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. 2007 Dec;81(24):13922-6.
doi: 10.1128/JVI.01517-07. Epub 2007 Sep 12.

DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication

Yasuo Ariumi  1 , Misao Kuroki, Ken-ichi Abe, Hiromichi Dansako, Masanori Ikeda, Takaji Wakita, Nobuyuki Kato
Affiliations

DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication

Yasuo Ariumi et al. J Virol. 2007 Dec.

Abstract

DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

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Figures

FIG. 1.
FIG. 1.
Requirement of DDX3 for HCV replication. (A to D) Effect of DDX3 knockdown on HCV RNA replication. (A) Inhibition of DDX3 expression by shRNA-producing lentivirus vector. The results of Western blot analysis of cellular lysates with anti-DDX3 (ProSci), anti-HCV core (CP-9; Institute of Immunology), or an anti-β-actin antibody (Sigma) in O cells expressing shRNA against DDX3 (DDX3i#3) as well as in O cells transduced with a control lentivirus vector (Con) are shown. (B) The level of genome-length HCV RNA was monitored by real-time LightCycler PCR (Roche). Experiments were done in duplicate, and bars represent the mean percentages of HCV RNA. (C) Efficiency of colony formation in DDX3 knockdown cells. In vitro-transcribed ON/C-5B K1609E RNA (2 μg) was transfected into the DDX3 knockdown Oc cells (DDX3i#3) or the Oc cells transduced with a control lentivirus vector (Con). G418-resistant colonies were stained with Coomassie brilliant blue at 3 weeks after electroporation of RNA. Experiments were done in duplicate, and representative results are shown. (D) The level of subgenomic replicon RNA was monitored by real-time LightCycler PCR. Experiments were done in duplicate, and bars represent the mean percentages of HCV RNA. (E to G) Effect of DDX3 knockdown on HCV infection. (E) Inhibition of DDX3 expression by shRNA-producing lentivirus vector. The results of Western blot analysis of cellular lysates with anti-DDX3 or an anti-β-actin antibody for RSc cells expressing the shRNA DDX3i#3 or DDX3i#7 and for RSc cells transduced with a control lentivirus vector (Con) are shown. (F) The level of genome-length HCV (JFH1) RNA was monitored by real-time LightCycler PCR after inoculation of the cell culture-generated HCVcc. Results from three independent experiments are shown. (G) The levels of the HCV core in the culture supernatants were determined by an enzyme-linked immunosorbent assay (Mitsubishi Kagaku Bio-Clinical Laboratories). Experiments were done in duplicate, and bars represent the mean HCV core protein levels.
FIG. 2.
FIG. 2.
Interaction of the HCV core with DDX3. (A) The HCV core colocalizes with DDX3. 293FT cells cotransfected with 100 ng of pCXbsr/core(HCV-O) or pcDNA3/core(JFH1) and 200 ng of pHA-DDX3 were examined by confocal laser scanning microscopy. Cells were stained with anti-HCV core (CP-9 and CP-11 mixture) and anti-DDX3 antibodies and were then visualized with fluorescein isothiocyanate (DDX3) or Cy3 (core). Images were visualized using confocal laser scanning microscopy (LSM510; Carl Zeiss). The right panels exhibit the two-color overlay images (Merged). Colocalization is shown in yellow. (B) The core binds to DDX3. 293FT cells were cotransfected with 4 μg of pHA-DDX3 and 4 μg of pCXbsr/core(HCV-O) (wt), pcDNA3/Δcore(HCV-O) (Δ), pcDNA3/core(JFH1) (wt), or pcDNA3/Δcore(JFH1) (Δ). The cell lysates were immunoprecipitated with an anti-HA antibody (3F10; Roche), followed by immunoblot analysis using either anti-HA (HA-7; Sigma) or anti-HCV core antibody (CP-9 and CP-11 mixture). (C) 293FT cells transfected with 4 μg of pHA-DDX3 (H), O cells (O), or RSc cells 3 days after inoculation of HCVcc (JFH1) (J) were lysed and immunoprecipitated (IP) with 1 μg of anti-HA antibody (3F10), followed by immunoblotting with anti-HA (HA-7), anti-core (CP-9 and CP-11 mixture), or anti-HCV NS5A (no. 8926) and anti-HCV NS5B. (D) 293FT cells transfected with 4 μg of pHA-DDX3 and 4 μg of pcDNA3/FLAG-core(HCV-O) (wt), pcDNA3/FLAG-Δcore(HCV-O) (Δ), pcDNA3/FLAG-core(JFH1) (wt), or pcDNA3/FLAG-Δcore(JFH1) (Δ) were lysed and immunoprecipitated with 1 μg of an anti-HA antibody (3F10), followed by immunoblotting with an anti-HA (HA-7) or anti-core (CP-9 and CP-11 mixture) antibody.
FIG. 3.
FIG. 3.
HCV core does not affect the DDX3-mediated synergistic activation of Rev function. (A) Schematic representation of HIV-1 Rev-dependent luciferase-based reporter plasmid pDM628 harboring a splicing donor (SD), splicing acceptor (SA), and RRE. (B) 293FT cells were cotransfected with 100 ng of pDM628, 200 ng of pcRev, 200 ng of pHA-DDX3, and/or 100 ng of either pcDNA3/core(HCV-O) (O) or pcDNA3/core(JFH1) (J). A luciferase assay was performed 24 h later. All transfections utilized equal total amounts of plasmid DNA owing to the addition of the empty vector pcDNA3 to the transfection mixture. The relative stimulation of luciferase activity (n-fold) is shown. The results shown are means from three independent experiments.
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