Volume 18, Issue 4, Pages 425-434 (May 2005) Identification of FBL2 As a Geranylgeranylated Cellular Protein Required for Hepatitis C Virus RNA Replication  Chunfu Wang, Michael Gale, Brian C. Keller, Hua Huang, Michael S. Brown, Joseph L. Goldstein, Jin Ye  Molecular Cell  Volume 18, Issue 4, Pages 425-434 (May 2005) DOI: 10.1016/j.molcel.2005.04.004 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Incorporation of [3H]Mevalonate into a Cellular Protein that Coimmunoprecipitates with the HCV NS5A Protein On day 0, Met-18b-2 cells were set up at 7 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 1 μg/dish of the indicated plasmid encoding Flag-tagged wild-type NS5A (WT) or mutant NS5A with the two COOH-terminal cysteines changed to serines (C447S;C448S). The total plasmid concentration was adjusted to 2 μg/dish with pcDNA3.1 (Invitrogen). Six hours after transfection, cells were radiolabeled for 16 hr with [3H]mevalonolactone as described in Experimental Procedures. On day 2, cells were harvested, and NS5A was immunoprecipitated with anti-Flag antibody. Pellet fractions (from two dishes of cells), and supernatant fractions (from 0.4 dish) from the immunoprecipitations were then subjected to 10% SDS-PAGE, followed by 3H-autoradiography and immunoblot analysis of NS5A using a human HCV patient serum. Filters for 3H-autoradiography and immunoblot analysis were exposed for 4 weeks and 2 s, respectively. The asterisk (*) denotes a putative cellular prenylated protein of ∼50 kDa that interacts with NS5A. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Identification of FBL2 as a NS5A-Interacting Protein (A) On day 0, Huh7 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, each dish of the cells were transfected with 0.5 μg of pCMV-Flag-HA-NS5A, 50 ng of pCMV-Myc-FBL2, 20 ng of pCMV-Myc-OAS1, and 10 ng of pCMV-Myc-ALDH7 as indicated. Total plasmid concentration was adjusted to 2 μg/dish with pcDNA3.1. On day 2, cells were harvested and subjected to immunoprecipitation with anti-Flag antibody. Pellet fractions (from 0.4 dish of cells) and supernatant fractions (from 0.08 dish) of immunoprecipitates were then subjected to SDS-PAGE, followed by immunoblot with anti-HA or polyclonal anti-Myc. Filters were exposed for 2–10 s. (B) Domain structure of human FBL2. The F box, 11 leucine-rich repeats (LRR), and CAAX motif (CVIL) are highlighted in yellow, orange, and blue, respectively. The diagram is drawn to scale. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Identification of FBL2 as a Geranylgeranylated Protein (A) On day 0, Met-18b-2 cells stably transfected without (lane 1) or with Myc-tagged wild-type FBL2 (lane 2) or mutant FBL2 (lane 3) were set up at 7 × 105 cells per 60 mm dish. On day 1, the cells were radiolabeled for 16 hr with [3H]mevalonolactone as described in Experimental Procedures. On day 2, the cells were harvested and subjected to immunoprecipitation with a polyclonal anti-Myc antibody. Pellet fractions (from two dishes of cells) and supernatant fractions (from 0.4 dish) of immunoprecipitations were then subjected to 10% SDS-PAGE, followed by 3H-autoradiography and immunoblot using monoclonal anti-Myc IgG-9E10. Filters for 3H-autoradiography and immunoblot analysis were exposed for 4 weeks and 2 s, respectively. (B) On day 0, CHO-7 cells were set up at 7 × 105 cells per 60 mm dish. On day 1, the cells were transfected without (lanes 1 and 2) or with 10 ng/dish Myc-tagged wild-type FBL2 (lanes 3–8) or Myc-tagged mutant FBL2(C420S) (lanes 9–14). Three hr after transfection, cells were treated with 10 μM GGTI-286 (GGTI) or 10 μM FPTI-III (FPTI) in medium containing 100 μM dithiothreitol for 16 hr. On day 2, cells were harvested and fractionated into cytosol (C) and membrane (M) fractions as described in Experimental Procedures. Aliquots of each fraction (0.2 dish of cells) were subjected to SDS-PAGE followed by immunoblot using monoclonal anti-Myc IgG-9E10. Filter was exposed for 5 s. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Coimmunoprecipitation of NS5A with Wild-Type and Mutant Versions of FBL2 (A) Transfected NS5A in Huh-7 cells. On day 0, Huh-7 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 0.5 μg/dish of pCMV-Flag-HA-NS5A and 50 ng/dish of pCMV-Myc-FBL2, as indicated. Total plasmid concentration was adjusted to 2 μg/dish with pcDNA3.1. On day 2, cells were harvested and subjected to immunoprecipitation with anti-Flag antibody. Pellet fractions (from 0.4 dish of cells) and supernatant fractions (from 0.08 dish) of immunoprecipitations were then subjected to SDS-PAGE, followed by immunoblot with anti-HA or polyclonal anti-Myc as indicated. Filters were exposed at 2–10 s. (B) Endogenous NS5A in Huh7-K2040 cells. On day 0, Huh7-K2040 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 0.4 μg/dish of pCMV-Flag-Myc-FBL2, as indicated. Total plasmid concentration was adjusted to 2 μg/dish with pcDNA3.1. On day 3, cells were harvested and subjected to immunoprecipitation with anti-Flag antibody. Pellet and supernatant fractions of immunoprecipitation were subjected to SDS-PAGE, followed by immunoblot with anti-NS5A or polyclonal anti-Myc, as indicated. For anti-NS5A immunoblots, pellet and supernatant fractions were derived from 0.5 and 0.1 dish of cells, respectively, and filters were exposed for 2 min. For anti-Myc immunoblots, pellet and supernatant fractions were derived from 0.1 and 0.02 dish of cells, respectively, and filters were exposed for 5 s. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Replication of HCV and West Nile Virus RNA in Huh7 Cells Transfected with Dominant-Negative Forms of FBL2 (A) On day 0, Huh7-K2040 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, the cells were transfected with indicated amount of pCMV-Myc-FBL2 (red), pCMV-Myc-FBL2(67–423) (green), or pCMV-Myc-FBL2(67–423; C420S) (blue). Total plasmid concentration was adjusted to 2 μg/dish with pcDNA3.1. On day 3, the cells were harvested, and total RNA pooled from 2 dishes was extracted. The level of HCV RNA was determined by real-time quantitative PCR (upper panel). Values from three independent experiments, each denoted by an individual dot, are presented relative to the empty vector-transfected control value, which was set at 1. Expression of FBL2 was examined by SDS-PAGE analysis of cell lysates (from 0.1 dish of cells) followed by immunoblot with anti-Myc IgG-9E10 (bottom panel). Filter was exposed for 1 s. (B) On day 0, Huh7-K2040 and Huh7-WNV-2 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 2 μg/dish of pcDNA3.1, pCMV-Myc-FBL2, pCMV-Myc-FBL2(67–423), or pCMV-Myc-FBL2(67–423;C420S) as indicated. On day 3, the cells were harvested, and total RNA pooled from 2 dishes was extracted. The level of HCV RNA and West Nile virus RNA from Huh7-K2040 and Huh7-WNV-2 cells, respectively, was determined by real-time quantitative PCR (upper panel). Values (mean ± SEM) from three independent experiments are presented relative to the empty vector-transfected control value, which was set at 1. Expression of FBL2 was examined by SDS-PAGE analysis of cell lysates (from 0.1 dish of cells) followed by immunoblot with anti-Myc IgG-9E10 (bottom panel). Filter was exposed for 1 s. (C) On day 0, Huh7-K2040 cells were set up at 3 × 105 cells per 60 mm dish. On day 1, the cells were cotransfected with the indicated amount of pCMV-Flag-HA-NS5A (red) or pCMV-Flag-HA-NS4B (blue) in the presence (left panel) or absence (right panel) of 0.5 μg/dish of pCMV-Myc-FLB2(67-423). Total plasmid concentration was adjusted to 2.5 μg/dish with pcDNA3.1. On day 3, cells were harvested, and total RNA pooled from 2 dishes was extracted. The level of HCV RNA was determined by real-time quantitative PCR. Values from three independent experiments, each denoted by an individual dot, are presented relative to the control value in cells not transfected with either NS5A or NS4B, which was set at 1. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Replication of HCV RNA in Huh7-HP Cells Transfected with siRNA Duplex Targeting FBL2 (A and B) On day 0, Huh7-HP cells were set up at 4 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 400 pmol/dish of siRNA duplex targeting GFP, FBL2, or a six-nucleotide mismatch control for siRNA targeting FBL2 (FBL2 mismatch) as described in Experimental Procedures. Four hr after transfection, the medium was switched to medium A supplemented with 10% FBS. On day 2, the cells were transfected with 1 μg/dish of empty vector pcDNA3.1 (A) or pCMV-Myc-FBL2(W) encoding a wobble mutant of FBL2 containing three neutral point mutations within the siRNA target sequence (B). On day 4, the cells were harvested, total RNA pooled from 2 dishes was extracted, and the RNA level was measured by real-time quantitative PCR. Values (mean ± SEM) from three independent experiments are shown relative to the GFP siRNA-transfected control, which was set at 1. The CT value for FBL2 mRNA in nontransfected Huh7-HP cells ranged from 22–24 in multiple experiments. Molecular Cell 2005 18, 425-434DOI: (10.1016/j.molcel.2005.04.004) Copyright © 2005 Elsevier Inc. Terms and Conditions