Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor–induced signal transduction by cleavage of the T‐cell protein tyrosine phosphatase†

Brenndörfer, Erwin Daniel; Karthe, Juliane; Frelin, Lars; Cebula, Patricia; Erhardt, Andreas; Esch, Jan Schulte Am; Hengel, Hartmut; Bartenschlager, Ralf; Sällberg, Matti; Häussinger, Dieter; Bode, Johannes G.

doi:10.1002/hep.22857

Cited by 67 publications

(72 citation statements)

References 26 publications

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“…8 The NS3 serine protease domain is cyan, with side chain atoms of the catalytic triad (His 57, Asp 81, and Ser 139) highlighted as purple spheres. The NS3 helicase domain is gray and the N-terminal transmembrane (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and central (amino acids 21-32) segments of NS4A are orange and light orange, respectively. Homology models of the GPx8 cytosolic tip and transmembrane segment are green, while the crystal structure of the ER luminal domain is light green (PDB entry 3KIJ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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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)

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Section: Resultsmentioning

confidence: 99%

“…11,14 Huh-7.5 cells were kindly provided by Charles M. Rice (Rockefeller University, New York, NY). 15 SILAC, Protein Separation, and MS. UNS3-4A-24 cells were heavy isotope labeled by culture in modified RPMI 1640 medium in which 13 C 6 -L-lysine and 13 C 6 15 N 4 -L-arginine (Cambridge Isotope Laboratories)…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

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“…In addition to MAVS, NS3/4A has also been reported to cleave the host cell proteins TRIF (13) and T-cell protein phosphatase (5). While it remains to be seen exactly how the cleavage of these molecules impacts viral persistence in vivo, it is clear that the HCV protease has the ability to cleave multiple cellular targets, likely to promote its replication and viral pathogenesis in the host, and these antiviral agents, which act directly against NS3/ 4A, will also inhibit these processes.…”

Section: Discussionmentioning

confidence: 99%

Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α ₀

Horner

Park

Gale

2012

J Virol

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Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix ␣ 0 of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix ␣ 0 are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix ␣ 0 is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix ␣ 0 impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix ␣ 0 of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates. Hepatitis C virus (HCV) is a major human pathogen and the leading cause of liver disease and liver cancer. Similar to other positive-strand RNA viruses, HCV harnesses cellular membranes as a scaffold for genome replication, protein translation, and assembly (21). During HCV infection, the cytosolic RNA sensor protein RIG-I detects specific sequences in the viral RNA and signals through its adaptor protein, MAVS (28,34). MAVS is the scaffold for a macromolecular signaling complex that assembles on innate immune synapses that are formed by mitochondrionassociated endoplasmic reticulum (ER) membrane (MAM) interactions with peroxisomes and mitochondria (9), with MAVS localized to all three of these subcellular locations (6,9,29). These interactions all drive intracellular innate immune signaling that culminates in the production of type I interferon (IFN), inflammatory cytokines, and IFN-stimulated genes to limit virus replication and spread (8, 11). However, HCV controls this downstream signaling, including IRF-3 activation, through HCV NS3/4A protease-mediated cleavage of MAVS on the MAM, which releases it from this intracellular membrane and also disrupts MAVS oligomerization (1,7,9,14,17,20).The 9.6-kb single-stranded RNA genome of HCV encodes a single polyprotein that is co-and posttranslationally processed by cellular and viral proteases, including the NS3/4A protease complex, into the structural and nonstructural proteins of the virus (21). The multifunctional NS3/4A protease processes the nonstructural proteins of the H...

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“…With such a fundamental role in the regulation of cell growth and proliferation, it is perhaps not surprising that a number of viruses have been found to interact directly with the PI3K/Akt pathway to effect greater control of their replication within the host cell. In hepatitis C virus (HCV), expression of viral proteins, including NS3/4A, NS4B, and NS5A, results in the activation of the PI3K/Akt pathway (reviewed in reference 3), which is required for efficient virus replication (4). Similarly, influenza A virus activates the PI3K/ Akt pathway through viral nonstructural protein 1 (13).…”

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confidence: 99%

Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

Eden

Sharpe

White

et al. 2011

J Virol

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Viruses commonly use host cell survival mechanisms to their own advantage. We show that Akt, an important signaling kinase involved in cell survival, phosphorylates the RNA-dependent RNA polymerase (RdRp) from norovirus, the major cause of gastroenteritis outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the more prevalent norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs at a residue (Thr33) located at the interface where the RdRp finger and thumb domains interact and decreases de novo activity of the polymerase. This finding provides fresh insights into virus-host cell interactions.

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Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor–induced signal transduction by cleavage of the T‐cell protein tyrosine phosphatase†

Cited by 67 publications

References 26 publications

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

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

Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α ₀

Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

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Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor–induced signal transduction by cleavage of the T‐cell protein tyrosine phosphatase†

Cited by 67 publications

References 26 publications

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

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

Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α 0

Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

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Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α ₀