Reduced Precore Transcription and Enhanced Core-Pregenome Transcription of Hepatitis B Virus DNA after Replacement of the Precore-Core Promoter with Sequences Associated with e Antigen-Seronegative Persistent Infections

Abstract

Hepatitis B virus variants harboring nucleotide alterations in the preC-C promoter have been detected in fulminant hepatitis B as well as in HBeAg-seronegative persistent infection. However, it has not been demonstrated that variants with nucleotide alterations in the preC-C promoter cause various disease states. We replaced the preC-C promoter region of a wild-type genome with the most frequent naturally occurring mutated form and introduced it into HepG2 cells. The mutant with coexisting A1762T and G1764A substitutions produced less than one-fifth of the wild-type level of HBeAg. Conversely, the mutant generated 2.4 times more core particle antigen and showed a high-replicator phenotype. RNase protection and quantitative 5' RACE showed a 16- to 32-fold reduction of preC transcripts and a 4-fold induction of C transcripts of the mutant compared to wild-type. The preC transcript of the mutant had a more heterogeneous 5' end than that of the wildtype. However, the mutations did not alter the initiation sites of C transcription. When the promoter region was cloned into CAT plasmids, the mutations had dual effects on preC and C promoter activities, decreasing and increasing them, respectively. These results suggest that these mutations are responsible for the reduced HBeAg production as well as the enhanced replication and core production. Analysis of revertants with either single point mutation showed that T at 1762 is critical for the mutant phenotype.

Different Hepatitis B Virus Genotypes Are Associated With Different Mutations in the Core Promoter and Precore Regions During Hepatitis B E Antigen Seroconversion

“…29 Further studies are needed to confirm if core promoter mutations are preferentially selected in HBV genotypes that cannot develop precore stop codon mutations and the exact mechanisms by which these mutations are selected. TA changes in the core promoter region have been reported to be associated with enhanced replication in in vitro studies, 16,17,19 but these findings have been refuted by other investigators. 18 TA changes have also been postulated to alter the secondary structure of the 3Ј-end of the pregenomic RNA, thus facilitating DNA minus-strand translocation to DR1, 30 but these effects have not been proven.…”

“…The TA changes decrease, but do not completely abolish, HBeAg secretion. [16][17][18][19][20] Thus, additional steps such as immune clearance or development of other mutations such as A1896 may be needed for HBeAg clearance. An isolated A1764 change has also been reported in a small percentage of patients by other investigators.…”

“…This hypothesis is supported by in vitro observations that the TA changes decrease transcription of precore mRNA and secretion of HBeAg. [16][17][18][19][20] However, TA changes have also been found in some HBeAg-positive patients, especially those with chronic hepatitis. 12,13 Thus, the role of core promoter mutations in HBeAg clearance is unclear.…”

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

“…14,[40][41][42][43][44][45][46][47][48][49] In this study, we report the development of a novel transient mechanism for studying HBV in the well differentiated human hepatoblastoma cell line HepG2. We conclude the following from our studies:…”

“…39 Transient transfection of HepG2 cells have been used to understand various aspects of HBV gene expression and replication at the molecular level. Some studies involve using greater than genome length HBV DNA sequences so that all HBV gene products can be produced 14,40,45,46,48,49 whereas others have concentrated on using HBV DNA sequences which encode restricted portions of the coding regions of the genome or enhancer or promoter sequences. 14,[40][41][42][43][44][45][46][47][48][49] Recently, transient transfection of HepG2 cells has been used to understand the function of the precore gene and HBeAg.…”