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

Abstract

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (deltaAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of deltaAg species that are nonfunctional or even inhibitory. A replication system was devised where all deltaAg 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 deltaAg, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express deltaAg. Thus, replication of this RNA was under control of the TET-inducible deltaAg. In the absence of TET, there was sufficient deltaAg to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both deltaAg 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 G1/G0 cells relative to both S and G2/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.

FIG. 1.

Expression in the 293-δAg cell line. (A) Monolayer cultures at 2 days after the addition of TET were examined for staining with DAPI and by immunocytochemistry to detect δAg. (B) Immunoblots were used to detect δAg in cells before and after TET induction relative to a standard of recombinant δAg. We thus deduced the number of molecules of δAg per average cell. (C and D) A similar quantitation was made for cells that were exposed to added TET either for 3 days in the presence of varied concentrations of TET (C) or for varied times but with a single concentration of TET (1 μg/ml) (D).

FIG. 2.

Accumulation of replicating HDV genomic RNA in the 293-δAg cell line after transfection with HDV RNA sequences. To initiate HDV genome replication, cells were transfected with a linear antigenomic HDV RNA that was greater than unit length and contained a 2-nucleotide deletion to disrupt the open reading frame for δAg. As indicated, cultures were incubated in the presence (open circles) or absence (filled circles) of TET (1 μg/ml). Cells were routinely subcultured every 3 to 4 days. After 8 and 52 weeks, some cultures were shifted from the absence to the presence of TET. At the times indicated, total RNA was extracted and examined by Northern analysis to quantitate the amount of HDV genomic RNA per average cell.

FIG. 3.

Immunocytochemistry of 293 cell cultures. The three rows show staining with DAPI, detection of δAg, and detection of SC35. Columns A and B represent 293-δAg cells that were transfected with HDV RNA maintained under Tet⁻ conditions and then TET induced for 2 and 14 days, respectively. Column C shows 293-HDV, a clone of cells that are replicating HDV RNA, as examined at day 2 after TET induction.

FIG. 4.

Immunocytochemistry of 293-δAg and 293-HDV cells. The three rows show staining with DAPI, detection of δAg, and detection of fibrillarin. Columns A and B show 293-δAg and 293-HDV cells, respectively, at day 1 after TET induction.

FIG. 5.

Prompt accumulation of replicating HDV genomic RNA after TET induction of 293-HDV cells. Northern analyses were used to quantitate the average number of genomic RNA molecules accumulated per average adherent cell. The data are the averages for two different clones, with the error bars representing the range of the averaged values.

FIG. 6.

Comparison of the effect of TET induction of 293-HDV relative to that of 293-δAg. The two cell clones were seeded and then cultured for 6 days in the absence or presence of TET, as indicated. Charge-coupled-device images were taken using regular light microscopy with a ×20 objective.

FIG. 7.

Cell cycle analyses of 293-δAg and 293-HDV cells. Data were obtained using propidium iodide staining followed by analytical cell sorting. This allowed separation of cells by DNA content as G₁/G₀, S, and G₂/M, as indicated. Columns A and B refer to 293-δAg and 293-HDV cells, respectively, with data shown for cells both without and with 2 days of TET induction.