2 CORE PROMOTOR MUTATIONS IDENTIFIED IN A HEPATITIS-B VIRUS-STRAIN ASSOCIATED WITH FULMINANT-HEPATITIS RESULT IN ENHANCED VIRAL REPLICATION

Viral mutations have been implicated in alteration of the biological p henotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulmi nant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more e fficient encapsidation of pregenomic RNA leading to highly enhanced re plication, To define the responsible mutation(s) for the enhanced repl ication, we introduced individual mutations of the FH strain into a wi ld-type construct by oligonucleotide-directed mutagenesis, Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication, Similar to the FH strain, this mutant disp layed the phenotype of enhanced encapsidation of pregenomic RNA. Funct ional studies in an encapsidation assay demonstrated that the identifi ed mutations resulted in a minor increase of pregenomic RNA transcript ion (two- to threefold) and a major transcription-independent enhancem ent (> 10-fold) of viral encapsidation, Our results demonstrate that t he two adjacent mutations in the HBV core promotor region are responsi ble for the enhanced replication of the FH strain, These two mutations , outside the previously described encapsidation signal, core, and pol ymerase polypeptides, appeared to affect a novel genetic element invol ved in viral encapsidation.