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. 2019 Apr 6;18(1):87.
doi: 10.1186/s12944-019-1022-7.

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver

Jin Zhang  1 , Xing Gao  1 , Yuan Yuan  1 , Chao Sun  2 , Yuanlin Zhao  1 , Liming Xiao  1 , Ying Yang  1 , Yu Gu  1 , Risheng Yang  1 , Peizhen Hu  1 , Lijun Zhang  3 , Chao Wang  4 , Jing Ye  5
Affiliations

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver

Jin Zhang et al. Lipids Health Dis. .

Abstract

Background: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit.

Methods: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A.

Results: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A.

Conclusions: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Keywords: Hepatitis C virus; Lipid droplet; Lipolysis; Nonstructural protein 5A; Perilipin 5.

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Conflict of interest statement

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committees of Fourth Military Medical University.

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All authors read and approve the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Plin5 deficiency aggravated HCV NS5A-induced hepatic injuries. Adenovirus containing NS5A (Ad-NS5A) or the control adenovirus (Ad-Null) were administrated to the wild-type and Plin5-null mice through tail veins respectively (n = 4). After 1 week, their livers were removed and the bloods were collected. a The expression of NS5A, Plin5 and ATGL in the livers was analysed by immunoblotting. b H&E staining showed that NS5A induced more severe hepatic injuries and more inflammatory cell infiltration in the Plin5-null livers than in wild-type livers. Scale bar = 50 μm. c & d Immunohistochemical staining indicated that NS5A significantly increased the infiltration of F4/80-positive macrophages (c) and CD3-positive T lymphocytes (d) in Plin5-null livers compared to that observed in wild-type livers. Scale bar = 50 μm. e & f The levels of IL-2 (e) and TNFα (f) in mouse sera were analysed by ELISA kits. *P < 0.05, **P < 0.01. Data are means ± SEs for triplicate samples for each representative experiment
Fig. 2
Fig. 2
Plin5 alleviated NS5A-induced lipid peroxidation in liver cells. a and b Livers were isolated and homogenized, and the levels of MDA and 4-hydroxynonenal (4-HNE), two lipid oxidation products, were determined in the homogenate (n = 4). c Primary hepatocytes were isolated from wild-type (Plin5+/+) and Plin5-null (Plin5−/−) mice and infected with control (Ad-Null) and NS5A-expressing (Ad-NS5A) adenoviruses. The expression of NS5A, Plin5 and ATGL in primary hepatocytes was analysed by immunoblotting. d After treatment with 200 μM oleic acid (OA) for 24 h, the ROS levels in hepatocytes with or without NS5A expression were determined by DCFH-DA staining. Scale bar = 50 μm. e and f The levels of MDA and 4-hydroxynonenal (4-HNE) were analysed in primary hepatocytes with or without NS5A expression.**P < 0.01, ***P < 0.001. Data are means ± SEs for triplicate samples for each representative experiment
Fig. 3
Fig. 3
Plin5 stimulated lipid accumulation and reduced the lipolysis induced by NS5A. a Oil red O (ORO) staining of liver tissues from wild-type (Plin5+/+) and Plin5-null (Plin5−/−) mice. Scale bar = 50 μm. b & c Triglyceride (TG) and non-esterified fatty acid (NEFA) content was quantified by commercial kits after extraction with hexane/isopropanol. d Primary hepatocytes were isolated from wild-type and Plin5-null mice and then infected with control (Ad-Null) and NS5A-expressing (Ad-NS5A) adenoviruses. The lipid droplets (LDs) in primary hepatocytes were stained with BODIPY 493/503 and observed under a fluorescence microscope. Scale bar = 20 μm. e After extraction with hexane/isopropanol, the TG content was quantified in wild-type (Plin5+/+) and Plin5-null (Plin5−/−) hepatocytes. f Primary hepatocytes with or without NS5A expression were treated with triacsin C (5 μM), an inhibitor of acyl-CoA synthase, and the NEFA content was determined over 8 h. g Wild-type (Plin5+/+) and Plin5-null (Plin5−/−) primary hepatocytes with or without NS5A expression were incubated with 200 μM OA for 24 h. The cells were then treated with 5 μM triacsin C to inhibit further triacylglycerol synthesis. Cellular lipids were extracted with hexane/isopropanol, and then the TG content was quantified. *P < 0.05, **P <0.01, ***P < 0.001. Data are means ± SEs for triplicate samples for each representative experiment
Fig. 4
Fig. 4
Plin5 overexpression blunted peroxidation injury induced by NS5A. a HepG2 cells expressing Plin5 (Plin5 OV) and empty vehicle (EV) were established by lentivirus infection. The expression of NS5A and Plin5 in HepG2 cells was analysed by immunoblotting. (b & c) The influence of NS5A on lipid content was observed in HepG2 cells with or without Plin5 overexpression after BODIPY 493/503 staining (b) and TG quantification (c). Scale bar = 10 μm. d Intracellular NEFAs were quantified by commercial kit after extraction with hexane/isopropanol. e HepG2 cells with or without Plin5 overexpression were infected with control (Ad-Null) and NS5A-expressing (Ad-NS5A) adenoviruses. After treatment with 200 μM oleic acid (OA) for 24 h, the ROS levels in HepG2 cells were observed by DCFH-DA staining. Scale bar = 50 μm. f & g The levels of MDA and 4-hydroxynonenal (4-HNE), two lipid oxidation products, were analysed in HepG2 cells with or without NS5A expression. h The triacylglycerol hydrolysis rate in HepG2 cells with or without NS5A expression was determined in the presence of triacsin C (5 μM). *P < 0.05, **P <0.01, ***P < 0.001. Data are means ± SEs for triplicate samples for each representative experiment
Fig. 5
Fig. 5
NS5A recruited Plin5 to LDs. a NS5A was expressed in primary hepatocytes, and exogenous HA-NS5A (red) and endogenous Plin5 (green) were observed by immunofluorescent staining. Scale bar = 20 μm. b LD-containing fractions were isolated from wild-type mice with or without NS5A expression by using sucrose density gradient ultracentrifugation, and the Plin5 content was determined in both the total cell lysate (TCL) and the lipid droplet (LD)-containing fraction by immunoblotting. c Co-IP assay was performed to determine the interaction of exogenous HA-tagged NS5A and FLAG-tagged Plin5 in 293 T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and then immunoblotted with an anti-HA antibody. d NS5A was expressed in wild-type (Plin5+/+) and Plin5-null (Plin5−/−) primary hepatocytes, endogenous Plin5 was immunoprecipitated with anti-Plin5 antibody, and NS5A was detected by immunoblotting with anti-HA antibody. e Co-IP assays to identify the specific domain of Plin5 responsible for the Plin5-NS5A interaction. Truncated GFP-tagged Plin5 constructs were overexpressed in 293 T cells with HA-tagged NS5A. NS5A was immunoprecipitated by anti-HA monoclonal antibody, and truncated Plin5 was detected by immunoblotting
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