Steven A. Weinman

Hepatitis C Virus Core Protein Inhibits Mitochondrial Electron Transport and Increases Reactive Oxygen Species (ROS) Production

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca2+ uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C.

Tumor Necrosis Factor-α-inducible IκBα Proteolysis Mediated by Cytosolic m-Calpain A MECHANISM PARALLEL TO THE UBIQUITIN-PROTEASOME PATHWAY FOR NUCLEAR FACTOR-κB ACTIVATION

The cytokine tumor necrosis factor α (TNF-α) induces expression of inflammatory gene networks by activating cytoplasmic to nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor. NF-κB activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, IκBα, through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible IκBα proteolysis in HepG2 liver cells mediated by cytosolic calcium-activated neutral protease (calpains). Pretreatment with either calpain- or proteasome-selective inhibitors partially blocks up to 50% of TNF-α-inducible IκBα proteolysis; pretreatment with both is required to completely block IκBα proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-α-inducible NF-κB-dependent promoter activity and IκBα proteolysis. In TNF-α-stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity is measured using a specific fluorescent assay. Inducible calpain proteolytic activity occurs coincidentally with the particulate-to-cytosol redistribution of the catalytic m-calpain subunit into the IκBα compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent IκBα proteolysis and NF-κB translocation. As additional evidence for calpain-dependent IκBα proteolysis and NF-κB activation, we demonstrate that this process occurs in a cell line (ts20b) deficient in the ubiquitin-proteasome pathway. Following inactivation of the temperature-sensitive ubiquitin-activating enzyme, IκBα proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-α activates cytosolic calpains, a parallel pathway that degrades IκBα and activates NF-κB activation independently of the ubiquitin-proteasome pathway.

Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earles balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.

Biophysical Properties of ClC-3 Differentiate It from Swelling-activated Chloride Channels in Chinese Hamster Ovary-K1 Cells

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl− > I− selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.

Intracellular Proton Conductance of the Hepatitis C Virus p7 Protein and Its Contribution to Infectious Virus Production

The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process.

Hepatitis C virus core protein increases mitochondrial ROS production by stimulation of Ca2+ uniporter activity

Many viruses have evolved mechanisms to alter mitochondrial function. The hepatitis C virus (HCV) produces a viral core protein that targets to mitochondria and increases Ca2+‐dependent ROS production. The aim of this study was to determine whether cores effects are mediated by changes in mito‐chondrial Ca2+ uptake. Core expression caused enhanced mitochondrial Ca2+ uptake in response to ER Ca2+ release induced by thapsigargin or ATP. It also increased mitochondrial superoxide production and mitochondrial permeability transition (MPT). Incubating mouse liver mitochondria with an HCV core (100 ng/mg) in vitro increased Ca2+ entry rate by ~ 2‐fold. Entry was entirely inhibited by the mitochondrial Ca2+ uniporter inhibitor, Ru‐360, but not influenced by an Na+/Ca2+ exchanger inhibitor or ROS scavengers. These results indicate that core directly increases mito‐chondrial Ca2+ uptake via a primary effect on the uniporter. This enhanced the ability of mitochondria to sequester Ca2+ in response to ER Ca2+ release, and increased mitochondrial ROS production and MPT. Thus, the mitochondrial Ca2+ uniporter is a newly identified target for viral modification of cell function.—Li, Y., Boehning, D. F., Qian, T., Popov, V. L., Weinman, S. A. Hepatitis C virus core protein increases mitochondrial ROS production by stimulation of Ca2+ uniporter activity. FASEB J. 21, 2474–2485 (2007)

GB Virus B Disrupts RIG-I Signaling by NS3/4A-Mediated Cleavage of the Adaptor Protein MAVS

ABSTRACT Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.

Antioxidants as therapeutic agents for liver disease.

Oxidative stress is commonly associated with a number of liver diseases and is thought to play a role in the pathogenesis of chronic hepatitis C, alcoholic liver disease, non‐alcoholic steatohepatitis (NASH), haemochromatosis and Wilsons disease. Antioxidant therapy has thus been considered to have the possibility of beneficial effects in the management of these liver diseases. Despite this promise, antioxidants have produced mixed results in a number of clinical trials of efficacy. This review summarizes the results of clinical trials of antioxidants as sole or adjuvant therapy of chronic hepatitis C, alcoholic liver disease and non‐alcoholic steatohepatitis (NASH). Overall, the most promising results to date are for vitamin E therapy of NASH but some encouraging results have been obtained with antioxidant therapy of acute alcoholic hepatitis as well. Despite evidence for small reductions of serum alanine aminotransferase, there is as yet no convincing evidence that antioxidant therapy itself is beneficial to patients with chronic hepatitis C. Problems such as small sample size, short follow up duration, inadequate endpoints, failure to demonstrate tissue delivery and antioxidant efficacy, and heterogeneous nature of the ‘antioxidant’ compounds used have complicated interpretation of results of the clinical studies. These limitations and their implications for future trial design are discussed.

Causes and consequences of mitochondrial reactive oxygen species generation in hepatitis C

Hepatitis C virus has developed mechanisms to alter the redox state of hepatocytes and this is associated with changes in mitochondrial structure and function. Chronic hepatitis C patients manifest hepatic oxidative stress and this is exacerbated by alcohol consumption and associated with fibrosis progression. Several viral proteins, including core and NS5a appear to contribute to reactive oxygen species (ROS) generation by mechanisms that involve both mitochondria and endoplasmic reticulum (ER). Hepatitis C virus (HCV) core protein localizes to both ER and mitochondria and has effects at both sites. At the mitochondria a chain of events is initiated by core binding, which consists of increased Ca2+ uptake, increased mitochondrial superoxide production, oxidation of the mitochondrial glutathione pool, inhibition of electron transport complex I activity, and sensitization of mitochondria to Ca2+‐ and ROS‐induced membrane permeability transition. These effects have been observed in isolated mitochondria, cells bearing full‐length HCV replicons, and liver mitochondria derived from HCV transgenic mice. In addition to these direct effects on mitochondria, core protein has been shown to causes a state of ER stress and an increase in the efficiency of ER to mitochondria Ca2+ transfer. The resulting oxidized redox state has a number of potential consequences for liver function. It interferes with the antiviral innate immune responses and potentiates fibrosis and carcinogenesis. Alcohol exacerbates these effects by increasing core‐induced ROS production, further oxidizing the mitochondrial glutathione pool. The resulting mitochondrial effects may contribute to liver injury and oxidative stress seen in chronic hepatitis C.

The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity

UNLABELLED Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870±180U/L) and centrilobular necrosis at 6h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. CONCLUSIONS the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.