Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C.

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.

Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes.

BACKGROUND/AIMS: The hepatitis C virus NS5A protein is phosphorylated by several cellular kinases, including casein kinase 2 (CK2). Little is known about CK2 phosphorylation of NS5A from different HCV genotypes and clinical isolates. METHODS: NS5A from patients with HCV-1a (24 cases), HCV-1b (9) or HCV-3 (16) was analyzed by direct sequencing and CK2 phosphorylation sites were defined using a well-validated prediction rule. In vitro phosphorylation assays were performed using recombinant CK2 and synthetic peptides or full-length NS5A. In vivo phosphorylation by endogenous CK2 of NS5A expressed in hepatoma cells was also investigated. RESULTS: The mean number of CK2 sites within full-length NS5A, was significantly higher in HCV-3 compared to HCV-1a (P<0.01) and HCV-1b (P<0.01). The number of CK2 sites was more homogeneous in HCV-3 variants compared to HCV-1a and HCV-1b variants (P<0.05). The number of predicted CK2 sites correlated with the degree of in vitro and in vivo phosphorylation of NS5A by CK2. CONCLUSIONS: CK2-dependent phosphorylation of NS5A is heterogeneous among different HCV genotypes and clinical isolates. This might have an influence on virus biology and pathogenicity.

Impact of NS5A sequences of Hepatitis C virus genotype 1a on early viral kinetics during treatment with peginterferon- alpha 2a plus ribavirin.

UNLABELLED: BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. METHODS: Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. RESULTS: At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. CONCLUSIONS: Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.

BACKGROUND & AIMS: Hepatic steatosis is frequent in chronic hepatitis C. Several mechanisms might be implicated, including metabolic cofactors and direct viral effects on intracellular lipid pathways. In a transgenic mouse model, hepatitis C virus (HCV) was shown to inhibit microsomal triglyceride transfer protein (MTP) activity, which is essential for hepatic lipoprotein assembly and secretion. No data are available on liver MTP activity in HCV-infected patients. We therefore investigated liver MTP gene expression and its lipid transfer activity in untreated cases infected with the major HCV genotypes showing variable degrees of hepatic steatosis. METHODS: MTP messenger RNA (mRNA) levels were measured by real-time polymerase chain reaction, and MTP activity was assessed by fluorescent assay in liver biopsy specimens of 58 HCV-positive patients. A set of metabolic and serum lipid markers was also measured at the time of liver biopsies. RESULTS: MTP mRNA levels showed a statistically significant (P = .001) inverse correlation with the degree of steatosis, independently of the HCV genotype. MTP mRNA levels also had an inverse correlation with serum insulin (P = .0002), homeostasis model assessment-insulin resistance (HOMA-IR) (P = .005), and body mass index (P = .02) in patients with HCV-1 and HCV-2 and with serum HCV-RNA (P = .02) in HCV-3 patients. Liver MTP-specific activity was significantly reduced in HCV-3 patients compared with those with other HCV genotypes (P = .004) and correlated with reduced serum cholesterol, apo B, and low-density lipoproteins. CONCLUSIONS: MTP may play a central role in HCV-related steatosis, being modulated by different genotype-specific mechanisms, mainly hyperinsulinemia in non-HCV-3 patients, and more profound and direct virus-related effects in HCV-3-infected individuals.

Hepatitis C minimal residual viremia (MRV) detected by TMA at the end of Peg-IFN plus ribavirin therapy predicts post-treatment relapse.

BACKGROUND/AIMS: Around 15-25% of chronic hepatitis C patients treated with Peg-IFN plus ribavirin become HCV-RNA negative by PCR during therapy but relapse after its withdrawal. We investigated whether minimal residual viremia (MRV) might be detected in these cases by Transcription-Mediated Amplification (TMA). METHODS: Two hundred and ninety-two consecutive patients (143 HCV-1, 82 HCV-2, 56 HCV-3 and 11 HCV-4) were prospectively treated with a standard schedule of Peg-IFNalpha 2b plus ribavirin combination and end-of-therapy response was assessed by conventional PCR using 2 protocol serum samples obtained 6-8h before the last two scheduled weekly injections of Peg-IFN. PCR negative samples were re-tested by TMA and the results were then correlated with the virological outcome after therapy withdrawal. RESULTS: Among 208 patients who were repeatedly HCV-RNA negative by PCR at the end-of-therapy, 26 (12.5%) were found HCV-RNA positive by TMA. Twenty-two of them, (96%) were PCR-relapsers after therapy withdrawal, compared to only 14% of the 182 TMA negative patients (P<0.0001). This virological profile was more frequent in HCV-1 and HCV-3 infected patients and correlated with a slower virological response during therapy. CONCLUSIONS: At the end of Peg-IFN plus ribavirin therapy, TMA is superior to PCR in identifying patients with sustained HCV-RNA clearance.

PKR gene expression and response to pegylated interferon plus ribavirin therapy in chronic hepatitis C.

Pegylated interferon (PEG-IFN) alpha combined with ribavirin is the current standard treatment for hepatitis C, but around 50% of patients do not respond for reasons that are not fully understood. To explore the regulation of IFN-inducible protein kinase (PKR), we have measured PKR mRNA levels in peripheral blood mononuclear cells (PBMCs) and in liver biopsies from patients with chronic hepatitis C. PBMCs were also analysed after in vitro incubation with IFN and during antiviral therapy. Non-responders to PEG-IFN plus ribavirin had pre-treatment PKR mRNA levels in PBMCs (0.1+/-0.0074) and in liver (0.102+/-0.051) that were significantly higher than those of responders (PBMCs: 0.023+/-0.014, P=0.0005; liver: 0.034+/-0.020; P=0.0002). On the other hand, PKR mRNA levels in PBMCs were similar in non-responders and in responders after in vitro exposure to IFN (0.434+/-0.301 vs 0.403+/-0.222; P=NS) and during therapy (0.31+/-0.10 vs 0.30+/-0.12; P=NS). These results indicate that in hepatitis C, non-responsiveness to IFN-alpha is associated with pre-treatment up-regulation of the PKR gene, evidence that the infecting hepatitis C virus is able to stimulate endogenous IFN production, being resistant to its antiviral effect. On the other hand, the PKR gene response to exogenous IFN was similar in responders and non-responders, at least in PBMCs, suggesting that variations in its activation are not major determinants of the outcome of antiviral treatment.

Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation.

BACKGROUND/AIMS: HCV-CORE protein has been implicated in the regulation of apoptosis of infected cells acting as full-length or C-terminus deleted forms and resulting in both proapoptotic and antiapoptotic effects in different experimental conditions. METHODS: We have fused full-length and C-terminus deleted CORE with GFP to assess intracellular localization in transiently transfected cell lines and primary hepatocytes. Apoptosis of cells expressing different levels of chimeric proteins was quantified by cytometry. RESULTS: Full-length CORE localized mainly in the cytoplasm, but nuclear staining was also observed, being more evident in primary human hepatocytes. Nuclear staining only was observed in cells expressing truncated CORE. Full-length CORE induced apoptosis in approximately 15-20% of transfected cells with low expression and in approximately 40-50% of those with high expression of viral protein. Interestingly, 40-50% of cells transfected with truncated CORE underwent apoptosis, independently of protein expression levels. CORE-induced apoptosis was significantly reduced in the presence of a protein kinase R (PKR) inhibiting peptide and truncated CORE was able to enhance translocation of PKR into nucleoli where CORE/PKR colocalization was observed. CONCLUSIONS: These results suggest that nuclear forms of HCV-CORE are generated in vivo in primary hepatocytes and induce PKR-dependent apoptosis, a mechanism that might have a relevant role during natural infection.