Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis

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Abstract
The capacities to induce the synthesis of hepatitis B virus (HBV) unit-length DNA were compared for two HBV DNAs with an overall sequence diversity of about 10%. They had been cloned from serum (DNA2) and from a hepatocellular carcinoma (DNA4), respectively. As a major difference, DNA4 carries a translational stop signal preventing the synthesis of precore protein. Progeny DNA yields obtained after transfection with respective pregenome transcription units allocated DNA2 to a low-replicator and DNA4 to a high-replicator phenotype. Cotransfection of DNA2 interfered with progeny DNA synthesis induced by DNA4. By mutual exchange of restriction fragments, the region on the viral genome responsible for the differing replicator phenotypes was confined to a sequence comprising the 3'-terminal part of the X gene, core promoter, encapsidation signal epsilon, precore/core gene, and 5'-terminal part of the pol gene. Point mutations in DNA2 abolishing proper expression of the precore gene strongly enhanced the yield of progeny DNA, whereas cotransfection of a precore expression plasmid with DNA4 or with the mutated DNA2 substantially lowered the amount of progeny DNA. Hence, precore expression acts as an inhibitory principle for HBV replication. The same stop mutation as in DNA4 has been found to arise frequently in virus carriers. Loss of precore expression and concomitant conversion to a more severe hepatitis, as observed in the course of a chronic infection, thus can be explained by a relaxation of replication-level control.