Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids

Abstract

We have previously shown that IFN-beta inhibits hepatitis B virus (HBV) replication by noncytolytic mechanisms that either destabilize pregenomic (pg)RNA-containing capsids or prevent their assembly. Using immortalized murine hepatocyte cell lines stably transfected with a doxycycline (dox)-inducible HBV replication system, we now show that replication-competent pgRNA-containing capsids are not produced when the cells are pretreated with IFN-beta before HBV expression is induced with dox. Furthermore, the turnover rate of preformed HBV RNA-containing capsids is not changed in the presence of IFN-beta or IFN-gamma under conditions in which further pgRNA synthesis is inhibited by dox removal. In summary, these results demonstrate that types 1 and 2 IFN activate hepatocellular mechanism(s) that prevent the formation of replication-competent HBV capsids and, thereby, inhibit HBV replication.

Figures

Fig. 1.

Structure of tetracycline-inducible HBV expression vectors pPGFPTREHBV and pPGFPTREHBV-V. An overlength genome of wild-type HBV ayw or HBV ayw containing a Met-to-Val substitution in the viral polymerase (amino acid position 539) was inserted into pBI-EGFP by fusing the viral transcriptional start site of the pgRNA (HBV position 1821) to the transcriptional start site in the tetracycline-inducible promoter TREminCMV in pBI-EGFP, as described in Materials and Methods. The pPGFPTREHBV(-V) vectors also contain an SV40 early promoter-driven expression cassette for the puromycin resistance gene (pac) for selection of stable transfectants in mammalian cells. The bidirectional TREminCMV promoter also drives expression of the EGFP to monitor tetracycline induction in cell cultures. Plasmid amplification is supported by the bacterial origin of replication (ori) and the β-lactamase gene (Ampr) providing ampicillin resistance. Numbers indicate nucleotide map positions in HBV.

Fig. 2.

Tetracycline-regulated expression and replication of HBV. TREHBV-V and TREHBV cells were cultured as described in Materials and Methods with or without dox, as indicated, and individual dishes were harvested at different days (d) during the experiment. (A) Tetracycline-regulated gene expression and accumulation of HBV pgRNA-containing capsids in TREHBV-V cells. Twenty micrograms of total cellular RNA was used for Northern blot analysis for viral pgRNA and viral envelope-encoding transcripts (envRNA) and the cellular gene for GAPDH as a control for loading differences (Upper). Encapsidated HBV RNA was isolated as described in Materials and Methods and subjected to HBV-specific Northern blotting (Lower). Twenty micrograms of total cellular RNA isolated from HBV transgenic (HBVtg) mice was included as size marker for HBV RNA. (B) Tetracycline-regulated expression and replication in TREHBV-V and TREHBV cells. Total cellular RNA isolated from TREHBV-V and TREHBV cells cultured in the presence of dox for the indicated time periods was subjected to HBV-specific Northern blotting (Top). Northern blot analysis of encapsidated HBV RNA extracted from parallel dishes of TREHBV-V and TREHBV cells (Middle). Twenty micrograms of total cellular DNA isolated from TREHBV-V and TREHBV cells was subjected to HBV-specific Southern blot analysis (Bottom). Tg, HBV transgene; DS, double-stranded HBV DNA replicative intermediates; SS, single-stranded HBV DNA replicative intermediates.

Fig. 3.

IFN-β inhibits the formation of replication-competent HBV RNA-containing capsids. TREHBV-V cells were preincubated with mIFN-β (100 units/ml) for 6 h before HBV gene expression was induced by the addition of dox (1 μg/ml). The cells were further incubated for 36 h in the presence of dox and mIFN-β.(A) HBV- and GAPDH-specific Northern blot analysis of total cellular RNA. (B) Northern blot analysis of encapsidated HBV RNA. (C) Northern blot analysis of total cellular RNA for the mIFN-β response gene 2′5′OAS. For experimental details and symbols, see Materials and Methods and the legend to Fig. 2, respectively.

Fig. 4.

IFNs do not shorten the half-life of HBV RNA-containing capsids. TREHBV-V cells were cultured in the presence of dox for 4 days, at which time dox was removed (time 0 h) and mIFN-β and -γ was added 84 h later, as indicated. Cells were harvested at the indicated time points. (A and D) HBV- and GAPDH-specific Northern blot analysis of total cellular RNA. (B and E) Northern blot analysis of encapsidated HBV RNA. (C) Northern blot analysis of total cellular RNA for the mIFN-β response gene 2′5′OAS. (F) Northern blot analysis of total cellular RNA for the mIFN-γ response gene TGTP. For experimental details and symbols, see Materials and Methods and the legend to Fig. 2, respectively.