Relation between viral fitness and immune escape within the hepatitis C virus protease
- J Söderholm1,
- G Ahlén1,
- A Kaul2,
- L Frelin3,
- M Alheim1,
- C Barnfield4,
- P Liljeström4,
- O Weiland5,
- D R Milich6,
- R Bartenschlager2,
- M Sällberg1
- 1Division of Clinical Virology, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden
- 2Division of Molecular Virology, University of Heidelberg, Heidelberg, Germany
- 3Division of Clinical Virology, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden, and Vaccine Research Institute of San Diego, San Diego, California, USA
- 4Swedish Institute for Infectious Disease Control and the Microbiology and Tumour Biology Centre, Karolinska Institutet, Stockholm, Sweden
- 5Division of Infectious Diseases, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden
- 6Vaccine Research Institute of San Diego, San Diego, California, USA
- Correspondence to:
Professor M Sallberg
Division of Clinical Virology, F 68, Huddinge University Hospital, S-141 86 Stockholm, Sweden;
- Accepted 3 August 2005
- Revised 21 July 2005
- Published Online First 16 August 2005
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.
Aims: 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.
Patients: 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.
- HCV, hepatitis C virus
- HBV, hepatitis B virus
- HLA, human leucocyte antigen
- NS, non-structural
- CTL, cytotoxic T lymphocyte
- PBMC, peripheral blood mononuclear cells
- SFV, semliki forest virus
- PCR, polymerase chain reaction
- DMEM, Dulbecco’s modified Eagle’s medium
- IFN-γ, interferon γ
Published online first 16 August 2005
Conflict of interest: None declared.