Hepatitis B virus and hepatitis C virus affect mitochondrial function through different metabolic pathways, explaining virus-specific clinical features of chronic hepatitis.

BACKGROUND: Hepatitis C virus (HCV) and hepatitis B virus (HBV) cause chronic hepatitis with important clinical differences. HCV causes hepatic steatosis and insulin resistance, while HBV confers increased risk of liver cancer. We hypothesised these differences may be due to virus-specific effects on mitochondrial function. METHODS: Seahorse technology was utilised to investigate effects of virus infection on mitochondrial function. Cell based assays were used to measure mitochondrial membrane potential and quantify pyruvate and lactate. Mass spectrometry was performed on mitochondria isolated from HBV expressing, HCV infected and control cells cultured with isotope-labelled amino acids, to identify proteins with different abundance. Altered expression of key mitochondrial proteins was confirmed by real time PCR and western blot. RESULTS: Reduced mitochondrial function and ATP production were observed with HCV infection and HBV expression. HCV impairs glycolysis and reduces expression of genes regulating fatty acid oxidation, promoting lipid accumulation. HBV causes lactate accumulation by increasing expression of lactate dehydrogenase A, which converts pyruvate to lactate. In HBV expressing cells there was marked enrichment of pyruvate dehydrogenase kinase, inhibiting conversion of pyruvate to acetyl-CoA and thereby reducing its availability for mitochondrial oxidative phosphorylation. CONCLUSIONS: HCV and HBV impair mitochondrial function and reduce ATP production. HCV reduces acetyl-CoA availability for energy production by impairing fatty acid oxidation, causing lipid accumulation and hepatic steatosis. HBV has no effect on fatty oxidation but reduces acetyl-CoA availability by disrupting pyruvate metabolism. This promotes lactic acidosis and oxidative stress, increasing the risk of disease progression and liver cancer.

PEMT Mediates Hepatitis C Virus-Induced Steatosis, Explains Genotype-Specific Phenotypes and Supports Virus Replication.

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.

Calsyntenin-1 mediates hepatitis C virus replication.

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host-targeting agents.

Direct-acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication, and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown upregulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell-culture model. Here, we investigated whether CB1 blockade inhibited HCV replication. The antiviral effect of a CB1 antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), was examined in HCV strain JFH1 cell-culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 short hairpin RNA (shRNA) was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70 %), viral protein (~80 %), the production of new virus particles (~70 %) and virus infectivity (~90 %). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARα). This effect was mediated by AMP-activated protein kinase (AMPK). Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA compared with controls. Thus, reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drug with activity against HCV.

Negative autoregulation of GTF2IRD1 in Williams-Beuren syndrome via a novel DNA binding mechanism.

The GTF2IRD1 gene is of principal interest to the study of Williams-Beuren syndrome (WBS). This neurodevelopmental disorder results from the hemizygous deletion of a region of chromosome 7q11.23 containing 28 genes including GTF2IRD1. WBS is thought to be caused by haploinsufficiency of certain dosage-sensitive genes within the deleted region, and the feature of supravalvular aortic stenosis (SVAS) has been attributed to reduced elastin caused by deletion of ELN. Human genetic mapping data have implicated two related genes GTF2IRD1 and GTF2I in the cause of some the key features of WBS, including craniofacial dysmorphology, hypersociability, and visuospatial deficits. Mice with mutations of the Gtf2ird1 allele show evidence of craniofacial abnormalities and behavioral changes. Here we show the existence of a negative autoregulatory mechanism that controls the level of GTF2IRD1 transcription via direct binding of the GTF2IRD1 protein to a highly conserved region of the GTF2IRD1 promoter containing an array of three binding sites. The affinity for this protein-DNA interaction is critically dependent upon multiple interactions between separate domains of the protein and at least two of the DNA binding sites. This autoregulatory mechanism leads to dosage compensation of GTF2IRD1 transcription in WBS patients. The GTF2IRD1 promoter represents the first established in vivo gene target of the GTF2IRD1 protein, and we use it to model its DNA interaction capabilities.

Impact of an Open Access Nationwide Treatment Model on Hepatitis C Virus Antiviral Drug Resistance.

Direct acting antivirals (DAAs) have revolutionized hepatitis C virus (HCV) treatment, but drug resistance could undermine proposed global elimination targets. Real-world studies are needed to inform the impact of widespread DAA treatment on antiviral resistance in the community. The prevalence and range of posttreatment resistance-associated substitutions (RASs) was determined in Australian patients with open access to DAAs through a wide range of prescribers. NS3, NS5A, and NS5B regions were amplified by polymerase chain reaction and analyzed by population sequencing. Clinically relevant RASs were identified using online databases (ReCALL and Geno2Pheno[hcv]). Of 572 samples, 60% were from genotype 3 and 27% from genotype 1a. Ninety-two percent of people failed a DAA regimen containing an NS5A inhibitor, including 10% with a pangenotype regimen. NS5A RASs were detected in 72% of people with genotype 1 and 80% with genotype 3. For genotype 1, there was a range of RASs across the NS5A region, while for genotype 3, the Y93H RAS predominated (72%). The prevalence of NS3 RASs was higher in people exposed to an NS3 inhibitor (35% vs. 3.9%; P < 0.0001). NS5B resistance was rare, with a single case of sofosbuvir resistance. Multiclass drug resistance was found in 33% of people exposed to both NS3 and NS5A inhibitors. Conclusion: The high prevalence of NS5A RASs among people failing DAA therapy reinforces the importance of specific retreatment regimens, ideally guided by resistance testing. The impact of multiclass drug resistance on retreatment in people exposed to both NS3 and NS5A inhibitors needs to be assessed in real-world studies. Surveillance for increasing antiviral resistance during treatment scale-up is essential to maintain the efficacy of current DAA regimens.

Expression of Gtf2ird1, the Williams syndrome-associated gene, during mouse development.

The gene GTF2IRD1 is localized within the critical region on chromosome 7 that is deleted in Williams syndrome patients. Genotype-phenotype comparisons of patients carrying variable deletions within this region have implicated GTF2IRD1 and a closely related homolog, GTF2I, as prime candidates for the causation of the principal symptoms of Williams syndrome. We have generated mice with an nls-LacZ knockin mutation of the Gtf2ird1 allele to study its functional role and examine its expression profile. In adults, expression is most prominent in neurons of the central and peripheral nervous system, the retina of the eye, the olfactory epithelium, the spiral ganglion of the cochlea, brown fat adipocytes and to a lesser degree myocytes of the heart and smooth muscle. During development, a dynamic pattern of expression is found predominantly in musculoskeletal tissues, the pituitary, craniofacial tissues, the eyes and tooth buds. Expression of Gtf2ird1 in these tissues correlates with the manifestation of some of the clinical features of Williams syndrome.

Regulation of alternative splicing of Gtf2ird1 and its impact on slow muscle promoter activity.

A human MusTRD (muscle TFII-I repeat domain (RD)-containing protein) isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641-6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1-13]. The human gene encoding MusTRD, GTF2IRD1 ( WBSCR11 / GTF3 ), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams-Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279-284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296-304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription-PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3' region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.

hMusTRD1alpha1 represses MEF2 activation of the troponin I slow enhancer.

The novel transcription factor hMusTRD1alpha1 (human muscle TFII-I repeat domain-containing protein 1alpha1; previously named MusTRD1; O'Mahoney, J. V., Guven, K. L., Lin, J., Joya, J. E., Robinson, C. S., Wade, R. P., and Hardeman, E. C. (1998) Mol. Cell. Biol. 18, 6641-6652) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnIslow). It has been proposed that hMusTRD1alpha1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nucleotides -977 to -960) within the hTnIslow upstream enhancer-containing region that is necessary for slow fiber-specific expression. In this study we demonstrate that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1alpha1 regulate hTnIslow through the Inr-like element. Co-transfection assays in C2C12 cells and Cos-7 cells demonstrate that hMusTRD1alpha1 represses hTnIslow transcription and prevents MEF2C-mediated activation of hTnIslow transcription. Gel shift analysis shows that hMusTRD1alpha1 can abrogate MEF2C binding to its cognate site in the hTnIslow enhancer. Glutathione S-transferase pull-down assays demonstrate that hMusTRD1alpha1 can interact with both MEF2C and the nuclear receptor co-repressor. The data support the role of hMusTRD1alpha1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and the nuclear receptor co-repressor.