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Perilipin-2 Is Critical for Efficient Lipoprotein and Hepatitis C Virus Particle Production Susan Lassen1, Cordula Grüttner1, Van Nguyen-Dinh1 and Eva Herker1,2,*

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Perilipin-2 Is Critical for Efficient Lipoprotein and Hepatitis C Virus Particle Production Susan Lassen1, Cordula Grüttner1, Van Nguyen-Dinh1 and Eva Herker1,2,* © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 RESEARCH ARTICLE Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production Susan Lassen1, Cordula Grüttner1, Van Nguyen-Dinh1 and Eva Herker1,2,* ABSTRACT as ADRP), the main LD coat protein in hepatocytes, is constitutively In hepatocytes, PLIN2 is the major protein coating lipid droplets (LDs), found on the LD surface and, if unbound, is rapidly degraded by the an organelle the hepatitis C virus (HCV) hijacks for virion proteasome (Takahashi et al., 2016). PLIN2 stabilizes and protects morphogenesis. We investigated the consequences of PLIN2 LDs from degradation by lipases or by the autophagic machinery deficiency on LDs and on HCV infection. Knockdown of PLIN2 did (Kaushik and Cuervo, 2015; Listenberger et al., 2007). PLIN2 as not affect LD homeostasis, likely due to compensation by PLIN3, but well as PLIN3 (also known as Tip47) are substrates for chaperone- severely impaired HCV particle production. PLIN2-knockdown cells mediated autophagy; degradation of both is a prerequisite for had slightly larger LDs with altered protein composition, enhanced breakdown of LDs by lipolysis or macrolipophagy (Kaushik and local lipase activity and higher β-oxidation capacity. Electron Cuervo, 2015, 2016). In line with this, overexpression of PLIN2 in micrographs showed that, after PLIN2 knockdown, LDs and HCV- cell lines causes an accumulation of neutral lipids and LDs owing to induced vesicular structures were tightly surrounded by ER-derived reduced TG turnover (Imamura et al., 2002; Listenberger et al., double-membrane sacs. Strikingly, the LD access for HCV core and 2007). In mice, reduced PLIN2 levels are associated with lower NS5A proteins was restricted in PLIN2-deficient cells, which levels of TGs and protection against diet-induced steatosis (Chang correlated with reduced formation of intracellular HCV particles that et al., 2006; Libby et al., 2016; McManaman et al., 2013; Tsai et al., were less infectious and of higher density, indicating defects in 2017a). maturation. PLIN2 depletion also reduced protein levels and One leading cause of liver diseases, such as liver cirrhosis and secretion of ApoE due to lysosomal degradation, but did not affect hepatocellular carcinoma, is hepatitis C virus (HCV) infection. the density of ApoE-containing lipoproteins. However, ApoE Approximately 71 million people are viraemic and 0.4 million overexpression in PLIN2-deficient cells did not restore HCV people die each year from HCV-related complications. Direct-acting ∼ spreading. Thus, PLIN2 expression is required for trafficking of core antivirals (DAAs) induce viral clearance in 95% of the patients, and NS5A proteins to LDs, and for formation of functional low-density a major improvement over interferon-based therapies, but the HCV particles prior to ApoE incorporation. treatment is extremely costly and the accessibility in high- prevalence countries is limited. Hepatic steatosis is frequently This article has an associated First Person interview with the first observed in patients suffering from chronic HCV infection, and author of the paper. virus replication is intertwined with lipid metabolism of the liver (Negro, 2014; Paul et al., 2014). The structural capsid protein core KEY WORDS: Hepatitis C virus, HCV, Lipid droplet, LD, Perilipin 2, and the non-structural protein 5A (NS5A) of HCV localize to PLIN2, ADRP, Apolipoprotein E, ApoE cytosolic LDs when expressed as single proteins in uninfected cells. In infected cells, they initiate HCV assembly at the close-by INTRODUCTION membranes of the ER (Barba et al., 1997; Miyanari et al., 2007; Shi Long considered as inert lipid storage organelles, lipid droplets et al., 2002). Trafficking of core and NS5A proteins to LDs is (LDs) have gained interest due to their critical involvement not only mediated by diacylglycerol acyltransferase 1 (DGAT1), which in lipid metabolism and metabolic disorders, but also in intracellular catalyzes the final step in TG biosynthesis; core protein additionally trafficking pathways, inflammatory responses and host–pathogen requires the cytosolic phospholipase A2 (cPLA2) enzyme (Camus interaction. LDs have an organic core of triglycerides (TGs) and et al., 2013; Herker et al., 2010; Menzel et al., 2012). Inhibition of sterol esters and are surrounded by an amphipathic phospholipid either enzyme leads to strong reduction in HCV particle production. monolayer with proteins embedded on the surface (Fujimoto and Upon infection, all components of the viral replication machinery Parton, 2011). The most abundant proteins associated to LDs are found in close proximity to LDs (Miyanari et al., 2007). PLIN3 belong to the perilipin (PLIN) protein family (PLIN1–PLIN5) directly interacts with HCV NS5A and has been shown to be vital (Kimmel et al., 2010). PLIN proteins are important regulators of for HCV RNA replication (Ploen et al., 2013a; Vogt et al., 2013). cellular lipid metabolism directly controlling how and when cells Infectious viral particles are lipoviroparticles containing neutral (and tissues) store, mobilize and utilize lipids. PLIN2 (also known lipids and apolipoproteins (Andre et al., 2002; Merz et al., 2011). Mobilization of lipids from LDs for lipoviroparticle formation is mediated by the lipase co-activator comparative gene identification 1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 58 (CGI-58, also known as ABHD5) (Vieyres et al., 2016) and Hamburg, Germany. 2Institute of Virology, Philipps University Marburg, 35043 Marburg, Germany. intact lipoprotein synthesis and secretion are essential for production of infectious particles (Gastaminza et al., 2008; Huang *Author for correspondence ([email protected]) et al., 2007; Jiang and Luo, 2009; Lee et al., 2014). In addition, E.H., 0000-0001-9644-2484 factors recruited to LDs upon HCV infection participate in virion morphogenesis, such as the phospholipid-binding protein annexin Received 20 February 2018; Accepted 7 December 2018 A3, which mediates re-routing of ApoE (Rösch et al., 2016). Similar Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 to what is seen upon PLIN2 overexpression, HCV core protein HCV is PLIN2 (Brasaemle et al., 1997; Fujimoto et al., 2004). causes lipid accumulation by inhibiting adipose triglyceride lipase Previous reports on the interaction of PLIN2 with HCV are (ATGL; also known as PNPLA2)-mediated lipid mobilization contradictory (Branche et al., 2016; Zhang et al., 2016). To (Camus et al., 2014; Harris et al., 2011). But core can also displace investigate whether PLIN2 itself or lipid metabolic processes PLIN2 from the LD surface (Boulant et al., 2008). Regarding HCV regulated by PLIN2 are required for HCV infection, we generated replication, overexpression of PLIN2 leads to an increase in HCV lentiviral constructs encoding six different shRNAs targeting replication while its knockdown using siRNAs has inconsistent PLIN2 and a non-targeting control (shNT) to transduce the effects (Branche et al., 2016; Zhang et al., 2016). hepatoma cell line Huh7.5 (Fig. 1A). The shRNAs induced Here, we revisited and investigated the consequences of PLIN2 variable PLIN2 knockdown levels and shRNA #3 and #4 reduced knockdown on lipid metabolism and on HCV replication. and increased cell growth of the transduced cells, respectively (Fig. 1B,C). Next, cells transduced with the four shRNAs that did RESULTS not affect cell growth were inoculated with a low multiplicity of PLIN2 is required for efficient HCV infection infection (MOI) of an HCV Jc1 reporter strain carrying an EGFP LDs are essential cellular organelles for HCV infection (Miyanari fluorescent reporter inserted between NS5A and NS5B with a et al., 2007; Paul et al., 2014). The most prominent protein on the duplicated protein cleavage site (Jc1NS5AB-EGFP) (Webster et al., surface of LDs in hepatocytes and hepatoma cells permissive for 2013), and analyzed by flow cytometry at up to 6 days post infection Fig. 1. PLIN2 is required for efficient HCV progeny production. (A) Scheme of the experiments. Huh7.5 cells were transduced with shPLIN2 or control shNT lentiviruses prior to infection with HCV Jc1NS5AB-EGFP or electroporation of HCV RNA. (B) Western blot analysis of the different shRNA constructs. (C) Viability assay for shRNA-transduced cells (mean±s.e.m., n=3). (D) HCV Jc1NS5AB-EGFP spreading kinetics in shRNA-transduced cells after infection with an MOI of ∼0.002 (mean±s.e.m., n=4). *P<0.05; **P<0.01 (Student’s t-test). shPLIN2 #1 was selected for follow-up experiments. (E) Western blot analysis of Huh7.5 cells at different days post transduction (dpt). (F) HCV RNA replication was determined after electroporation with envelope-deleted luciferase reporter RNA (Jc1ΔE1E2NS5AB-Luc) by measuring luciferase activity several days post electroporation (dpe). Shown is the luciferase activity (relative light units, RLU) per μg protein standardized to value at the 4 h time point (mean±s.e.m., n=3). *P<0.05 (Welch’s t-test). (G) Cells were electroporated with in vitro transcribed HCV RNA (Jc1NS5AB-EGFP) and equal transfection rates were verified by flow cytometry. At the time points indicated, we harvested the supernatant and measured HCV RNA copy numbers (GE, genome equivalents) through qRT-PCR, measured secretion of the HCV core protein by ELISA, and determined the released infectivity (TCID50) by limiting dilution titration on Huh7.5 RFP–NLS–IPS reporter cells. Shown are the absolute values and values normalized to control for each time point (mean±s.e.m., nqPCR,d2=5, nqPCR,d4,d6=8, nELISA,d2=4, nELISA,d4,d6=6, nTCID50=5). *P<0.05, **P<0.01, ***P<0.001 (Welch’s t-test and one sample Student’s t-test for percentage infectivity). Journal of Cell Science 2 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 (dpi). HCV spreading was impaired in shPLIN2-transduced cells consistently CGI-58, increased in LD fractions after knockdown of and correlated with the PLIN2 knockdown levels (Fig.
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