Michael J. McGarvey

MUTATION PREVENTING FORMATION OF HEPATITIS B e ANTIGEN IN PATIENTS WITH CHRONIC HEPATITIS B INFECTION

Some patients with chronic hepatitis B virus (HBV) infection are HB e antigen (HBeAg) negative, have circulating HBV particles, and often have especially severe chronic hepatitis. To test the hypothesis that the absence of HBeAg production may be due to a change in the nucleotide sequence of the pre-core region of the genome, 18 Greek and 3 non-Greek patients positive for HB surface antigen underwent direct sequencing of HBV-DNA amplified from sera. In 7 out of 8 HBeAg negative patients, two mutations (guanosine to adenosine) were found in the terminal two codons of the pre-core region, giving the sequence TAGGACATG. The remaining patient had the first mutation only. The sequence TGGGGCATG was found in 4 of 5 of the HBeAg positive patients. The first mutation results in a translational stop codon that is predicted to result in failure to produce HBeAg. The rest of the pre-core region in the HBeAg negative patients was otherwise homologous to that of the HBeAg positive patients and to known sequences.

Intracellular localization of the hepatitis B virus HBx protein

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.

Gene Expression Profiling Indicates the Roles of Host Oxidative Stress, Apoptosis, Lipid Metabolism, and Intracellular Transport Genes in the Replication of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.

Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein.

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 Mg2+ 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.

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors

There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required.

Respiratory syncytial virus RNA in cells from the peripheral blood during acute infection

It is not known whether respiratory syncytial virus spreads beyond the respiratory tract. With the use of reverse transcriptase polymerase chain reaction, we found viral and transcribed RNA in cells from the arterial blood of four children with bronchiolitis but none in serum or cerebrospinal fluid. Respiratory syncytial virus might therefore spread outside the respiratory tract.

Complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice

The baculovirus Autographa californica nucleo‐polyhedrosis virus (AcNPV) is an alternative to other viral vectors for hepatic gene delivery. A barrier to AcNPV being used in vivo is its susceptibility to inactivation by serum complement 1 . In vivo utility has only been demonstrated using methods that avoid contact with serum 2–5 . We have studied the complement pathways involved in baculovirus inactivation in vitro and the systemic administration of baculovirus vectors in vivo, with the co‐administration of the complement inhibitor, soluble complement inhibitor 1 (sCR1).

Hepatitis G virus infection: clinical characteristics and response to interferon

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‐α, 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.

Subcellular localisation of NS3 in HCV-infected hepatocytes

Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti‐NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV‐infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection. J. Med. Virol. 59:456–462, 1999.

The GB hepatitis viruses

Summary. The genomes of three new flavi‐like viruses, GBV‐A, GBV‐B and GBV‐C have been identified. Nucleic acid molecules corresponding to the genomes of GBV‐A and GBV‐B were isolated from tamarins with hepatitis which had been infected with the GB agent. RNA sequences corresponding to GBV‐C have been shown to be present in sera from humans with non‐A‐E hepatitis. Sequence comparisons show that these three viruses are more closely related to each other and to hepatitis C virus (HCV) than to any other known viruses. Together with HCV they appear to form a discrete cluster of related viruses within the larger genus of flaviviridae. The pathological significance of these viruses and their association with hepatitis is currently emerging.