GRIM-19 Restricts HCV Replication by Attenuating Intracellular Lipid Accumulation

Abstract

Gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) targets multiple signaling pathways involved in cell death and growth. However, the role of GRIM-19 in the pathogenesis of hepatitis virus infections remains unexplored. Here, we investigated the restrictive effects of GRIM-19 on the replication of hepatitis C virus (HCV). We found that GRIM-19 protein levels were reduced in HCV-infected Huh7 cells and Huh7 cells harboring HCV replicons. Moreover, ectopically expressed GRIM-19 caused a reduction in both intracellular viral RNA levels and secreted viruses in HCVcc-infected cell cultures. The restrictive effect on HCV replication was restored by treatment with siRNA against GRIM-19. Interestingly, GRIM-19 overexpression did not alter the level of phosphorylated STAT3 or its subcellular distribution. Strikingly, forced expression of GRIM-19 attenuated an increase in intracellular lipid droplets after oleic acid (OA) treatment or HCVcc infection. GRIM-19 overexpression abrogated fatty acid-induced upregulation of sterol regulatory element-binding transcription factor-1 (SREBP-1c), resulting in attenuated expression of its target genes such as fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC). Treatment with OA or overexpression of SREBP-1c in GRIM-19-expressing, HCVcc-infected cells restored HCV replication. Our results suggest that GRIM-19 interferes with HCV replication by attenuating intracellular lipid accumulation and therefore is an anti-viral host factor that could be a promising target for HCV treatment.

Keywords: anti-viral host factor; hepatitis C virus; intracellular lipid accumulation; lipogenesis; viral replication.

FIGURE 1

Hepatitis C virus (HCV) infection and viral replication reduced GRIM-19 expression. (A,B) Protein levels of GRIM-19 were evaluated using western blot analysis in Huh7 cells infected with HCVcc at days 3, 6, 9, and 12 post-infection (A), FGR cells (B), and SGR cells (B). The relative protein expression was normalized to β-actin as a reference. (C) The endogenous GRIM-19 level was assessed by western blot analysis in Huh7.5.1 cells or Huh7.5.1-derived HCV genotype 3 full genomic replicon cells. The relative protein expression was normalized to β-actin as a reference. (D) Relative levels of endogenous GRIM-19 mRNA in SGR cells, FGR cells, and HCVcc-infected Huh7 cells compared to that in Huh7 cells. β-actin was used as a reference gene. (E) Protein levels of GRIM-19 in liver tissues from patients with chronic liver diseases (CLD) caused by persistent HCV infection (n = 4) were analyzed by western blot analysis. Tissue lysates from liver without viral hepatitis were used as a control (normal; n = 4). β-actin was used as a loading control. The values of the GRIM-19 protein levels were expressed relative to the level in control tissues (right). All data represent the mean ± SEM (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to control.

FIGURE 2

Anti-HCV activity of ectopically expressed GRIM-19. (A) Overexpression of GRIM-19 was induced by transfection with pcDNA3_GRIM-19 in Huh7 cells. At 48 h post-transfection, GRIM-19 overexpression was detected by western blot analysis. (B) Huh7 cells were transfected with pEGFP-C1 or pEGFP-C1-GRIM-19. After 48 h, the cells were subjected to flow cytometry to evaluate the transfection efficiency. (C) HCVcc-infected Huh7 cells at day 9 post-infection were transfected with pcDNA3, pcDNA3_GRIM-19 or pcDNA3_EGFP. After 48 h, the intracellular levels of HCV RNA were evaluated by rqRT PCR (left), and the protein level of HCV NS5A was detected by western blot analysis (right). The values of the HCV RNA levels were expressed relative to the level in cells transfected with pcDNA3. (D) At day 9 post-infection, HCVcc-infected Huh7 cells were transfected with pcDNA3 or pcDNA3_GRIM-19. After 48 h, the culture media was used to evaluate the extracellular level of HCV RNA (left) as well as for re-infection. After another 48 h, the HCV RNA levels in re-infected Huh7 cells were evaluated by rqRT PCR. (E) Levels of HCV RNA and HCV core protein in FGR cells were analyzed after transfection with pcDNA3, pcDNA3_GRIM-19 or pcDNA3_EGFP as in (C). (F) The values of the HCV RNA levels are analyzed in SGR cells transfected with pcDNA3 or pcDNA3_GRIM-19 as in (C). (G) To examine the inhibitory effect of repeated transfection with pcDNA3_GRIM-19, Huh7 cells were infected with HCVcc at an MOI of 0.3. After 24 h, cells were transfected with pcDNA3 or pcDNA3_GRIM-19. The cells were maintained at 3-day intervals and transfected with pcDNA3 or pcDNA3_GRIM-19 repeatedly. At 3, 6, 9, and 12 days post-infection, cells were harvested, and HCV RNA levels were evaluated. (H) At day 9 post-infection, HCVcc-infected Huh7 cells were transfected with pcDNA3 or pcDNA3_GRIM-19. After 24 h, the cells were transfected with scrambled siRNA or GRIM-19-siRNA. Changes in GRIM-19 levels were assessed by western blot analysis 48 h post-transfection with siRNAs (bottom). In addition, HCV RNA levels were evaluated by rqRT-PCR (top). (I) HCV-IRES activity in Huh7 transfected with pcDNA3 or pcDNA3_GRIM-19. The data represent the means ± SEM (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to control.

FIGURE 3

GRIM-19 overexpression did not alter STAT3 transcriptional activity. (A) The expression and phosphorylation of STAT3 after transfection with pcDNA3 or pcDNA3_GRIM-19 were evaluated by western blot analysis. β-actin was used as an internal control for loading. (B) To evaluate subcellular localization of phospho-STAT3, STAT3, and GRIM-19, HCVcc-infected Huh7 cells were transfected with pEGFP-C1 or pEGFP-C1_GRIM-19. After 48 h, the cells were subjected to subcellular fractionation followed by western blot analysis. PARP and SOD1 were used as markers for the nucleus and cytosol. (C) The mRNA levels of bcl2 and mmp2 were assessed by RT-PCR with a gene-specific primer set. The mRNA expression was normalized to β-actin as a reference and expressed relative to the density of Huh7 cells. Values represent means ± SD. (D) Examination of the induction of apoptosis by overexpression of GRIM-19 in Huh7 cells or FGR cells. The cells were transfected with pcDNA3 or pcDNA3_GRIM-19. After 48 h, apoptosis was determined by a Annexin V/PI staining.

FIGURE 4

GRIM-19 overexpression inhibited lipid accumulation. (A) Huh7 cells were transfected with pcDNA3 or pcDNA3_GRIM-19. After 24 h, cells were treated with 100 μM OA. After an additional 24 h, intracellular lipid accumulation was visualized by Nile Red staining and observed under a confocal microscope (left). Red, lipid droplets (LDs); blue, DAPI; original magnification 200×. Fluorescence densities of Nile Red were quantified using a microplate reader and normalized to the cellular DAPI content. The values of intracellular lipid level were expressed relative to the level in the cells transfected with pcDNA3 and without OA treatment (right). The data represent the mean ± SEM (n = 3). ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to control. (B) Huh7 cells were transfected with pEGFP-C1 or pEGFP-C1-GRIM-19. After 24 h, cells were treated with 100 μM OA. After an additional 24 h, intracellular lipid accumulation was assessed by Nile Red staining. Red, LDs; Green, EGFP or EGFP-fused GRIM-19; blue, DAPI; original magnification 800×. (C) HCV core was immunostained in Huh7 cells infected with HCVcc at day 9 post-infection. Red, HCV core; blue, DAPI; original magnification 400×. (D) Changes of intracellular lipid accumulation by GRIM-19 overexpression was assessed in Huh7 cells infected with HCVcc at day 9 post-infection as in (A). (E) Smaller size and number of LDs in GRIM-19 overexpressing cells were visualized in HCVcc infected Huh7 cells as in (B).

FIGURE 5

Effect of GRIM-19 overexpression on the expression levels of genes involved in lipid metabolism. (A) Examination of the mRNA levels of three transcription factors that regulate the intracellular lipid level in Huh7 cells treated with OA or transfected with pcDNA3 or pcDNA3_GRIM-19. The mRNA expression was normalized to β-actin as a reference and the values of mRNA level were expressed relative to the level in cells transfected with pcDNA3 and without OA treatment. (B) mRNA levels of target genes of SREBP-1c were analyzed as in (A). (C) Protein levels of SREBP-1c and its target genes in Huh7 cells treated as in (A) were analyzed using western blot analysis. β-actin was used as an internal control for loading. (D,E) Protein levels of SREBP-1c and its target genes after transfection with pcDNA3 or pcDNA3_GRIM-19 in FGR cells (D) and Huh7 cells infected with HCVcc at day 9 post-infection (E). β-actin was used as an internal control for loading. (F) mRNA levels of DGAT1, DGAT-2, and MTP were analyzed as in (A). The data represent the mean ± SEM (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to control.

FIGURE 6

Restoration of GRIM-19 inhibition of HCV replication. At day 9 post-infection, HCVcc-infected Huh7 cells were transfected with pcDNA3 or pcDNA3_GRIM-19. After 24 h, the cells were treated with OA (A) or transfected with SREBP-1c-overexpressing plasmids (B). After an additional 48 h, intracellular levels of HCV RNA were evaluated by rqRT PCR (top), and the overexpressed protein levels was detected by western blot analysis (bottom). The values of the HCV RNA levels were expressed relative to the level in cells transfected with pcDNA3. The data represent the mean ± SEM (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to control.

FIGURE 7

A proposed model of the mechanism underlying the anti-HCV activity of GRIM-19. After HCV infection, lipid biosynthesis is upregulated via SREBP-1c activation. SREBP-1c is a transcription factor that enhances the expression of genes involved in fatty acid biosynthesis such as ACC, FAS, and SCD, causing increased levels of triglycerides that help create an appropriate microenvironment for persistent HCV infection. Conversely, GRIM-19 function is downregulated through the reduction of its protein levels in HCV-replicating cells. However, restoration of GRIM-19 expression can disrupt this suitable microenvironment by downregulating SREBP-1c expression.