Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease

Abstract

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. Conclusion: 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. (HEPATOLOGY 2014;59:423-433)

Oxidative stress, a trigger of hepatitis C and B virus-induced liver carcinogenesis

“…A similar calcium-mediated effect was recently observed for endogenous production of ROS as a result of the ER overload response in the HCV-infected cells [157]. Knock-down of GPx1 and GPx8, as well as of SOD1 or SOD2, in the infected cells had no significant impact on HCV replication [145, 154]. Another effect that ROS appear to have on HCV is an increase in HCV genome heterogeneity, which may contribute to the evolutional survival of the virus by ensuring viral escape from the immune system (during establishment of infection as well as during treatment) [158, 159].…”

“…HCV infection also increases the expression levels of GPx1 and GPx4 [145], implicated in protection against lipid peroxides. In addition, HCV NS3/4A protease was recently shown to cleave GPx8 [154]. This enzyme scavenges hydrogen peroxide produced in the ER by Ero1α (see above) [95, 128]).…”

“…This enzyme scavenges hydrogen peroxide produced in the ER by Ero1α (see above) [95, 128]). Interestingly, NS3/4A cleaves a small cytoplasmic tip of GPx8 molecule, however, this appears not to affect its enzymatic activity [154]. …”

Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-β induction by efficient MAVS cleavage

“…U-2 OS cells were chosen for the present study because they are highly permissive for transfection of NS3-4A expression constructs (unlike hepatocyte-derived cell lines), which is an important prerequisite for solid quantitative analyses of MAVS cleavage. Furthermore, U-2 OS cells express relatively high levels of endogenous MAVS [6]. U-2 OS cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Darmstadt, Germany) containing 10% heat-inactivated fetal calf serum.…”

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity

“…S1A) led us to hypothesize that these molecules may target a cellular protease. We used quantitative proteomics to search for putative cellular substrates, the processing of which could be impaired in the presence of NFR (31). To avoid cell death, we performed the experiment in HeLa as a model cell line that is not competent for inflammasome formation and subsequent pyroptosis.…”