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Updated: Jun 24, 2026

A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Localization determines function: N-terminally truncated NS5A fragments accumulate in the nucleus and impair HCV
Daniel Sauter1, Kiyoshi Himmelsbach, Malte Kriegs
1Department of Internal Medicine II, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.
Background/Aims:
The Hepatitis C Virus (HCV) nonstructural protein 5A (NS5A) is an essential part of the ER-localized HCV-replicon complex. Although NS5A harbours a conserved NLS in its C-terminal domain, NS5A is associated with the cytoplasmic face of the ER by an amphipathic helix close to its N-terminus.
Methods:
Intracellular distribution of NS5A in HCV replicating cells was analyzed by confocal microscopy and subcellular fractionation. The effect on HCV replication was analyzed using the JFH-1-based infection/replication system.
Results:
During viral life cycle N-terminally truncated NS5A fragments are caspase-dependent formed that lack the ER-attachment signal and are localized within the nucleus. These N-terminally truncated fragments inhibit HCV replication. If their formation is blocked by inhibition of caspases HCV replication is increased. The C-terminal domain of NS5A binds to c-Raf and thereby localizes it to the replicon complex. This interaction is essential for HCV replication. The N-terminally truncated NS5A fragments are still able to bind c-Raf. However, due to their nuclear localization they withdraw c-Raf from the replicon complex into the nucleus resulting in an impaired HCV replication.
Conclusions:
Formation of N-terminally truncated NS5A fragments could represent a mechanism to regulate HCV replication by withdrawal of essential factors from the replicon complex.
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