Development of a novel system to study hepatitis delta virus genome replication

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (delta Ag). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of delta Ag species that are nonfunctional or even inhibitory. A replication system was devised where all delta Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small delta Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express delta Ag. Thus, replication of this RNA was under control of the TET-inducible delta Ag. In the absence of TET, there was sufficient delta Ag to allow a low level of HDV replication that could be maintained for at least I year. When TET was added, both delta Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G(1)/G(0) cells relative to both S and G(2)/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.