Hepatitis C virus hijacks P-body and stress granule components around lipid droplets

Abstract

The microRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of the microRNA effectors DDX6, Lsm1, Xrn1, PATL1, and Ago2, but not the decapping enzyme DCP2, and dynamically redistributed these microRNA effectors to the HCV production factory around lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed the stress granule components GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), ataxin-2 (ATX2), and poly(A)-binding protein 1 (PABP1) to the HCV production factory. In this regard, we found that the P-body formation of DDX6 began to be disrupted at 36 h postinfection. Consistently, G3BP1 transiently formed stress granules at 36 h postinfection. We then observed the ringlike formation of DDX6 or G3BP1 and colocalization with HCV core after 48 h postinfection, suggesting that the disruption of P-body formation and the hijacking of P-body and stress granule components occur at a late step of HCV infection. Furthermore, HCV infection could suppress stress granule formation in response to heat shock or treatment with arsenite. Importantly, we demonstrate that the accumulation of HCV RNA was significantly suppressed in DDX6, Lsm1, ATX2, and PABP1 knockdown cells after the inoculation of HCV-JFH1, suggesting that the P-body and the stress granule components are required for the HCV life cycle. Altogether, HCV seems to hijack the P-body and the stress granule components for HCV replication.

Fig. 1.

Dynamic recruitment of DDX3 and DDX6 around lipid droplets (LDs) in response to HCV-JFH1 infection. (A) HCV-JFH1 disrupts the P-body formation of DDX6. Cells were fixed at 60 h postinfection and were then examined by confocal laser scanning microscopy. Cells were stained with anti-HCV core (CP-9 and CP-11 mixture) and either anti-DDX3 (54257 and 54258 mixture) or anti-DDX6 (A300-460A) antibody and then visualized with FITC (DDX3 or DDX6) or Cy3 (core). Images were visualized by using confocal laser scanning microscopy. The two-color overlay images are also exhibited (merged). Colocalization is shown in yellow. (B) HCV-JFH1 recruits DDX3 or DDX6 around LDs. Cells were stained with either anti-DDX3 or anti-DDX6 antibody and were then visualized with Cy3 (red). Lipid droplets and nuclei were stained with BODIPY 493/503 (green) and DAPI (blue), respectively. A high-magnification image is also shown. (C) Colocalization of DDX3 with DDX6. HCV-JFH1-infected RSc cells at 60 h postinfection were stained with anti-DDX3X (LS-C64576) and anti-DDX6 (A300-460A) antibodies. 293FT cells cotransfected with 100 ng of pcDNA3-FLAG-DDX6 and 100 ng of pHA-DDX3 (41) were stained with anti-FLAG-Cy3 and anti-HA-FITC antibodies (Sigma). (D) Association of DDX3 and DDX6 with LDs in response to HCV-JFH1 infection. The LD fraction and whole-cell lysates (WCL) were collected from uninfected RSc cells (control) or HCV-JFH1-infected RSc cells at 5 days postinfection. The results of Western blot analyses of DDX3, DDX6, and the HCV core protein as well as the LD marker ADFP and the endoplasmic reticulum (ER) marker calnexin in the LD fraction are shown.

Fig. 2.

HCV disrupts the P-body formation of microRNA effectors. Uninfected RSc cells and HCV-JFH1-infected RSc cells at 72 h postinfection were stained with anti-human AGO2 (011-22033) and anti-DDX6 (A300-460A) antibodies. The cells were also stained with anti-HCV core and anti-Lsm1 (LS-C97364), anti-Xrn1 (A300-443A), anti-PATL1, or anti-DCP2 (A302-597A) antibodies and were examined by confocal laser scanning microscopy.

Fig. 3.

Dynamic redistribution of ataxin-2 (ATX2) around LDs in response to HCV-JFH1 infection. (A) ATX2 is a stress granule component. RSc cells were incubated at 37°C or 43°C for 45 min. Cells were stained with anti-G3BP1 (A302-033A) and anti-ATX2 (A93520) antibodies and were examined by confocal laser scanning microscopy. (B) Dynamic redistribution of DDX6 and ATX2 in response to heat shock or HCV infection. RSc cells after heat shock at 43°C for 45 min or 72 h after inoculation with HCV-JFH1 were stained with anti-DDX6 and anti-ATX2 (A93520) antibodies. (C) HCV relocalizes ataxin-2 to LDs. HCV-JFH1-infected RSc cells at 72 h postinfection were stained with anti-ATX2 (A93520) antibody and BODIPY 493/503. (D) ATX2 colocalizes with the HCV core protein. HCV-JFH1-infected RSc cells at 72 h postinfection were stained with anti-ATX2/SCA2 (A301-118A) and anti-HCV core antibodies.

Fig. 4.

Dynamic redistribution of DDX6 and G3BP1 in response to HCV-JFH1 infection. RSc cells at the indicated times (hours) after inoculation with HCV-JFH1 were stained with anti-HCV core and either anti-DDX6 (A300-460A) or anti-G3BP1 (A302-033A) antibodies.

Fig. 5.

HCV suppresses stress granule formation in response to heat shock or treatment with arsenite. Naïve RSc cells or HCV-JFH1-infected RSc cells at 72 h postinfection were incubated at 37°C or 43°C for 45 min. Cells were also treated with 0.5 mM arsenite for 30 min. Cells were stained with anti-HCV core and anti-G3BP1 (A), anti-ATX2 (B), or anti-PABP1 (ab21060) (C) antibodies and were examined by confocal laser scanning microscopy.

Fig. 6.

Host protein expression levels in response to HCV-JFH1 infection. The results of the Western blot analyses of cellular lysates with anti-ATX2/SCA2 antibody (A301-118A), anti-PABP1 (ab21060), anti-HSP70 (610607), anti-HCV core, anti-β-actin, anti-DDX3 (54257 [NT] and 5428 [IN] mixture), anti-DDX6 (A300-460A), anti-G3BP1 (611126), or anti-LSM1 (LS-C97364) antibody in HCV-JFH1-infected RSc cells at 72 h postinfection as well as in naïve RSc cells are shown.

Fig. 7.

Requirement of P-body and stress granule components for HCV replication. (A) Inhibition of DDX3 or DDX6 mRNA expression by the shRNA-producing lentiviral vector. Real-time LightCycler RT-PCR for DDX3 or DDX6 was also performed for β-actin mRNA in RSc cells expressing shRNA targeted to DDX3 (DDX3i) or DDX6 (DDX6i) or the control nontargeting shRNA (shCon) in triplicate. Each mRNA level was calculated relative to the level in RSc cells transduced with the control nontargeting lentiviral vector (shCon), which was assigned as 100%. Error bars in this panel and other panels indicate standard deviations. (B) Levels of intracellular genome-length HCV-JFH1 RNA in the cells at 24 h postinfection at an MOI of 4 were monitored by real-time LightCycler RT-PCR. Results from three independent experiments are shown. Each HCV RNA level was calculated relative to the level in RSc cells transduced with a control lentiviral vector (shCon), which was assigned as 100%. (C) The levels of HCV core in the culture supernatants from the stable knockdown RSc cells 24 h after inoculation of HCV-JFH1 at an MOI of 4 were determined by ELISA. Experiments were done in triplicate, and columns represent the mean core protein levels. (D) The infectivity of HCV in the culture supernatants from stable-knockdown RSc cells 24 h after inoculation of HCV-JFH1 at an MOI of 4 was determined by a focus-forming assay at 24 h postinfection. Experiments were done in triplicate, and each virus titer was calculated relative to the level in RSc cells transduced with a control lentiviral vector (shCon), which was assigned as 100%. (E) Inhibition of ATX2, PABP1, Lsm1, Xrn1, G3BP1, or PATL1 protein expression by 72 h after transient transfection of RSc cells with a pool of control nontargeting siRNA (siCon) or a pool of siRNAs specific for ATX2, PABP1, Lsm1, Xrn1, G3BP1, or PATL1 (25 nM), respectively. The results of Western blot analyses of cellular lysates with anti-ATX2, anti-PABP1, anti-Lsm1, anti-Xrn1, anti-G3BP1, anti-PATL1, or anti-β-actin antibody are shown. (F) Levels of intracellular genome-length HCV-JFH1 RNA in the cells at 48 h postinfection at an MOI of 1 were monitored by real-time LightCycler RT-PCR. RSc cells were transiently transfected with a pool of control siRNA (siCon) or a pool of siRNAs specific for ATX2, PABP1, Lsm1, Xrn1, G3BP1, and PATL1 (25 nM). At 48 h after transfection, the cells were inoculated with HCV-JFH1 at an MOI of 1 and incubated for 2 h. The culture medium was then changed and incubated for 22 h. Experiments were done in triplicate, and each HCV RNA level was calculated relative to the level in RSc cells transfected with a control siRNA (siCon), which was assigned as 100%. Asterisks indicate significant differences compared to the control treatment (*, P < 0.01).