Duck hepatitis B virus integrations in LMH chicken hepatoma cells: identification and characterization of new episomally derived integrations

While the cytoplasmic phase of the hepadnavirus replication cycle is well understood, very little is known about the nuclear phase. In contrast to retroviruses, proviral integration is not required for hepadnavirus replication; however, some of the viral DNAs in the nucleus are diverted into an integration pathway. Under certain conditions these integrations function as carcinogenic agents. In order to study the integration process, we have utilized LMH-D2 cells, which replicate wild-type duck hepatitis B virus (DHBV), to develop the first protocol to detect and characterize integrations of DHBV originating from episomal viral DNAs. Contrary to expectations, our results showed that stable new integrations are readily detectable in subclones of LMH-D2 cells. Complete characterization of one integration revealed a single-genome-length integrant with the structure of double-stranded linear (DSL) DHBV DNAs which are produced by in situ priming during viral replication. The integration contained a terminal redundancy of 6 bp from the r region of the virus DNA minus strand as well as a direct repeat of 70 bp of cellular DNA. On the basis of the structure of the integrant and the cellular DNA target site, we propose a molecular model for the integration mechanism that has some similarities to that of retroviruses. Identification of DSL hepadnavirus DNA integration suggests the possibility that modified DSL viral DNAs may be the precursors to a class of simple, unrearranged hepadnavirus integrations.