the level of gene expression. Thus, oligodeoxynucleotides The hepatitis B virus (HBV) nucleocapsid consists of were successfully used to block protein expression and inhibit 240 viral core proteins that are arranged in a highly hepadnaviral replication in vivo. 3 Moreover, hepadnaviral symmetrical structure. HBV replication can only take RNA was shown to be a suitable target for cleavage by riboplace inside intact nucleocapsids. In the present study, nucleic acid enzymes (ribozymes) in vitro. 4 While promising, we investigated whether genetically engineered core these nucleic acid-based strategies are limited by the inhermutants can inhibit viral replication by interfering with ent sequence variability of HBV and the coexistence of multithe formation of intact nucleocapsids. Using the duck ple related but nonidentical virus strains (viral quasispecies) hepatitis B virus (DHBV) model, a series of core protein in a single host. 5 In this context, the functional inactivation mutants was generated. Polymerase chain reaction-amof viral proteins by modified viral gene products, also termed plified fragments from the bacterial lacZ gene expressdominant negative (DN) mutants, has gained substantial ining up to 282 amino acids were added either to the terest. DN mutants have been successfully applied to inhibit amino-or carboxy-terminus of the DHBV core protein.
replication of herpes simplex virus 6 and human immunodefi-In addition, carboxy-terminal extensions were generciency virus. 7 DN mutants have also been described for hepated by fusing the DHBV core protein with the DHBV adnaviruses. [8][9][10] In particular, a preliminary study by Delasmall surface protein or various fragments of the viral ney et al. 8 and a recent report by Scaglioni et al. 10 showed polymerase. Finally, the green fluorescent protein (GFP) that the core proteins of the woodchuck hepatitis virus and was fused in-frame to the carboxy-terminus of the DHBV HBV, when fused at their truncated C-terminus to the viral core protein. In this chimeric protein, GFP is still funcsmall surface antigen, efficiently inhibit viral replication. The tional and can act as a reporter molecule. The various hepadnaviral core protein represents a particularly promiscore protein mutants were tested for their potential antiing target for DN mutants because it is central to several viral activity by cotransfection with a replication-comfunctions critical for viral replication: it is the building block petent DHBV construct into the avian hepatoma cell line of the viral nucleocapsid and plays an essential role in both LMH. Carboxy-terminal, but not amino-terminal, DHBV packaging of the RNA pregenome into core particles and the core mutants inhibited DHBV replication by up to 90% subsequent maturation of viral DNA. 11-16 In the present reat an effector-to-target ratio of 1:10, thus displaying a port, we addressed the question whether DN core proteins dominant negative phenotype. Antiviral activity was are functional among all hepadnaviruses and determined species-specific and caused by posttranslational interstructural and sequence requirements for their design. ference with viral replication. The DHBV core-GFP fusion protein should be an ideal tool to assess the antivi-MATERIALS AND METHODS ral potential of dominant negative core proteins in vivo. (HEPATOLOGY 1996;24:294-299.)
Constructs Fusion Proteins of DHBV Core and Fragments of the Bacterial lacZ
Hepatitis B virus (HBV) infection is endemic throughout
Protein. Fragments spanning amino acids (aa) 7-70, 7-125, 7-185, much of the world, with an estimated 400 million persistently and 7-284 of the b-galactosidase gene of Escherichia coli (lacZ) 17 infected people. HBV infection is associated with a wide specwere amplified by polymerase chain reaction. Primer sequences and trum of clinical presentations, ranging from the healthy caramplification conditions are available from the authors upon request. rier state to acute or chronic hepatitis, as well as cirrhosis and A single 5-primer containing an Avr II and EcoR V site was used hepatocellular carcinoma. 1 Therapy of chronic HBV infection for all polymerase chain reaction reactions. Individual 3-primers still poses a major problem. Currently, interferon alfa is the used for the amplification of lacZ fragments for C-terminal addition treatment of choice, yielding long-term suppression of viral to the duck hepatitis B virus (DHBV) core protein (subtype F1-6) 18 contained an in-frame stop codon and a BamH I cloning site. Amplifi-replication in 30% to 40% of selected patients. 1,2 Hence, other cation of lacZ fragments for N-terminal addition to the DHBV core therapeutic strategies are clearly needed. Recent experimenprotein were performed with 3-primers containing DHBV core setal approaches include interference with viral replication at quences from EcoR V (pos. 2650) up to and including the XbaI site (pos. 2662).
Expression vectors encoding DHBV core proteins with N-or Cterminal lacZ additions were constructed as follows: for N-terminal Abbreviations: HBV, hepatitis B virus; DN, dominant negative; aa, amino acid; DHBV, additions, the expression vector for DHBV core (''wt''; plasmid T7 duck hepatitis B virus; pTC, plasmid T7 promoter cytomegalovirus; GFP, green fluorescent protein; HTD, head-to-tail dimer; CAT, chloramphenicol acetyl transferase.
promoter cytomegalovirus [pTC]-Dcore) 19 was opened at the N-ter-From the