We have used linker scanning and site-directed mutagenesis in an attempt to distinguish among the known functions of the duck hepatitis B virus large envelope protein, p36. We found that linker-encoded amino acid substitutions in at least one region of the pre-S envelope protein p36 produced defects in both the production of enveloped virus and the regulation of covalently closed circular DNA (cccDNA) synthesis. Most linker substitutions, typically in the 5' two-thirds of the pre-S region of the p36 gene did not affect either cccDNA regulation or enveloped virus production but did destroy the infection competence of the enveloped particles produced. Single amino acid substitutions of residues 128 and 131 demonstrated a similar correlation between defects in the ability of p36 to support enveloped virus production and to control cccDNA levels. We concluded from these studies that virus production and cccDNA regulation probably require a common activity of p36.

Hepadnaviruses cause persistent noncytopathic infections of hepatocytes in humans and other animals. Virus replication depends on the pool of viral covalently closed circular DNA (cccDNA) molecules, which serve as transcriptional templates in the nuclei of infected cells. The size of this pool of cccDNA molecules is regulated by the ability of the large envelope protein of the virus to direct newly synthesized viral DNAs into a pathway for viral secretion and thereby inhibit their utilization for viral cccDNA synthesis. In this study, we showed that single amino acid changes in the large envelope protein could cause profound changes in cccDNA levels in transfected permissive cells or in infected cultured hepatocytes. While defects in cccDNA regulation were accompanied by a decrease of enveloped virus production in transfected cells, primary hepatocytes infected by such mutant viruses transiently produced wild-type or higher levels of enveloped virus. Moreover, high levels of cccDNA were always associated with cytopathic effects in the infected hepatocytes. The results demonstrate that the large envelope protein promotes persistent noncytopathic infection of hepatocytes by acting as an overall repressor of virus replication.

Several examples of human hepatitis B virus strains with enhanced replication in vitro have been described. To understand whether this characteristic could be a cause of liver disease, we have studied a variant of the closely related duck hepatitis B virus (DHBV) that had enhanced levels of cccDNA accumulation, previously shown to be cytopathic in vitro, as a model for the pathogenesis of analogous viruses in humans. In vivo liver damage caused by this variant (G133E) occurred only during the first 2 weeks p.i., after which time cccDNA levels and liver histology returned to near normal despite continued virus replication. To determine whether recovery was due to the emergence of noncytopathic revertant, we tested whether wild-type virus would have a selective advantage in competition with the cytopathic mutant in a fully infected liver. In a mixed infection of ducklings with G133E and a small amount of wild-type virus, the wild-type virus was detected as the predominant genotype after recovery of normal liver histology. Two candidate revertant viral genomes were cloned directly from the serum virus of G133E-infected birds after recovery and tested for (i) control of cccDNA levels in primary hepatocyte cultures and (ii) their ability to compete with wild-type virus in a mixed infection. At least one noncytopathic revertant was identified by these two criteria. The results support the conclusion that the recovery from liver damage in G133E-infected ducklings was due to the emergence of spontaneous noncytopathic revertants rather than to host suppression of virus cytotoxicity. The results indicate that acute liver injury may result from infection with a cytopathic hepadnavirus but that such viruses may be rapidly replaced by noncytopathic variants during persistent infection.

A variant avian hepadnavirus that has been shown to destroy hepatocytes in vitro was found to be cytopathic in vivo. A single amino acid change of glycine to glutamic acid at position 133 (G133E) in the preS protein of duck hepatitis B virus (DHBV) caused an increase in the intranuclear pool of viral covalently closed circular DNA (cccDNA), resulting in a transient elevation of viral replication and eventual hepatocyte destruction. In vivo viral infection with the G133E virus was compared with infection with wild-type virus over a 72-day period. Birds were inoculated with virus at day 2 post-hatch to ensure a high percentage of infected hepatocytes and potential persistence of virus. Birds infected with the G133E virus had increased periportal cellular proliferation and numerous lysed apoptotic hepatocytes following 100% infection of hepatocytes. The liver damage within G133E virus-infected birds subsided over time, resulting in mild chronic hepatitis that was similar to that observed within wild-type virusinfected birds. The subsidence of liver damage in G133E virus-infected birds coincided with a reduction of viral cccDNA to wild-type virus levels in the liver. Our study indicates that maintenance of wild-type levels of viral cccDNA promotes persistence of virus infection by establishing a noncytopathic infection. (HEPATOLOGY 1999;29:563-571.)Hepadnaviruses cause persistent productive infections of each infected hepatocyte. During the initiation of infection, the relaxed circular DNA (rcDNA) genome in the virion is converted to covalently closed circular DNA (cccDNA) in the nucleus. Viral cccDNA is used as the template for viral RNA transcription, resulting in the production of a viral RNA molecule (pregenome) that is greater than one genome in length. The RNA pregenome is encapsidated within a viral core particle, where it is reverse-transcribed by the viralencoded DNA polymerase to produce viral minus-strand DNA. 1 Plus-strand DNA synthesis is then carried out by the same viral DNA polymerase using the viral DNA minusstrand as a template. The final DNA product contained with the capsid is a double-stranded molecule that is held in a circular conformation by a short region of base-pairing between the 5Ј end of the two strands. This form of viral DNA is referred to as relaxed circular DNA, or rcDNA. The viral capsids that contain rcDNA associate with the viral envelope proteins at the endoplasmic reticulum and are secreted as virions. Alternatively, rcDNA molecules may enter into the nucleus of the infected hepatocyte, where they are converted to additional molecules of cccDNA and cause increased levels of viral replication. The production of new viral cccDNA is regulated by the viral large envelope protein (preS) by a mechanism that has not been biochemically defined. 2 Presumably, an interaction between the preS protein and rcDNAcontaining capsids is necessary for enveloped virus formation and promotes secretion of viral rcDNA from the hepatocyte as virions. Because defects in preS that specifically inhibit ...

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