Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation

Abstract

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

Figures

Fig. 1.

Effects of lovastatin on HCV RNA and protein abundance. (A) The HCV genome and its genomic and subgenomic HCV RNA replicons. The polyprotein cleavage products and 5′ and 3′ nontranslated regions are indicated. The bicistronic replicon genomes encode a fusion protein consisting of the first 12 aa of the HCV core protein (ΔC) fused to the amino terminus of the neomycin phosphotransferase gene (neo), the translation of which is directed by the HCV IRES. Translation of the HCV polyprotein is directed from the second cistron by the encephalomyocarditis virus (EMCV) IRES. Representative Huh7 cell lines harboring the genomic and subgenomic HCV RNA replicons are shown at the right. Sites of adaptive (point) mutations in the Huh7-HP replicon are denoted by dots; the site of the lysine (K) insertion in the Huh7-K2040 replicon is denoted by the vertical arrow. (B and C) Huh7-K2040 (4 × 105), Huh7-HP (2 × 105), or Huh7-C5B3 (2 × 105) cells were cultured in 60-mm dishes. At time 0, the culture medium was replaced with medium containing 5% FCS (medium A) or medium A containing 50 μM lovastatin, and cultures were further incubated for the indicated time at which point cells were harvested for analysis of RNA and protein. (B) Total RNA from Huh7-K2040 cells cultured without (▵) or with (▴) lovastatin for the indicated time were subjected to real-time RT-PCR for quantification of HCV RNA. The relative level of HCV RNA at each time point is presented as a proportion of the time 0 level, which was assigned a value of 1.0. (C) Immunoblot analysis of HCV NS3, NS4B, and NS5A protein abundance in Huh7-K2040 cells (lanes 1–8) or HCV NS3 protein abundance in Huh7-C5B3 and Huh7-HP cells (lanes 9–16). Lanes 1 and 9 show protein levels in control Huh7 cells; the rest of the lanes show protein levels in cells harboring the indicated HCV replicon RNA cultured in the absence (-) or presence (+) of lovastatin for the indicated time. NS3, NS4B, and NS5A in Huh7-K2040 cells, NS3 in Huh7-HP and Huh7-C5B3 cells, and actin were detected by immunoblot analysis with a human HCV patient serum (1:1,000 dilution), monoclonal anti-NS3 antibody (1:1,000 dilution), and anti-human actin serum (1:2,000 dilution), respectively. The positions of the basal phosphorylated (NS5A) and hyperphosphorylated (NS5A-Pi) isoforms (2) are indicated. (D) Huh7-K2040 cells (2 × 105) were cultured in 37-mm culture dishes. At time 0, the medium was replaced with medium A alone (gray bars) or with medium A containing 50 μM lovastatin (filled bars), 100 μM aphidicolin (hatched bars), or 10 mM hydroxyurea (open bars). After 24 h, one culture set was harvested and the relative level of HCV RNA was analyzed by quantitative real-time RT-PCR. Cells in the remaining cultures were pulse labeled with [3H]thymidine, [3H]uridine, or [35S]methionine for the respective analysis of DNA, mRNA, or protein synthesis as described in Material and Methods. For each analysis, the results are normalized to the value obtained from the medium A control culture, which was set as 100%. The 100% control values for DNA, RNA, and protein synthesis were 174, 482, and 21,100 cpm/mg protein, respectively. Each value is the mean of three replicate incubations.

Fig. 2.

Mevalonate and geranylgeraniol restore HCV RNA replication in lovastatin-treated cells. Huh7-K2040 cells (4 × 105) were cultured for 16 h in 60-mm dishes. The cultures were then subjected to the following treatments. (A) Cultures were changed to medium A with (▴) or without (▵) 100 μg of protein per ml of LDL. Except for the nontreated control (not plotted), all other cultures received 50 μM lovastatin and increasing amounts of sodium mevalonate as indicated. (B) Cultures were changed to medium A containing 100 μg of protein per ml of LDL. Except for the nontreated control (not plotted), cells were treated with 50 μM lovastatin and increasing amounts of geranylgeraniol (▴), farnesol (○), or oleate (▪) as indicated. (C) Cultures were changed to medium A containing increasing amounts of lovastatin as indicated. In all treatments, cultures were continued for a further 24 h, after which the cells were harvested and total RNA was extracted. The HCV RNA level was determined by real-time quantitative RT-PCR analysis. Values are presented as a proportion of the nontreated control, which was set at 1.0. (B Inset and C Inset) Cultures in B Inset were changed to medium A containing no addition (lane a), 50 μM lovastatin (lanes b–d), 10 μM geranylgeraniol (lane c), or 10 μM farnesol (lane d). Cultures in C Inset were changed to medium A containing the indicated amount of lovastatin. Forty-eight hours later, cells were harvested and 20-μg aliquots of cell lysate were subjected to SDS/PAGE and immunoblot analysis with anti-Rap1a antibody (0.4 μg/ml). U and P denote unprenylated and prenylated forms of Rap1a, respectively.

Fig. 3.

Geranylgeraniol, but not farnesol, restores the HCV replication complex in lovastatin-treated cells as visualized by immunofluorescence. (A–T) Huh7-K2040 cells (5 × 104) were cultured for 16 h in the wells of a 1.8 × 0.9-cm chamber slide. Cells were fixed, and multicolor immunofluorescence microscopy analysis was performed as described in Materials and Methods. (A–C, E–G, I–K, M–O, and Q–S) Cells were counterstained with the nucleic acid probe 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) or the indicated antibody. (M–O) Cells were transfected with 5-bromouridine 5′-triphosphate by using FuGENE 6 reagent as described (6) and cultured in the presence of actinomycin D for 2 h to facilitate the specific incorporation of bromouridine into HCV RNA. HCV RNA was visualized by immunostaining of bromouridine. (D, H, L, P, and T) These images are merged fluorescent composite images of panels A–C, E–G, I–K, M–O, and Q–S, respectively. Within each merged composite image, yellow denotes protein colocalization and the position of the HCV replication complex. (U–Z) Huh7-K2040 cells (105) were cultured for 16 h in the wells of a 1.8 × 0.9-cm chamber slide, after which the culture medium was switched to medium A containing the indicated additions and cultures were continued for a further 48 h. Cells were then fixed and immunostained with monoclonal anti-NS5A and FITC-labeled secondary antibody. Shown is NS5A protein localization in cells that were cultured with medium A with no addition (U), 50 μM lovastatin (V), 10 μM geranylgeraniol (W), 50 μM lovastatin plus 10 μM geranylgeraniol (X), 10 μM farnesol (Y), or 50 μM lovastatin plus 10 μM farnesol (Z). No fluorescence signal was detected in parallel cultures of control Huh7 cells (data not shown).

Fig. 4.

Disruption of HCV replication through specific inhibition of geranylgeranyl transferase I. (A–C) Huh7-K2040 cells (4 × 105) were seeded in 60-mm dishes on day 0 and cultured for 16 h. (A) Cells were then switched to medium A containing 100 μM DTT. Except for the nontreated control, all dishes received increasing amounts of GGTI-286 (▪) or FPTI-III (○) as indicated. After 24 h the cells were harvested and total RNA was extracted. HCV RNA level was determined by quantitative real-time RT-PCR analysis. Values are presented as a proportion of the nontreated control, which was set at 1.0. (B) Cells were transfected on day 1 with 0.5 μg of the indicated plasmid (2 μgof total DNA per dish, using pcDNA3.1 empty vector). Six hours later the cells were treated with 10 μM FPTI-III or GGTI-286. On day 2, the cells were harvested, and 20 μg of cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-H-Ras antibody (0.2 μg/ml) as described (20). U and P denote unprenylated and prenylated form of H-Ras, respectively. (C) Cells were treated with the indicated concentrations of FPTI-III (lanes 2–4) or GGTI-286 (lanes 5–7). On day 2, the cells were processed for immunoblot analysis with anti-Rap1a antibody (0.4 μg/ml) as described in B. (D) NS5A protein localization was analyzed in Huh7-K2040 cells. Cells (105) were cultured for 16 h in the wells of a 1.8 × 0.9-cm chamber slide, after which the culture medium was replaced with medium A containing one of the following additions: none (Left), 20 μM GGTI-286 (Center), or 20 μM FPTI-III (Right). Cultures were continued for a further 24 h, after which the cells were fixed and immunostained with monoclonal anti-NS5A and a FITC-conjugated secondary antibody. No fluorescent signal was detected in control Huh7 cells (data not shown).