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Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A
Nicole Appel1, Ulrike Herian, Ralf Bartenschlager
1Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
Journal of Virology
|December 23, 2004
Summary
Hepatitis C virus (HCV) nonstructural gene mutations were studied using trans-complementation. Only NS5A mutants were rescued, revealing functional organization of the HCV replication complex.
Area of Science:
- Virology
- Molecular Biology
- Hepatology
Background:
- Subgenomic replicons enable studies of Hepatitis C virus (HCV) RNA replication.
- The viral replication complex components are poorly defined.
Purpose of the Study:
- Investigate if lethal mutations in HCV nonstructural genes can be rescued by trans-complementation.
- Analyze the functional organization of the HCV replication complex.
Main Methods:
- Transfection of replicon RNAs with mutations in NS3, NS4B, NS5A, and NS5B into Huh-7 cells harboring helper RNAs.
- Established a transient complementation assay using cotransfection of helper and mutant RNAs.
Main Results:
- Only NS5A mutants were efficiently rescued, unlike mutations in NS3, NS4B, and NS5B.
- No RNA recombination or reversion of transfected mutants was observed.
- Inactivating NS5A mutations in the amino-terminal amphipathic helix could not be complemented in trans; helper RNA replication was not required; NS3-NS5A sequence was minimal for complementation.
Conclusions:
- The study provides the first insight into the functional organization of the HCV replication complex.
- NS5A plays a critical role in HCV RNA replication and cannot be complemented in trans if essential domains are mutated.