A prenylation inhibitor prevents production of infectious hepatitis delta virus particles

Abstract

Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.

FIG. 1.

Production of HDV virions. Northern analysis of RNAs from supernatants of Huh7 cells transfected with HDV and/or HBV genome-encoding constructs. Following transfection of Huh7 cells with pSVLD3 alone (lanes 1, 4, 7, and 10), pGEM4ayw.2x alone (lanes 2, 5, 8, and 11), or both constructs (lanes 3, 6, 9, and 12), culture media were collected on day 3 (lanes 1 to 3), day 6 (lanes 4 to 6), and day 9 (lanes 7 to 9) and processed for HDV RNA analysis, as described in Materials and Methods. Lanes 10 to 12 contain RNA isolated from the underlying cells at day 9. Arrows indicate sizes of circular HDV genomic RNA.

FIG. 2.

HDV infection of human primary hepatocytes. In vitro-generated HDV inocula were used to infect monolayer cultures of hepatocytes as described in Materials and Methods. One week after inoculation, cells were fixed and stained with a human serum containing antibodies against delta antigen. Epitopes recognized by this serum were visualized with a rhodamine-conjugated anti-human antibody.

FIG. 3.

FTI-277 inhibits production of HDV genome-containing particles. (A) Following transfection with both HDV and HBV genome-encoding constructs, as described in Fig. 1 and in Materials and Methods, Huh7 cells were maintained in a daily changed medium containing carrier (0.2% DMSO and 400 μM DTT) alone (lanes 2 and 8) or carrier plus 0.5 μM (lanes 3 and 9), 1 μM (lanes 4 and 10), 5 μM (lanes 5 and 11), 10 μM (lanes 6 and 12), or 20 μM (lanes 7 and 13) FTI-277. On day 10 after transfection, cells (lanes 1 to 7) and supernatants (lanes 8 to 13) were processed for Northern analysis of HDV RNA, as described in Materials and Methods. Lane 1 corresponds to total RNA extracted from nontransfected cells subjected to carrier-containing medium. Molecular weight markers are indicated at the left of the figure. (B) The amount of HDV RNA in the culture medium of cells treated with the indicated amount of FTI-277 was quantitated using a phosphorimager and plotted as percentage of the untreated control (0 μM) (black bars). Prior to total RNA extraction, cells were monitored for viability (XTT assay) (grey bars) and supernatants were analyzed for protein expression and secretion (HBV surface antigen released into the medium) (empty bars).

FIG. 4.

FTI-277 inhibits the production of infectious HDV particles. Huh7 cells were transfected as in Fig. 3 and treated with carrier alone or carrier plus 5 μM FTI-277. On day 10 after transfection, culture media were concentrated and inoculated onto duplicate cultures of primary human hepatocytes, as described in Materials and Methods. (A) After overnight exposure to these inocula, hepatocytes were washed with fresh medium and processed for Northern analysis of HDV genomic RNA either immediately (day 0, lanes 1, 2, 5, and 6) or after culture for 10 more days (day 10, lanes 3, 4, 7, and 8). MW markers are indicated at the left of the figure. (B and C) Similarly treated cells were also analyzed on day 10 for large delta antigen by immunofluorescence as in Fig. 2. Panel C shows a higher power magnification of the corresponding quadrants in panel B.

FIG. 5.

FTI-277 inhibits production of HDV genotype III genome-containing particles. (A) Following transfection with both HDV genotype III and HBV genome-encoding constructs, as described in Fig. 1 and in Materials and Methods, Huh7 cells were maintained in a daily changed medium containing carrier (0.22% DMSO and 400 μM DTT) alone (lanes 2 and 8) or carrier plus 0.1 μM (lanes 3 and 9), 0.5 μM (lanes 4 and 10), 1 μM (lanes 5 and 11), or 5 μM (lanes 6 and 12) FTI-277. On day 10 after transfection, cells (lanes 1 to 6) and supernatants (lanes 7 to 12) were processed for Northern analysis of HDV RNA, as described in Materials and Methods. Lanes 1 and 7 correspond to nontransfected cells subjected to carrier-containing medium. MW markers are indicated at the left of the figure. (B) HDV RNA in the culture medium of cells treated with the indicated amounts of FTI-277 was quantitated using a phosphorimager and plotted as a percentage of the no-drug control (0 μM) (black bars). Also shown are the results from total RNA quantitation (Total RNA) (grey bars) and HBV surface antigen synthesis and secretion (HBsAg) (empty bars).