Limitations to replication of hepatitis delta virus in avian cells

Abstract

Human hepatitis delta virus (HDV) is a natural subviral agent that uses hepatitis B virus as a helper. Experimentally, HDV can be made to replicate in woodchucks, using woodchuck hepatitis B virus as a helper virus. Also, independent of such helper activity, replication of the HDV RNA genome can be achieved in many mammalian cells. In this study we examined whether such replication could also be achieved in avian cells. We used cotransfection strategies and initially found no detectable genome replication in chicken LMH cells relative to the mammalian cell line Huh7, used as a positive control. We also found that, in contrast to transfected Huh7 cells, the avian cell line was readily and efficiently killed by expression of the delta protein. Three strategies were used to reduce such killing: (i) the delta protein was expressed from a separate expression vector, the amount of which was then reduced as much as 33-fold; (ii) the protein was expressed transiently, using a promoter under tetracycline control; and (iii) the transfected cells were treated with Z-VAD-fmk, a broad-spectrum caspase inhibitor, which reduced cell killing. This last result indicated that cell killing occurred via an apoptotic pathway. After application of these three strategies to reduce cell killing, together with a novel procedure to improve the signal-to-noise ratio in Northern analyses, replication of the HDV genome was then detected in LMH cells. However, even after removal of obvious signs of toxicity, the amount was still >50 times lower than in the Huh7 cells. Our findings explain previous unsuccessful attempts to demonstrate replication of the HDV genome in avian cells and establish the precedent that in certain situations HDV replication can be cytotoxic.

FIG. 1

Assays of HDV RNA processing in avian cells. LMH (lanes 2 to 4) or Huh7 cells (lanes 5 to 7) were transfected with pCMV(D3) (lanes 2 and 5) or with both pCMV(D3) and pTW198 (lanes 3 and 6) or left untransfected (lanes 4 and 7). After 4 days the RNA was extracted and assayed by Northern analysis to detect genomic RNA. The gel electrophoretic conditions were such as to resolve linear and circular forms of unit-length HDV RNA, as indicated at the right side (4). Lane 1 contains 5′-labeled DNA size markers.

FIG. 2

Variation in the expression of GFP reveals the toxic effect of delta protein in transfected avian cells. Cultures of LMH cells (A to D) and Huh7 cells (E to H) were cotransfected with fixed amounts of plasmids pGG119 and pJC110 along with variable amounts of pTW198. The ratios of pTW198 to pJC110 are indicated. At 4 days after transfection, fluorescence microscopy, coupled with a charge-coupled device camera, was used to record the levels of GFP fluorescence.

FIG. 3

Assays of HDV replication in avian and mammalian cells during reduced expression of small delta protein. Cultures of LMH (lanes 1 to 5) or Huh7 cells (lanes 6 to 10) were cotransfected with fixed amounts of pGG119 and pJC110 along with various amounts of pTW198. The ratios of pTW198 to pJC110 are as follows: lanes 1 and 6, 0:1; lanes 2 and 7, 1:1; lanes 3 and 8, 0.3:1; lanes 4 and 9, 0.03:1; lanes 5 and 10, untransfected. At 4 days after transfection, RNA and protein were extracted. RNA was analyzed by Northern blot for antigenomic RNA (A) or genomic RNA (B). Protein was assayed by immunoblot for delta protein (C).

FIG. 4

Assays of HDV replication in avian and mammalian cells in the presence of tetracycline-controlled expression of small delta protein. Cultures of LMH (lanes 3 to 6) or Huh7 cells (lanes 7 to 10) were cotransfected in the presence of tetracycline with fixed amounts of pGG119, pJC110, and pPB106, expressing the small form of the delta protein under control of tetracycline. Tetracycline was removed after day 1 for 0.5 days (lanes 3 and 7), 3 days (lanes 4 and 8), and 5 days (lanes 5 and 9). Lanes 6 and 10 are untransfected controls. Lane 1 contains 5′-labeled DNA size markers. Lane 2 is a standard of unit-length HDV cDNA (200 pg). At 6 days after transfection, RNA and protein were extracted. RNA was analyzed by Northern blot for antigenomic RNA (A) or genomic RNA (B). Protein was assayed by immunoblot for delta protein (C).

FIG. 5

Assays of HDV replication in avian and mammalian cells during expression of small delta protein in the presence of the antiapoptotic compound Z-VAD-fmk. Cultures of LMH (lanes 3 to 10) or Huh7 cells (lanes 11 and 12) were cotransfected with fixed amounts of pGG119 and pJC110 along with various amounts of pTW198. The ratios of pTW198 to pJC110 are as follows: lane 3, 0:1; lanes 4, 5, 11, and 12, 1:1; lanes 6 and 7, 0.3:1; lanes 8 and 9, 0.03:1. Lane 1 contains 5′-labeled DNA size markers. Lane 2 is a standard of unit-length HDV cDNA (200 pg). Lane 10 is a sample from untransfected cells. Lanes 5, 7, 9, and 12 correspond to cultures treated, beginning 1 h prior to cotransfection, with 40 μM Z-VAD-fmk. At 4 days after transfection, RNA and protein were extracted. RNA was analyzed by Northern blot for antigenomic RNA (A) or genomic RNA (B). Protein was assayed by immunoblot for delta protein (C).