Localization determines function: N-terminally truncated NS5A fragments accumulate in the nucleus and impair HCV replication.
OBJECTIVE
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.
Sauter
Daniel
D
Department of Internal Medicine II, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.
Himmelsbach
Kiyoshi
K
Kriegs
Malte
M
Carvajal Yepes
Monica
M
Hildt
Eberhard
E
D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012093:Replicon / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
D019908:Proto-Oncogene Proteins c-raf; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase