The Role And Mechanism Of Perilipin 5 In The Development Of HCV NS5A Associated Hepatic Lipid Dysmetabolism

Hepatitis C virus(HCV)infection is one of the most important causes of chronic liver disease,with approximately 2.5% of the world’s population infected with HCV.Chronic HCV infection causes a spectrum of liver diseases,including hepatic steatosis,virus-associated steatohepatitis(VASH),fibrosis,cirrhosis,and even hepatocellular carcinoma(HCC).Among them,hepatic steatosis is an important clinicopathological manifestation of chronic HCV infection.Current research shows that the life cycle of HCV depends greatly on the disordered lipid metabolism in liver.The virus hijacks the lipid metabolism in the host,but the details and mechanism are still unclear.HCV RNA translates a variety of structural proteins(Core,E1,E2)and non-structural proteins(p7,NS2,NS3,NS4 A,NS4B,NS5 A,NS5B)in the host cells,which are crucial for life cycle of HCV.In the HCV infected hepatocytes,the viral proteins interact with the host proteins,and form an endoplasmic reticulum-derived “membranous web” to facilitate its own replication and assembly.In liver cells,lipid droplets(LDs)serve as “scaffold” for the assembly of infectious virions beyond their roles in fat storage.Moreover,NS5 A occupies the central role in the viral replication complex on LDs,and it manipulates the host lipid metabolism by interacting with various fatty acid synthetases and lipid droplet transport proteins.The perilipin family is the most important group of lipid droplet-assosiated proteins.Perilipin 5(Plin5)is usually expressed in tissues and organs that utilize mitochondrial β-oxidation for energy supply,such as liver,skeletal muscle,myocardium and brown adipose tissue(BAT).Plin5 serves as an energy sensor to regulate lipolysis of LDs and fatty acid β-oxidation in mitochondria according to the intracellular level of non-esterified fatty acid(NEFA).Our previous works indicted that Plin5 deficiency significantly reduced the hepatic triglyceride(TG)content,and elevated the lipid peroxidation level,resulting in liver injury.However,the roles of Plin5 in the lipid metabolic disorder induced by HCV infection remains to be further studied.AIMS:(1)Using Plin5-knockout mice and primary hepatocytes,we aimed to investigate the specific effects of Plin5 deficiency on oxidative stress and lipid peroxidation levels and hepatic injury induced by HCV NS5 A.(2)To determine the function of Plin5 in the NS5A-induced lipid metabolic disorder,we observed the effects of Plin5 deficiency on liver lipid content,crucial lipid metabolites and TG hydrolysis rate in the liver or cells with NS5 A expression.(3)To clarify the role of Plin5 in NS5A-induced lipid metabolic disorder,we explored the interaction between Plin5 and NS5 A and mapping the critical interacting domain.METHODS:To investigate the role of Plin5 in NS5A-induced hepatic lipid metabolic disorder and injury,we first constructed adenoviruses expressing HCV NS5 A in the livers of wild-type and Plin5-null mice through tail vein injection.The levels of lipid content,oxidative stress,lipid peroxidation and inflammatory biomarkers were further determined.NS5 A was also expressed in both primary hepatocytes and Plin5-overexpressing HepG2 cells,then the morphology of LDs,lipid content,TG hydrolysis rate and oxidative stress level were detected.Finally,the interaction between NS5 A and Plin5 and its specific binding domain of Plin5 were determined by immunofluorescence and co-immunoprecipitation(Co-IP).RESULTS:(1)One week after adenovirus injection,the H&E and immunohistochemitry(IHC)staining showed that the Plin5 deficiency aggravated the NS5A-induced hepatic injury and increased the number of F4/80-positive macrophage infiltrating in the liver.The blood examination showed that NS5 A significantly elevated the transaminases in Plin5-knockout mice,suggesting that Plin5 deficiency aggravated NS5A-induced hepatic injury.At the same time,both in vivo and in vitro experiments confirmed that Plin5 inhibited lipid peroxidation and lipotoxicity induced by NS5 A.Moreover,NS5 A significantly increased the levels of ROS,MDA and 4-HNE in both liver and primary hepatocytes of Plin5-deficient mice,but had little effects on the liver and primary hepatocytes of wild-type mice.Furthermore,Plin5 overexpression inhibited NS5A-induced ROS,MDA and 4-HNE elevation in HepG2 cells.These results confirmed that Plin5 inhibited oxidative stress and hepatic injury induced by NS5 A.(2)Furthermore,we also found that NS5 A promoted lipid accumulation in livers and primary hepatocytes of wild-type mice,but not in Plin5-deficient mice.In addition,NS5 A expression significantly increased the levels of non-esterified fatty acids(NEFAs)in the Plin5-deficient liver and hepatocytes,but not in the wild-type liver and hepatocytes.Consistent with the previous results,in Plin5-deficient hepatocytes NS5 A accelerated the hydrolysis rate of TG,but no distinguishable alteration was observed in wild-type hepatocytes.In HepG2 cells,NS5 A also increased intracellular NEFA level and accelerated TG hydrolysis rate,but Plin5 overexpression significantly inhibited TG hydrolysis and increased intracellular TG content in NS5A-expressing HepG2 cells.These results indicated that Plin5 inhibited the lipolysis and increased the intracellular lipid content in the NS5A-expressing cells.(3)To clarify the mechanism,we found that NS5 A and Plin5 co-localized on the surface of LDs using immunofluorescence.By isolating subcellular fractions and immunoblotting,we found that NS5 A recruited Plin5 from the cytoplasm onto the surface of LDs without altering the expression of Plin5 in liver.Furthermore,Co-IP assay indicated that Plin5 interacted with NS5 A,and the Co-IP with truncated Plin5 showed that the N-terminus(32-128aa)of Plin5 was the key binding domain for its interaction with NS5 A.CONCLUSION:NS5A recruited Plin5 from the cytoplasm to the surface of LDs by interacting with Plin5.On the surface of LDs,Plin5 inhibited the lipolysis,promoted lipid accumulation and alleviated NS5A-induced liver damage.This study not only helps us understand the specific role of Plin5 in LD metabolism,but also provides an experimental basis for the mechanism of disordered lipid metabolism associated with HCV infection.