Infection with hepatitis E virus (HEV) is a significant cause of morbidity and mortality, representing an important global health problem. Our understanding of HEV has changed completely over the past decade. Previously, HEV was thought to be limited to certain developing countries. We now know that HEV is endemic in most high-income countries and is largely a zoonotic infection. Given the paradigm shift in our understanding of zoonotic HEV and that locally acquired HEV is now the commonest cause of acute viral hepatitis in many European countries, the focus of these Clinical Practice Guidelines will be on HEV genotype 3 (and 4).
The development and clinical implementation of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C. Infection with any hepatitis C virus (HCV) genotype can now be eliminated in more than 95% of patients with short courses of all-oral, well-tolerated drugs, even in those with advanced liver disease and liver transplant recipients. DAAs have proven so successful that some now consider HCV amenable to eradication, and continued research on the virus of little remaining medical relevance. However, given 400,000 HCV-related deaths annually important challenges remain, including identifying those who are infected, providing access to treatment and reducing its costs. Moreover, HCV infection rarely induces sterilizing immunity, and those who have been cured with DAAs remain at risk for reinfection. Thus, it is very unlikely that global eradication and elimination of the cancer risk associated with HCV infection can be achieved without a vaccine, yet research in that direction receives little attention. Further, over the past two decades HCV research has spearheaded numerous fundamental discoveries in the fields of molecular and cell biology, immunology and microbiology. It will continue to do so, given the unique opportunities afforded by the reagents and knowledge base that have been generated in the development and clinical application of DAAs. Considering these critical challenges and new opportunities, we conclude that funding for HCV research must be sustained.
Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. Alpha interferon (IFN-α) therapy of chronic hepatitis C leads to a sustained response in 10 to 20% of patients only. The mechanisms of viral persistence and the pathogenesis of hepatitis C are poorly understood. We established continuous human cell lines, allowing the tightly regulated expression of the entire HCV open reading frame under the control of a tetracycline-responsive promoter. Using this in vitro system, we analyzed the effect of HCV proteins on IFN-induced intracellular signaling. Expression of HCV proteins in these cells strongly inhibited IFN-α-induced signal transduction through the Jak-STAT pathway. Inhibition occurred downstream of STAT tyrosine phosphorylation. Inhibition of the Jak-STAT pathway was not restricted to IFN-α-induced signaling but was observed in leukemia inhibitory factor-induced signaling through Stat3 as well. By contrast, tumor necrosis factor alpha-induced activation of the transcription factor NF-κB was not affected. Interference of HCV with IFN-α-induced signaling through the Jak-STAT pathway could contribute to the resistance to IFN-α therapy observed in the majority of patients and may represent a general escape strategy of HCV contributing to viral persistence and pathogenesis of chronic liver disease.
The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.
Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.
Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.
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