Hepatitis C virus (HCV) infection induces a state of oxidative stress that is more pronounced than that in many other inflammatory diseases. In this study we used well-characterized cell lines inducibly expressing the entire HCV open-reading frame to investigate the impact of viral protein expression on cell bioenergetics. It was shown that HCV protein expression has a profound effect on cell oxidative metabolism, with specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, increased production of reactive oxygen and nitrogen species, as well as loss of the Pasteur effect. Importantly, all these effects were causally related to mitochondrial calcium overload, as inhibition of mitochondrial calcium uptake completely reversed the observed bioenergetic alterations. H epatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting more than 170 million people worldwide. 1 There is no protective vaccine, and current therapies show limited efficacy for many patients with chronic hepatitis C. 2 HCV is an enveloped, positive-strand RNA virus. Its genome encodes a polyprotein of more than 3,000 amino acids that is cleaved co-and posttranslationally at the endoplasmic reticulum (ER) by host and viral proteases, yielding 3 structural (core, E 1 , and E2) and 7 nonstructural (p7, NS2 to NS5B) proteins. 3 HCV nonstructural proteins, together with replicating viral RNA and altered cellular membranes, form a membrane-associated replication complex. 3 A minor fraction of the viral proteins has been found to localize to mitochondria, 4-7 where the NS3-4A complex inactivates a newly discovered mitochondrion-dependent innate immunity antiviral signaling pathway. 8,9 A mounting body of evidence points toward alterations of mitochondrial oxidative metabolism by HCV, albeit the mechanism has remained elusive. 10,11 In the present study we used tetracycline-regulated cell lines 12 to analyze the impact of HCV protein expression on mitochondrial bioenergetics. 13 These cell lines allow the regulated expression and correct processing of all viral proteins in a well-defined cellular context. The data reported here extend our previous preliminary observations 14 and provide new mechanistic insight, correlating mitochondrial oxidative stress to the deregulation of ER stress-dependent Ca 2ϩ homeostasis by HCV proteins. Materials and Methods Cell

Hepatitis C virus (HCV) infection induces

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The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H 2 O 2 and O 2 . in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.Deficiency of complex I (NADH ubiquinone oxidoreductase, EC 1.6.5.3) of the respiratory chain is a major cause of inborn mitochondrial disease (1-6). Leigh syndrome (early onset fatal neurodegenerative disorder) and Leigh syndrome-like disease are the most common clinical phenotypes associated with complex I deficiency. Impairment of complex I has also been reported in Parkinson (7), Alzheimer (8, 9), and Huntington (10) diseases.Complex I is the largest of the respiratory chain enzymes, being composed of seven mitochondrial DNA and at least 39 nuclear DNA-encoded subunits (11). Mutations in structural subunits have been found in ϳ40% of the patients with inborn deficiency of complex I. Reported mutations include all of the mitochondrial DNA-encoded subunits (12) and twelve nuclear-encoded subunits (3, 6, 13-15).The nuclear NDUFS4 gene codes for an 18-kDa subunit of the complex (11), which in high eukaryotes contains potential phosphorylation sites for cAMP-dependent protein kinase in both the presequence and the carboxyl-terminal region (EMBL Data Bank). In mammalian (16 -18) and human (19) cell cultures cAMP promotes the phosphorylation of the NDUFS4 protein and enhances the functional capacity of complex I. Three recessive mutations in the nuclear NDUFS4 gene have been identified in three unrelated children affected by Leigh syndromelike syndrome with deficiency of complex I, including an AAGTC duplication at position 466 -470 in exon 5 (20), a single base deletion at position 289/290 in exon 3 (21), and a G44A nonsense mutation in the first exon of the gene, introducing a premature termination codon in the sequence coding for the mitochondrial leader peptide (13). All three mutations resulted in the disappearance of the 18-kDa subunit and defect in both the activity and assembly of the complex (22). In the 289/290 deletion in exon 3, whic...

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.