Effect of basal core promoter and pre-core mutations on hepatitis B virus replication

1 Department of Medicine, Imperial College, St Mary's Campus, London, UK 2 Department of Virology, Imperial College, St Mary's Campus, London, UK Correspondence Peter Karayiannis p.karayiannis{at}imperial.ac.uk There are two hypotheses explaining a fulminant outcome after hepatitis B virus (HBV) infection, both of which may be applicable at the same time: (i) basal core promoter (BCP) mutations increase viral replication, allowing rapid spread of the virus through the liver, and (ii) pre-core (pre-C) mutations abrogating hepatitis B e antigen (HBeAg) synthesis remove its tolerogenic effect, leading to a vigorous immune response. This study investigated the effect of these mutations on virus replication efficiency and HBeAg production. Substitutions A1762T/G1764A and T1753C, C1766T and T1768A in the BCP region, and G1896A and G1899A in the pre-C region, were examined either alone or in combination, using a common genetic background. Huh7 cells were transfected with these constructs and real-time PCR was used to quantify released virion-associated and intracellular HBV DNA, pregenomic RNA and pre-C mRNA. In addition, culture supernatants were tested for hepatitis B surface antigen (HBsAg) and HBeAg. The double BCP mutation (A1762T/G1764A) and the pre-C mutations (G1896A, G1899A), either alone or in combination, had no appreciable effect on the replication capacity of the virus. In contrast, clones with mutations at positions 1766/1768, 1762/1764/1766 and 1753/1762/1764 exhibited increased-replication phenotypes. HBeAg was undetectable in all cultures transfected with constructs bearing the G1896A stop-codon mutation, as expected. In contrast, constructs with additional mutations in the BCP region had appreciably lower levels of HBeAg expression than the wild type. Thus, core promoter mutations other than those at 1762/1764 appear to upregulate viral DNA replication and, at the same time, greatly reduce HBeAg production. A supplementary table showing oligonucleotides used in site-directed mutagenesis to generate the mutated plasmid constructs is available with the online version of this paper.