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

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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 substantial subpopulation (≈50%) of genomes from one patient did not express pre-S2/S mRNA and HBsAg. Site-directed mutagenesis identified a single G-A mutation within the S gene (position 458) to be responsible for this effect. The G458A mutation was also effective if the S gene was placed under control of a heterologous promoter. Furthermore, nuclear run-on transcription showed that the G458A mutation acts at the posttranscriptional level. The mutation affected a 5′ splice site and prevented splicing of the pre-S2/S mRNA from position 458 to 1305. In conclusion , HBV latency may be characterized by viruses with reduced replication competence and antigen expression. In one patient, HBsAg expression was terminated by an as yet undescribed posttranscriptional mechanism. A single mutation inactivated a 5′ splice site that is obviously essential for pre-S2/S mRNA accumulation. Supplementary material for this article can be found on the Hepatology website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html ). (Hepatology 2005;42:93–103.)