No abstract

The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.

Hepatitis C virus (HCV) isHepatitis C virus (HCV) is a leading cause of chronic liver disease, which affects around 170 million people worldwide. Infections are initially acute, and in many cases the symptoms are mild. However, around 80% of patients eventually develop a persistent chronic infection which can result in steatosis, fibrosis, cirrhosis, liver failure, and, in some cases, hepatocellular carcinoma. The main treatments currently available for chronically infected patients use a combination of pegylated alpha interferon and ribavirin, but these still result in a sustained antiviral response in only about 50% of genotype 1 infections (4). Consequently, more effective antivirals that target either the virus proteins directly or the host cell proteins required during HCV replication are currently being developed. In order to ensure that successful antivirals are generated, it is important that all aspects of the HCV life cycle and HCV-associated pathology are well understood.One way in which the different host processes that are an essential part of the HCV replication cycle can be studied is to investigate the effect that HCV infection has on cellular gene expression. RNA microarray hybridization is routinely used to investigate host gene expression and allows the entire transcriptomic profile of the cell to be characterized. Microarray analysis of HCV-infected cells can provide an insight into the genes involved in host cell antiviral responses, genes that are essential for the HCV replication cycle, and genes that contribute to HCV-associated liver pathology. Microarray expression profiling has already been used to study host gene expression in cells transfected with RNA encoding either individual HCV genes, HCV subgenomic replicons, or the full-length HCV genome. These studies have demonstrated that the replication of the HCV genome results in the regulation of a small number of host genes involved in lipid metabolism, cellular immunity, proliferation, apoptosis, and molecular transport (2,5,15,32). These studies have provided interesting insights into the HCV replication cycle. However, the biological significance of gene expression patterns identified is less clear, since the full virus replication cycle, including the processes of viral entry, assembly, and exit, does not take place.The recent discovery of JFH-1, a genotype 2a HCV clone that can undergo a complete infection cycle in cell culture, provides the opportunity to characterize the true effect of HCV infection on host gene expression (51). A recent study investigated the effects that a J6/JFH-1 chimera had on the gene expression profile of Huh7.5 cells during a time course infection with time points of 24, 48, 72, 96, and 120 h (53). The number of host genes regulated during infection was much higher than that previously observed for cells permitting only genome replication, indicating that the full replication cycle has additional effects on host gene expression.In this study, we present the results from an investigation

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication ; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg 2M and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV-C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV-A or GBV-B sequences, two other recently described flaviviruses. Forty-five per cent of the HGV-infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co-infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon-alpha, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV-C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

Transgene expression was demonstrated following intraportal infusion of recombinant baculovirus vectors in combination with sCR1; however, our experiments suggest a significant associated toxicity.

This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.