Chronic persistent infection andpol gene expression by duck hepatitis B virus

Hepadnaviruses can cause not only acute but also chronic infections. The regulation of the copy number of the viral transcriptional template, covalently closed circular (CCC) DNA, is believed to play an important role in maintenance of chronic infections. To distinguish between an intracellular and an extracellular pathway for DHBV CCC DNA amplification in infected primary duck hepatocyte cultures, we employed anti-viral agents to block the extracellular route. We found that CCC DNA amplification still occurred when the extracellular reinfection pathway was blocked. This result indicates that CCC DNA amplification in chronically infected cultures and by inference, the maintenance of persistent infection, involves primarily an intracellular regulatory pathway. The role of viral polymerase and precore proteins in CCC DNA amplification was next investigated by genetic analysis. DHBV with a mutated genome was produced by transfection of LMH cells with cloned DHBV DNAs, and was used to infect primary duck hepatocytes. Our results provided genetic evidence that CCC DNA is synthesized by the viral polymerase and that precore is not essential for CCC DNA amplification in hepatocyte cultures. The synthesis of CCC DNA was also studied in LMH sublines stably-transformed with DHBV DNAs. An LMH subline (D2) stably-transformed with wild-type DHBV, accumulated a large amount of CCC DNA (30-50 copies per cell), just as the chronically infected liver does. A similar level of CCC DNA synthesis was observed in an LMH subline (1S-5) stably-transformed with a DHBV envelope mutant. The mechanisms by which less-than-full-length pol gene products are produced and their role in viral replication are not clear. We introduced frameshift or stop codon insertion mutations in the spacer region of the DHBV pol gene to address this problem. Our results demonstrated a leaky viral DNA synthesis phenotype. The leakiness of most of these mutants appeared to attributable to translational suppression. However, the +2 frameshift mutants appeared to use a novel way to express the viral pol gene, by synthesizing the viral reverse transcriptase as a fusion protein with the amino-terminal portion of preS protein. This result implies that less-than-full-length pol gene products are functional in viral DNA synthesis