatitis C, Chronic / Q000276:immunology / Q000821:virology; D006801:Humans; D007108:Immune Tolerance; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008875:Middle Aged; D009154:Mutation; D010446:Peptide Fragments / Q000276:immunology; D012367:RNA, Viral / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000276:immunology; D014779:Virus Replication / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases