The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo

Abstract

Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.

FIG. 1.

(A) Amino acid sequences of the C-terminal transmembrane domain (amino acids 571 to 591) of HCV and BVDV NS5Bs (23, 34). Underlined boldface letters correspond to residues observed in more than 10% of the 307 HCV isolates (34). The rest occurred infrequently. Sequences are listed in decreasing order of observed frequency from top to bottom. Numbers above amino acid sequences represent amino acid positions in HCV and BVDV NS5Bs. (B) Schematic diagrams of the NS5B mutants in various replicons used in this study. Open and gray boxes represent HCV and BVDV NS5B transmembrane domains (TMD), respectively. Angled and straight lines represent the deletion and a noncoding RNA, respectively.

FIG. 2.

Deletion of the transmembrane domain of HCV NS5B abolishes replication of subgenomic replicons in Huh-7 cells. In vitro-transcribed replicons were transfected into Huh-7 cells, and G418-resistant colonies were stained with 0.2% crystal violet 3 weeks after transfection. For Rep/GDD, the GDD catalytic motif of HCV NS5B was changed to AAA.

FIG. 3.

Intracellular localization of the cell-permeable peptides. The various synthetic peptides were labeled with rhodamine at the N terminus. Huh-7/replicon cells were plated onto chamber slides and treated with the indicated peptides at 2.5 μM for 48 h. Cells were then washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. Peptides were visualized by fluorescence microscopy.

FIG. 4.

The C-terminal transmembrane peptide affects the membrane association of HCV NS5B in Huh-7/replicon cells. Cells were treated with the indicated peptide at 25 μM or with DMSO alone for 48 h. Postnuclear lysates were analyzed by a membrane flotation assay. Fractions were collected from the top to the bottom of the gradient, and each fraction was analyzed for HCV NS5B (A) or calnexin (B) by immunoblotting. Experiments were repeated three times, and the results of representative experiments are shown.

FIG. 5.

The C-terminal transmembrane peptide inhibits HCV RNA replication in Huh-7/replicon cells. Cells were treated with the peptides as in the experiments shown in Fig. 4. Total cellular RNA was analyzed for HCV and GAPDH RNA by real-time Taqman PCR. Relative HCV RNA copies per GAPDH RNA are presented. The RNA level in 1% DMSO-treated cells is represented as 100%. Experiments were repeated four times, and the average values for the experiments are shown.

FIG. 6.

A related transmembrane domain from BVDV NS5B cannot replace the transmembrane domain of HCV NS5B. (A) The transmembrane domains of HCV and BVDV NS5Bs were fused to the C terminus of GFP and transfected into Huh-7 cells. A membrane flotation assay was performed 48 h after transfection. The membrane fractions (M; fractions 1 to 4) and the cytosolic fractions (C; fractions 5 to 9) were separately pooled and analyzed by immunoblotting with an anti-GFP rabbit polyclonal antibody. (B) Replication of the chimeric HCV-BVDV NS5B (Fig. 1B, Rep/BVDVTM). A colony formation assay was performed as described in the legend to Fig. 2.