Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication

Abstract

We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.

FIG. 1.

Effects of BFA and GCA treatments on levels of HCV RNA (A and B) and protein (C) in FLRP1 replicons. (A and B) Total cellular RNA was harvested, and levels of HCV and Arf1 RNA were determined by quantitative RT-PCR, as described in Materials and Methods. Data are means ± standard errors for three independent experiments. (A) Cells were treated with either BFA, GCA, or an equivalent volume of DMSO, as described in Materials and Methods. (B) In some experiments, cells were treated with GCA for only 1 h or for 20 h, and immediately thereafter, cells were incubated in drug-free growth medium for an additional 16 h (washout). (C) Equal protein levels were loaded, and after BFA or GCA treatment, levels were analyzed by Western blotting using specific antibodies directed against the indicated proteins.

FIG. 2.

NS5A and NS3, but not core, are redistributed from speckle-like structures to the peripheries of LDs following BFA and GCA treatments. (A and B) FLRP1 cells were treated with either BFA, GCA, or DMSO (see Materials and Methods for details). In another experiment, cells were first treated with BFA or GCA and were then incubated for 16 h in drug-free medium (washout). Cells were immunostained with anti-NS5A (A), anti-NS3, or anti-core (B) primary antibodies and Alexa 488-labeled secondary antibodies (green). Oil Red O and 4′,6-diamidino-2-phenylindole (DAPI) were used to label LDs (red) and nuclei (blue), respectively. Cells were visualized by confocal microscopy. Images are representative of three independent experiments. (C) LDs whose peripheries were labeled with the viral protein (A, arrow) and LDs that did not show associated staining of viral proteins (A, arrowhead) were scored. A total of 50 to 100 LDs were counted in each experiment. The fraction of LDs (percentage of total) exhibiting peripheral viral protein labeling was determined per microscopic field. Results are means ± standard errors for six fields of view.

FIG. 3.

Effects of BFA or GCA treatment on viral RNA levels (A), NS5A distribution (B), and the level of infectious viral particles (C). (A) (Top) GCA and BFA reduce HCV and Arf1 RNA levels. J6/JFH1-expressing cells were treated with BFA or GCA. Arf1 and HCV RNA levels were determined by RT-PCR. The results are means ± standard errors for three independent experiments. (Bottom) GCA inhibits HCV replication. Huh7.5 cells were electroporated with J6/JFH (5′C19Rluc2AUbi), a monocistronic reporter virus expressing Renilla luciferase, and were cultured in the presence or absence of 10 μM GCA for 4, 24, and 48 h. Control cells were treated identically with DMSO. Cells electroporated with a replicon containing the GND inactivating mutation in the viral polymerase (Pol⁻) served as a negative control. Duplicate samples were harvested for luciferase assays at the indicated time points posttreatment. Values were normalized to the input RNA levels measured at 4 h. Results are means ± standard errors. (B) NS5A is redistributed from speckle-like structures to the peripheries of LDs following GCA treatment. (Top) J6/JFH1-expressing cells were treated with GCA and stained with anti-NS5A, Oil Red O, and DAPI, as for Fig. 2A. (Bottom) NS5A associated with the peripheries of LDs was quantified as for Fig. 2C. (C) Effects of BFA and GCA on infectious viral particle levels. Huh7.5 cells were electroporated with J6/JFH1 RNA, and 72 h later, they were treated with either BFA (12 h), GCA (20 h), or an equivalent volume of DMSO as a control. Titers of infective HCV particles were determined in the cell medium (Extracellular) and lysates (Intracellular) by the TCID₅₀ method (see Materials and Methods). Results are means ± standard errors for three experiments performed independently, each in triplicate. P, <0.001 by a two-tailed t test of treated cells versus untreated controls.

FIG. 4.

Ectopic expression of NS5A reduces GTP-Arf1 levels. Huh7 cells were either transfected with pEF6-NS5A, mock transfected with the pEF6 vector, or left untreated (cont.). Seventy-two hours after transfection, cells were lysed and subjected to a pulldown assay using GGA-GST bound to glutathione-Sepharose beads, as described in Materials and Methods. (Top) Representative Western blot. Equal amounts of cell lysates of the pulled-down material were probed with anti-Arf1, anti-NS5A, and anti-actin antibodies. (Bottom) Quantitative analyses of three experiments. Results are means ± standard errors for three independent experiments (P, <0.001 by a two-tailed t test of NS5A- versus mock-transfected cells).

FIG. 5.

NS5A colocalizes with Arf1 T31N-GFP at the peripheries of LDs. (A to C) FLRP1 cells were transfected with the indicated Arf1-GFP constructs. (D to F) Huh7 cells were cotransfected with Arf1-GFP constructs and pEF6-NS5A. Cells were immunostained with anti-NS5A antibodies (blue) and with Oil Red O (red). Cells were then visualized by confocal microscopy. Arrows indicate sites of NS5A and Arf colocalization at the peripheries of LDs. (G) Quantitative analysis of GFP-Arf and NS5A associated with the peripheries of LDs was performed as for Fig. 2C.

FIG. 6.

Arf1 knockdown by siRNA treatment decreases HCV RNA replication and the levels of infectious viral particles. (A and B) FLRP1 cells. Cells were transfected with Arf1 siRNA (siArf1) or with a control nontargeting siRNA (siControl), as described in Materials and Methods. (A) (Top) Cells were then lysed and subjected to Western blot analysis using antibodies directed against NS5A, NS3, Arf1, or actin. (Bottom) In parallel experiments, total cellular RNA was extracted and used to determine HCV and Arf1 RNA levels by quantitative RT-PCR. Results are means ± standard errors for three experiments, each performed in triplicate. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.0001) by a two-tailed t test. (B) (Top) Cells treated with siArf1 or siControl were immunostained with anti-NS5A (green) and Oil Red O (red). (Bottom) NS5A associated with LDs was scored as for Fig. 2C. (C and D) J6/JFH1-expressing cells. Huh7.5 cells were electroporated with J6/JFH1 and were transfected with siArf1 or siControl. (C) Total RNA was extracted, and quantitative RT-PCR was used to determine the levels of HCV and Arf1 RNA. The results are means ± standard errors for three independent experiments (P < 0.0001). (D) Levels of infectious HCV particles were determined in the cell medium (Extracellular) and cell lysates (Intracellular). Results are means ± standard errors for three experiments performed independently, each in triplicate. P, <0.0001 by a two-tailed t test for siArf1-treated versus siControl-treated cells.