Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.
The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication.


CONCLUSIONS
We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease.
From the Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Kenichi
K
Morikawa
Jérôme
J
Gouttenoire
Céline
C
Hernandez
Viet Loan
VL
Dao Thi
Huong T L
HT
Tran
Christian M
CM
Lange
Michael T
MT
Dill
Markus H
MH
Heim
Olivier
O
Donzé
François
F
Penin
Manfredo
M
Quadroni
Darius
D
Moradpour
D000595:Amino Acid Sequence; D001706:Biopsy; D002460:Cell Line; D016174:Hepacivirus / Q000187:drug effects; D019698:Hepatitis C, Chronic / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010544:Peroxidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014771:Virion / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010447:Peptide Hydrolases; D010544:Peroxidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C558642:GPX8 protein, human