Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

<div><p>Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation.</p></div

PC interacts and colocalizes with the HCV NS5A protein.

<p>PC interacts with NS5A derived from both genotype 1b and 2a. Either Huh7.5 cells (A) or HEK293T cells (B) were cotransfected with Myc-tagged NS5A of genotype 1b or 2a in the absence or presence of Flag-tagged PC. Cell lysates harvested at 24 h after transfection were immunoprecipitated with anti-Myc antibody and then bound proteins were immunoblotted with anti-Flag antibody. Protein expressions of NS5A and PC were verified using the same cell lysates by immunoblotting with anti-Myc antibody and anti-Flag antibody, respectively. (C) The biotin carboxylase domain of PC is the binding site for NS5A. Schematic diagram of both wild type and mutants of PC (upper panel). HEK293T cells were cotransfected with Myc-tagged NS5A and Flag-tagged mutant constructs of PC. Cells were harvested at 24 h after transfection and were immunoprecipitated with anti-Myc antibody. The coprecipitated proteins were immunoblotted with anti-Flag antibody (lower panel). BC, biotin carboxylase; CT, carboxyltransferase; PT, PC tetramerization; BCCP, biotin-carboxyl carrier protein. (D) PC interacts with the domain I of NS5A. Schematic diagram shows both wild type and mutants of NS5A (upper panel). HEK293T cells were cotransfected with Flag-tagged PC and Myc-tagged constructs of NS5A. Cell lysates harvested at 24 h after transfection were immunoprecipitated with anti-Flag antibody and then bound proteins were immunoblotted with anti-Myc antibody. Protein expressions of both NS5A and PC were confirmed using the same lysates by immunoblotting with anti-Myc and anti-Flag antibodies, respectively. (E) PC colocalizes with NS5A. Huh7.5 cells were infected with HCV Jc1 for 4 h. At two days postinfection, cells were fixed and then incubated with anti-NS5A, anti-PC antibody, and anti-VDAC antibody, respectively. Cells were further incubated with the appropriated secondary antibodies and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. (F) NS5A localizes to mitochondria. Huh7.5 cells were infected with Jc1 for 4 h and were cultured for 2 days. The cells were fractionated into cytosol, mitochondria, nuclei & cell debris, and microsomal fraction. The purities of the each subcellular fraction were confirmed by using GAPDH, VDAC, and Lamin A antibody, respectively.</p

HCV proteins modulate transcriptional activity of PC.

<p>(A) Schematic diagram of two PC promoters, P1 and P2, used in this study. (B) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with the indicated HCV protein expression plasmid. At 24 h after transfection, cells were harvested and luciferase activities were determined (top panels). Protein expressions were determined by anti-Myc antibody as shown in arrows (bottom panels). Vector indicates pGL3 empty vector. (-) denotes pGL3-PC in the absence of HCV protein (C) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with increasing amounts of NS5A expression plasmid (genotype 1b). Cells were harvested at 24 h after transfection and then luciferase activities were determined (top panels). NS5A expressions were determined by anti-NS5A antibody (bottom panels). (D) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with Myc-tagged mutant constructs of NS5A. At 24 h after transfection, cells were harvested and luciferase activities were determined. (E) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with NS5A plasmid derived from either genotype 1b or 2a. At 24 h after transfection, cells were harvested and luciferase activities were determined. (F) Huh7.5 cells were either mock infected or infected with HCV Jc1 for 4 h. At 5 days postinfection, cells were transfected with either vector or P1-Luc (left panel), or vector or P2-Luc (right panel) reporter plasmid. Cells were further cultured for 2 days and then luciferase activities were determined. Protein expressions were verified by immunoblot analysis using anti-NS3 and anti-actin antibody, respectively. Luciferase activities were normalized based on β-galactosidase activities. Asterisks indicate significant differences (*, <i>P</i><0.05, **, <i>P</i><0.01) from the activity for the control.</p

NS5A regulates PC expression level.

<p>(A–C) PC expression level is decreased, whereas FAS expression level is increased in cells expressing NS5A protein. (A) Total cell lysates harvested from either IFN-cured cells or replicon cells were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from either IFN-cured cells or replicon cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel) by qRT-PCR. (B) Huh7.5 cells were either mock infected or infected with Jc1. Total cell lysates harvested at 5 days postinfection were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from either mock infected or Jc1 infected cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel) by qRT-PCR using data from three independent experiments. Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. Error bars indicate standard deviations. (C) Equal amounts of cell lysates harvested from either vector stable or NS5A stable cells derived from genotype 1b or 2a were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from the indicated stable cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel). Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. (D) Huh7.5 cells were transfected with siRNAs of either negative or PC. At 3 days after transfection, cells were harvested and analyzed by immunoblotting with the indicated antibodies (top panel). Actin was used as a loading control. Total RNAs isolated from the indicated cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel). Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. (E) Huh7.5 cells were transiently transfected with either pEF6 empty vector or pEF6-NS5A-Myc expression plasmid. Cell lysates harvested at 48 h or 72 h after transfection were immunoblotted with the indicated antibodies.</p

Silencing of PC impairs production of infectious HCV.

<p>(A) Knockdown of PC had no effect on viral protein levels in HCV replicon cells. Huh7 cells harboring HCV replicon were transfected with 10 nM of negative (Neg), positive (Pos), or the indicated siRNA duplexes for 3 days. Total RNAs were extracted and intracellular HCV RNA was analyzed by qRT-PCR. Negative, irrelevant siRNA pool; positive, HCV-specific siRNAs. (B) Total replicon cell lysates harvested at 3 days after siRNA transfection were immunoblotted with the indicated antibodies. (C) Huh7.5 cells were transfected with 10 nM of the indicated siRNA. At 2 days after siRNA transfection, cells were infected with Jc1 for 4 h. At 48 h postinfection, cell proliferation was assessed by the MTT assay. Huh7.5 cells were treated as described in (C). At 48 h postinfection, both intracellular HCV RNA (D) and extracellular HCV RNA isolated from culture supernatants (F) were analyzed by qRT-PCR. (E) Total cell lysates harvested at 48 h after Jc1 infection were immunoblotted with the indicated antibodies. (G) Intracellular infectious HCV particles were prepared by 4 rounds of freeze and thaw treatments from cells treated as in figure legend to C. Neg indicates cells infected with intracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with intracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with intracellular HCV isolated from the Positive siRNA-transfected cells. (H) Extracellular infectious HCV particles were prepared from the culture media in cells treated as in figure legend to C. Neg indicates cells infected with extracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with extracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with extracellular HCV isolated from the Positive siRNA-transfected cells. (G, H) Naïve Huh7.5 cells were then infected with intracellular infectious HCV (G) and extracellular infectious HCV (H). Total cell lysates harvested at 2 days postinfection were immunoblotted to determine the indicated protein levels, and HCV RNA levels were analyzed by qRT-PCR (top panels). Naïve Huh7.5 cells treated as described above were analyzed for immunofluorescence using anti-NS5A antibody (bottom panels). Cells were counterstained with DAPI to label nuclei. Samples were analyzed for immunofluorescence staining using a Zeiss LSM 700 laser confocal microscopy system. Asterisks indicate significant differences (*, <i>P</i><0.05, **, <i>P</i><0.01) from the value for the negative control. Error bars indicate standard deviations.</p