Doris Fernholz

Replicating and virion secreting hepatitis B mutant virus unable to produce preS2 protein

Mutant HBV genomes unable to produce preS2 protein were identified in the serum of a highly viremic chronic carrier. By direct sequencing of the amplified preS region and by sequencing of 50 cloned amplified preS DNA fragments all molecules were found to have deleted the preS2 translation initiation codon and three amino acids 54 nucleotides downstream thereof. In addition, numerous amino acid changes, predominantly located between both deletions, were revealed. HBV-DNA genomes containing the mutated preS sequences were shown to be replication competent and to secrete efficiently virions that were morphologically and by the type of DNA encapsidated not distinguishable from wild-type virus. These data demonstrate that HBV with mutated preS sequences and unable to express preS2 protein can occur as a dominant or exclusive virus population in a highly viremic chronic carrier. The data also show that expression of the preS2 protein is not essential for HBV replication, virion morphogenesis and secretion.

Mechanism, kinetics, and role of duck hepatitis B virus e-antigen expression in vivo

No duck hepatitis B virus (DHBV) pre-C transcript has been identified so far, and neither the interrelationship of e-antigen (DHBeAg) with the expression of other viral antigens or virus replication nor its function is known. In this study we identified in infected livers a minor transcript from which the precursor protein of DHBeAg could be synthesized. Mutation of the first AUG on this transcript abolished expression of DHBeAg. DHBV genomes containing this mutation were infectious in Pekin ducks, the kinetics of pre-S envelope protein expression and virus secretion were not significantly different from wild-type, and the mutant genomes did not revert to wild-type to a detectable level after several passages. In contrast to pre-S protein, the level of DHBeAg in the serum was independent of the level of viremia, accumulated gradually to a high and constant level after a lag phase, and was also easily detectable in a mixed infection containing less than 0.1% of wild-type in a pre-C mutant virus containing inoculum. These data indicate that precore protein is synthesized from a minor pre-C mRNA with translation initiation at the pre-C AUG codon, and leads to high levels of DHBeAg rather late in infection. High levels of DHBeAg can even be produced efficiently by a very small subpopulation of wild-type virus in a mixed infection with predominantly pre-C mutant virus. Lack of DHBeAg appears to have no effect on DHBV viability and kinetics of virus secretion into the bloodstream when ducklings are infected with the pre-C AUG mutant virus a few days after birth.

Characterization of infectious and defective cloned avian hepadnavirus genomes

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.