Functional Analysis of Hepatitis B Virus Reactivating in Hepatitis B Surface Antigen-Negative Individuals *

Abstract

The biological properties of latent or occult hepatitis B virus (HBV) have been poorly characterized as a result of the extremely low virus concentration. This report describes the phenotype of HBV reactivating in two patients after an HBsAg-negative latency period. One patient had latent HBV infection for at least 12 years without detectable viremia and symptoms of liver disease. Several full-length HBV genomes were cloned at reactivation, sequenced, and functionally tested by transfection into HuH7 cells. Genomes from both patients showed a low replication phenotype. It was caused at the level of RNA encapsidation or HBV DNA synthesis, but was not attributable to uncommon mutations in the terminal protein domain of P protein.

A Novel Target of Hepatitis B Virus Mutations: Splicing of Surface RNA * #

“…Starting from this clinical presentation, Hass et al cloned several full-length HBV genomes from the time of HBV reactivation and functionally characterized the sequenced genomes by transfection into human hepatoma cells. 12 Interestingly, a large fraction of genomes from one of the two patients did not express pre-S2/S mRNA and HBsAg, suggesting the presence of mutations resulting in an altered S gene expression. Sequence analysis and sitedirected mutagenesis revealed a single G3 A mutation within the S gene at position 458 (G458A) to be responsible for this effect.…”

“…In this issue of HEPATOLOGY, Hass et al describe a novel mutation affecting HBsAg expression by a posttranscriptional mechanism. 12 Aiming to study the functional role of HBV mutations in HBsAg-negative HBV infection, Hass et al functionally characterized viral strains from two HBsAg-negative patients with reactivation. Starting from this clinical presentation, Hass et al cloned several full-length HBV genomes from the time of HBV reactivation and functionally characterized the sequenced genomes by transfection into human hepatoma cells.…”

“…Sequence analysis and sitedirected mutagenesis revealed a single G3 A mutation within the S gene at position 458 (G458A) to be responsible for this effect. 12 To further characterize the mechanism of this mutant-induced downregulation of preS2/S transcription and HBsAg expression, the authors per-formed a series of experiments demonstrating that the G458A mutation exerts a posttranscriptional effect on preS2/S mRNA processing. By studying the viral sequences, the authors noticed that the G458A mutation is adjacent to a 5Ј splice site of the S mRNA.…”

“…By studying the viral sequences, the authors noticed that the G458A mutation is adjacent to a 5Ј splice site of the S mRNA. 12 Although functional subgenomic S transcripts have not been previously shown to undergo splicing, splicing of HBV pregenomic RNA has been described. [13][14][15] Splicing of HBV transcripts does not appear to be essential for HBV replication, [13][14][15] but whether this process may affect other aspects of viral life cycle has not been rigorously studied.…”

Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

“…Several mechanisms have been proposed to underlie OBI, including multiple amino acid substitutions in the S protein affecting HBsAg detection with commercial immunoassays (23,24) and mutations in the HBV genomes that regulate S protein expression (22,(25)(26)(27).…”