Background: The hepatitis C virus (HCV) NS3-4A protease is not only
an essential component of the viral replication complex and a prime
target for a ntiviral intervention but also a key player i n the persistence
and pathogenesis of HCV. It cleaves and thereby inactivates two crucial
adaptor proteins in viral RNA sensing and innate immunity (MAVS and
TRIF) as well as a phosphatase involved in growth factor signaling (TCPTP).
T he aim of this study was to identify novel cellular substrates o f
the N S3-4A protease and to investigate their role in the replication and
pathogenesis of HCV.
Methods: Cell lines inducibly expressing t he NS3-4A protease were
analyzed in basal as well as interferon-α-stimulated states by stable
isotopic l abeling using amino acids in cell culture (SILAC) coupled with
protein separation and mass spectrometry. Candidates fulfilling stringent
criteria for potential substrates or products of the NS3-4A protease were
further i nvestigated in different experimental systems as well a s in liver
biopsies from patients with chronic hepatitis C.
Results: SILAC coupled with protein separation and mass spectrometry
yielded > 5000 proteins of which 18 candidates were selected for further
analyses. These allowed us to identify GPx8, a membrane-associated
peroxidase involved in disulfide bond formation in the endoplasmic
reticulum, as a n ovel cellular substrate of the H CV NS3-4A protease.
Cleavage occurs at cysteine in position 11, removing the cytosolic tip of
GPx8, and was observed in different experimental systems as well as in
liver biopsies from patients with chronic hepatitis C. Further functional
studies, involving overexpression and RNA silencing, revealed that GPx8
is a p roviral factor involved in viral particle production but not in HCV
entry or HCV RNA replication.
Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A
protease. Studies investigating the consequences of GPx8 cleavage for
protein function are underway. The identification of novel cellular
substrates o f the HCV N S3-4A protease should yield new insights i nto
the HCV life cycle and the pathogenesis of hepatitis C and may reveal
novel targets for antiviral intervention.