Volume 118, Issue 1, Pages 152-162 (January 2000) Cell cycle regulation of hepatitis C virus internal ribosomal entry site–directed translation  Masao Honda, Shuichi Kaneko, Eiki Matsushita, Kenichi Kobayashi, Geoffrey A. Abell, Stanley M. Lemon  Gastroenterology  Volume 118, Issue 1, Pages 152-162 (January 2000) DOI: 10.1016/S0016-5085(00)70424-0 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Organization of the transcriptional unit of plasmid pRL-HL. Plasmid pRL-HL contains a dicistronic CMV transcriptional cassette in which an upstream Renilla luciferase gene and a downstream firefly luciferase gene are separated by the complete 5'NTR and 66-nucleotide core sequence of HCV (nucleotides 1-407, strain 1b) placed within the intercistronic space. CMV, cytomegalovirus promoter; T7, bacteriophage T7 RNA polymerase promoter; BGH-pA, bovine growth hormone polyadenylation signal. (B) In vitro translation of pRL-HL. The 36-kilodalton Renilla luciferase is produced from the upstream reading frame in this transcript, and the 63-kilodalton protein representing the HCV core fusion with firefly luciferase is produced from the downstream reading frame. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Organization of the transcriptional unit of plasmid pRL-HL. Plasmid pRL-HL contains a dicistronic CMV transcriptional cassette in which an upstream Renilla luciferase gene and a downstream firefly luciferase gene are separated by the complete 5'NTR and 66-nucleotide core sequence of HCV (nucleotides 1-407, strain 1b) placed within the intercistronic space. CMV, cytomegalovirus promoter; T7, bacteriophage T7 RNA polymerase promoter; BGH-pA, bovine growth hormone polyadenylation signal. (B) In vitro translation of pRL-HL. The 36-kilodalton Renilla luciferase is produced from the upstream reading frame in this transcript, and the 63-kilodalton protein representing the HCV core fusion with firefly luciferase is produced from the downstream reading frame. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Southern blotting of RCF-26 cellular DNA after either single ApaI or double ApaI/BglII digestions. Digestion of pRL-HL generated a fragment  ̃8.8 kb long, whereas a double digestion of this DNA with ApaI and BglII generated a 4.2-kb band, consistent with the length of the segment containing the T7/CMV promoter, Renilla luciferase, HCV cDNA, and firefly luciferase sequences. We electrophoresed 150 pg of pRL-HL (which corresponds to 10 copies of integrated genes) in the positive control lane. Huh-7 cell DNA was used as the negative control. (B) Northern blot analysis of RNA extracted from the RCF-26 cell line showed a single transcript of the expected 3.3-kb length. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Southern blotting of RCF-26 cellular DNA after either single ApaI or double ApaI/BglII digestions. Digestion of pRL-HL generated a fragment  ̃8.8 kb long, whereas a double digestion of this DNA with ApaI and BglII generated a 4.2-kb band, consistent with the length of the segment containing the T7/CMV promoter, Renilla luciferase, HCV cDNA, and firefly luciferase sequences. We electrophoresed 150 pg of pRL-HL (which corresponds to 10 copies of integrated genes) in the positive control lane. Huh-7 cell DNA was used as the negative control. (B) Northern blot analysis of RNA extracted from the RCF-26 cell line showed a single transcript of the expected 3.3-kb length. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Cycloheximide treatment of RCF-26 cell lines. Cells were treated with cyclohexamide (2500 μg/mL) and harvested at 3-hour intervals for testing in the dual luciferase assay. ■, Renilla luciferase activity; 2, firefly luciferase activity. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Poliovirus infection of RCF-26 cells. (A) Renilla (■) and firefly (2) luciferase activities at 3-hour intervals after infection. (B) Relative IRES activity (firefly luciferase/Renilla luciferase activities). Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Cell growth and differences in cap-dependent translation and HCV IRES–directed translation. (A) Luciferase activities measured in replicate cultures beginning 24 hours after the seeding of cells into culture wells and then daily for a total of 7 days. ■, Renilla luciferase activity; 2, firefly luciferase activity. Cell confluency is shown at bottom. (B) Renilla and firefly luciferase activities normalized to cell number. (C) Relative IRES activity. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 HCV IRES activity in cells grown in different concentrations of serum. RCF-26 cells were grown in 10% FBS until 10% confluent and then refed with media having different serum concentrations at time 0:1% (♢), 5% (2), 10% (▴), and 20% (●) fetal bovine serum. (A) Cell number. (B) Renilla and firefly luciferase activities normalized to cell number. (C) Relative IRES activity (F/R, firefly luciferase/Renilla luciferase activities). **P < 0.01. NS, not significant. Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Influence of cell cycle on HCV IRES activity in synchronized cell cultures. Luciferase activities measured at 3-hour intervals in cultures of (A) RCF-26 and (B) RCF-1 cells after release from aphidicoline block at the G1/S interface. ■, Renilla luciferase activity; 2, firefly luciferase activity. Relative IRES activity at 3-hour intervals in (C) RCF-26 and (D) RCF-1 cells after release from aphidicoline block. Cell cycle distributions in each cell population were determined by flow-cytometric measurement of DNA content of individual cells. ■, Relative IRES activities; ●, proportion of cells in S phase; ○, proportion of cells in G2/M phase. (E) Northern blot analysis of RCF-26 cells 6, 18, and 32 hours after release from the aphidicoline block. An intact 3.3-kb transcript was detected at each point in time (see text for details). Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Influence of cell cycle on HCV IRES activity in synchronized cell cultures. Luciferase activities measured at 3-hour intervals in cultures of (A) RCF-26 and (B) RCF-1 cells after release from aphidicoline block at the G1/S interface. ■, Renilla luciferase activity; 2, firefly luciferase activity. Relative IRES activity at 3-hour intervals in (C) RCF-26 and (D) RCF-1 cells after release from aphidicoline block. Cell cycle distributions in each cell population were determined by flow-cytometric measurement of DNA content of individual cells. ■, Relative IRES activities; ●, proportion of cells in S phase; ○, proportion of cells in G2/M phase. (E) Northern blot analysis of RCF-26 cells 6, 18, and 32 hours after release from the aphidicoline block. An intact 3.3-kb transcript was detected at each point in time (see text for details). Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Influence of cell cycle on HCV IRES activity in synchronized cell cultures. Luciferase activities measured at 3-hour intervals in cultures of (A) RCF-26 and (B) RCF-1 cells after release from aphidicoline block at the G1/S interface. ■, Renilla luciferase activity; 2, firefly luciferase activity. Relative IRES activity at 3-hour intervals in (C) RCF-26 and (D) RCF-1 cells after release from aphidicoline block. Cell cycle distributions in each cell population were determined by flow-cytometric measurement of DNA content of individual cells. ■, Relative IRES activities; ●, proportion of cells in S phase; ○, proportion of cells in G2/M phase. (E) Northern blot analysis of RCF-26 cells 6, 18, and 32 hours after release from the aphidicoline block. An intact 3.3-kb transcript was detected at each point in time (see text for details). Gastroenterology 2000 118, 152-162DOI: (10.1016/S0016-5085(00)70424-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions