Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

Abstract

The pregenomic RNA directs replication of the hepatitis B virus (HBV) genome by serving both as the messenger for core protein and polymerase and as the genome precursor following its packaging into the core particle. RNA packaging is mediated by a stem-loop structure present at its 5 end designated the signal, which includes the core gene initiator AUG. The precore RNA has a slightly extended 5 end to cover the entire precore region and, consequently, directs the translation of a precore/core protein, which is secreted as e antigen (HBeAg) following removal of precore-derived signal peptide and the carboxyl terminus. A naturally occurring G1862T mutation upstream of the core AUG affects the bulge of the signal and generates a "forbidden" residue at the ؊3 position of the signal peptide cleavage site. Transfection of this and other mutants into human hepatoma cells failed to prove their inhibition of HBeAg secretion but rather revealed great impairment of genome replication. This replication defect was associated with reduced expression of core protein and could be overcome by a G1899A covariation, or by nonsense or frameshift mutation in the precore region. All these mutations antagonized the G1862T mutation on core protein expression. Cotransfection of the G1862T mutant with a replication-deficient HBV genome that provides core protein in trans also restored genome replication. Consistent with our findings in cell culture, HBV genotype A found in African/Asian patients has T1862 and is associated with much lower viremia titers than the European subgroup of genotype A.The hepatitis B virus (HBV) primarily infects the liver and causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. It is an enveloped DNA virus with a small double-stranded genome of 3.2 kb. Inside hepatocytes, viral genomic and subgenomic RNAs are transcribed from a covalently closed circular DNA template in the nucleus and exported to the cytoplasm for translation into viral proteins. The core protein assembles into the core particle, packaging both the pregenomic RNA and DNA polymerase. Subsequently, the DNA polymerase synthesizes the negative-strand DNA via reverse transcription from the RNA template, followed by RNA degradation and synthesis of positive-strand DNA. The core particle with the double-stranded DNA genome is enveloped by host-derived lipids and viral envelope proteins and secreted as an infectious virus particle (for a review, see reference 7). Since the 3.5-kb pregenomic RNA is also the mRNA for the expression of both core protein and DNA polymerase, it is the sole component required for genome replication. Consequently, increased transcription of pregenomic RNA will lead to enhanced HBV replication, as exemplified by the naturally occurring core promoter mutants (3,20).The AUG initiator of the core gene (position 1901 to 1903) is located approximately 80 nucleotides (nt) downstream of the 5Ј end of its mRNA, while the initiation codon of polymerase is 400 nt further downstream. Therefore, core pro...

Hepatitis B Virus Genotype C Isolates with Wild-Type Core Promoter Sequence Replicate Less Efficiently than Genotype B Isolates but Possess Higher Virion Secretion Capacity

“…Intracellular envelope proteins were detected by direct Western blot analysis (8,9,32). Briefly, 50 g protein from cell lysate was separated overnight in an SDS-12% polyacrylamide gel and transferred to polyvinylidene difluoride membranes.…”

“…Detailed procedures for transfection of Huh7 cells and subsequent detection of genome replication, protein expression, protein secretion, and virion secretion have been described previously (8,9,32,44). Huh7 cells seeded at 6 ϫ 10 5 /wells in six-well plates were transfected with 1.5 g of circularized HBV DNA or 1 g of SphI dimer DNA using TransIT-LT1 reagent (Mirus).…”

Identification and Characterization of Clevudine-Resistant Mutants of Hepatitis B Virus Isolated from Chronic Hepatitis B Patients

“…A susceptibility assay was performed using Southern blotting as described previously (7). HBV replication was quantified using a phosphorimager and Multi-Gauge v3.2 software (Fujifilm, Tokyo, Japan).…”

Hepatitis B virus replication

“…An illustrating example are the HBV precore variants in which HBeAg synthesis is prevented by stop mutations in the ε overlapping precore region. The only mutations causing this phenotype found in nature are those which maintain a stable ε secondary structure [66,67] . It should be noted that RNA secondary structure (2D) analysis provides a mere description of the base-pairing pattern.…”