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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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

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(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. 1D). PLIN2 in both uninfected and HCV-infected cells without Therefore, PLIN2 expression is required for efficient HCV concomitant changes in the input fractions (Fig. 2B). Therefore, infection. As shPLIN2 #2 slightly impacted cell viability, we knockdown of PLIN2 is sufficient to cause relocalization of the chose shPLIN2 #1 (from now on referred to as shPLIN2) for lipase complex even though PLIN3 levels increase in LD fractions. mechanistic studies. shPLIN2 induced a stable knockdown for at least 15 days (Fig. 1E). To avoid adaptation, we performed most of Lack of PLIN2 increases local lipolysis rates of isolated LDs the experiments at 5 days after transduction with the lentiviral and increases mitochondrial β-oxidation capacity constructs. To investigate whether increased levels of LD-localized ATGL increases local lipolysis, we performed in vitro self-digestion Production of infectious HCV particles depends on PLIN2 experiments with isolated LDs (Fig. 2C). After isolation and expression quantification of TG and protein content, LDs were incubated in the Next, we investigated the impact of PLIN2 silencing on different presence of fatty-acid-free bovine serum albumin (BSA) to steps of HCV replication. To probe viral RNA replication, we accelerate lipolysis rates. After self-digestion, levels of released transfected shRNA-transduced cells with in vitro transcribed RNA free fatty acids (FFA) were determined. Indeed, we detected higher of an envelope-deleted Jc1 strain encoding a firefly luciferase amounts of fatty acids released from LDs isolated from PLIN2- reporter (Jc1ΔE1E2NS5AB-Luc). Knockdown of PLIN2 only slightly knockdown cells compared to LDs isolated from control cells reduced HCV RNA replication early after transfection of viral RNA (Fig. 2C). To examine whether the increased local lipolysis rate (Fig. 1F). Therefore, HCV RNA replication and translation are affects steady-state lipid content, we measured the TG content of the mostly independent of PLIN2 expression. To investigate the cells, but did not detect significant differences (Fig. 2D). To address production of infectious virions, we electroporated full-length how PLIN2-deficient cells handle excess fatty acids, cells were Jc1NS5AB-EGFP RNA, verified equal transfection rates 3 days post incubated with oleate. TG levels increased in both shNT- and electroporation (dpe), and harvested the supernatant to determine shPLIN2-expressing cells after oleate loading, as expected, but the HCV RNA copy number, release of the viral capsid protein core PLIN2-knockdown cells had slightly less TGs indicating a reduced and the infectivity in the supernatant (50% tissue culture infectious capacity to store TGs (Fig. 2D). To analyze whether the excess fatty dose, TCID50). The amount of HCV RNA, of the capsid protein, acids are degraded by β-oxidation, we incubated cells with 14 14 and infectivity were all significantly reduced in the supernatant of C-labeled palmitate and measured the production of CO2 and cells lacking PLIN2 (Fig. 1G). In rescue experiments, production of 14C-containing acid-soluble metabolites (ASMs). Intriguingly, cells infectious viral progeny was partially restored in PLIN2- depleted of PLIN2 had significantly increased maximal levels of knockdown cells upon expression of an shRNA-resistant mutant β-oxidation (Fig. 2E). To confirm that all fatty acids are channeled to but not upon expression of wild-type PLIN2 (Fig. S1). Hence, β-oxidation, cells were treated with a DGAT1 inhibitor (DGAT1i) to expression of the LD protein PLIN2 is required for efficient prevent esterification of palmitate. β-oxidation rates were essentially production of HCV progeny. the same with and without DGAT1i and elevated in PLIN2- knockdown cells. In conclusion, PLIN2-deficient cells have increased LD-associated lipase activities and preferentially PLIN3 and ATGL levels increase at LDs in degrade excess fatty acids rather than store them as TGs. PLIN2-knockdown cells PLIN2 is the most abundant protein coating LDs in the liver (Brasaemle et al., 1997; Fujimoto et al., 2004). In contrast, the PLIN2-deficient cells have slightly larger LDs without exchangeable PLIN3 predominantly localizes to LDs under changes in total LD content conditions of LD biogenesis (Bulankina et al., 2009; Wolins Overexpression of PLIN2 in Huh7 cells alters LD morphology et al., 2001). PLIN3 protein levels increase at LDs in oleate-loaded (Branche et al., 2016; Zhang et al., 2016). We investigated the PLIN2-knockdown cells (Bell et al., 2008; Sztalryd et al., 2006) and impact of PLIN2 depletion on LDs by performing 3D spinning disk in lipid-rich fractions of HCV-replicon cells owing to direct confocal microscopy (Fig. 3A). To knockdown PLIN2 we used an interaction with the viral NS5A protein (Vogt et al., 2013). We shRNA construct expressing a puromycin-resistance gene that investigated how loss of PLIN2 affects the protein composition of efficiently suppressed PLIN2 expression (Fig. 3B). In addition, cells LDs under steady-state conditions and during HCV infection. We were transduced with the HCV RFP–NLS–IPS reporter to monitor transduced HCV-infected and uninfected cells with lentiviral HCV infection (Jones et al., 2010). This reporter indicates shRNAs, isolated LDs by density-gradient centrifugations and uninfected cells through mitochondrially localized RFP–NLS– analyzed the protein levels of LD-associated proteins (Fig. 2A). To IPS. After infection, the viral protease NS3–4A cleaves the IPS verify equal protein levels for LD fractions, which are devoid of target site and RFP–NLS translocates to the nucleus. We recorded z- reliable markers, we performed silver staining prior to western blot stacks and determined individual LD volumes as well as the number of analysis. The PLIN2 shRNA reduced PLIN2 protein levels in input LDs per cell (Fig. 3C). PLIN2 knockdown caused a significant increase and in LD fractions (Fig. 2B). In the input, we did not detect changes in average LD volumes (mean, shNT=0.132 µm3 versus in PLIN3 protein levels. In contrast, LDs isolated from PLIN2- shPLIN2=0.156; median, shNT=0.088 versus shPLIN2=0.103). In knockdown cells had more PLIN3 compared to the control in both non-infected cells, this was accompanied by a slight decrease of the HCV-infected and uninfected cells. This indicates that PLIN3 is numbers of LDs per cell, resulting in no change in total LD volume per recruited to LDs lacking PLIN2 even in the absence of oleate cell. PLIN2 deficiency led to a similar increase in LD volumes in HCV- loading. Both PLIN2 and PLIN3 can counteract the breakdown of positive as in HCV-negative cells (mean, shNT=0.139 µm3 versus TGs (Kaushik and Cuervo, 2015), and simultaneous knockdown of shPLIN2=0.152; median, shNT=0.090 versus shPLIN2=0.100) both proteins leads to increased recruitment of the TG lipase ATGL without significant changes in the number or total LD volume per and its co-activator CGI-58 (Bell et al., 2008). ATGL, and less cell. Therefore, even though PLIN2 deficiency enhances local fatty acid Journal of Cell Science

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Fig. 2. LDs lacking PLIN2 harbor more PLIN3 and ATGL, and display increased local lipolysis and total β-oxidation rates. (A) Scheme of the experimental procedure. (B) LDs were isolated by sucrose-density gradient centrifugation from HCV-infected and uninfected Huh7.5 cells transduced with lentiviral shRNAs. Input and LD fractions were analyzed by western blotting. We used silver staining to confirm equal protein amounts loaded on the gels for LD fractions. The # mark indicates the PLIN3 band from prior antibody incubation and asterisks indicate unspecific bands. Band intensities were quantified in Fiji and are presented relative to control (mean±s.e.m.,

nuninfected=4–6, nHCV-Infected=3). (C) After LD isolation, equal amounts of TGs were self-digested in vitro before the amount of released FFAs was determined as shown in the diagram (left). Right, LD-associated lipase activity as raw data (FFA/µg protein×h) and normalized to that in control cells (mean±s.e.m., n=6, *P<0.05, one sample Student’s t-test). (D) Cellular TG content was

determined in the presence or absence of oleate (mean±s.e.m., n−oleate=5, 14 n+oleate=6). (E) Cells were incubated with C-labeled palmitate to determine 14 mitochondrial β-oxidation by quantifying C-containing CO2 and acid-soluble metabolites (ASMs). 20 µM DGAT1 inhibitor (DGAT1i) (Herker et al., 2010) treatment prevented esterification of FFAs into TGs. Shown are the counts per minute (cpm) normalized to μg protein (mean±s.e.m., n=4). *P<0.05, **P<0.01 (Welch’s t-test).

cells overexpressing Rab18 (Ozeki et al., 2005). Mechanistically, this was linked to PLIN2 levels at LDs as the close apposition of LDs with ER cisterna was recapitulated by depleting PLIN2 in 3T3 cells. Here, we used Huh7.5 cells expressing the EGFP–NLS–IPS HCV reporter that were transduced with shRNAs targeting PLIN2 prior infection with HCV Jc1. HCV-infected and uninfected shRNA-transduced (mCherry-positive) target cells were identified by epifluorescence and the samples were fixed and processed for EM with a modified osmium-thiocarbohydrazide-osmium (OTO) method (Seligman et al., 1966) for enhancing the contrast of membranes and LDs (Fig. 4A, Figs S2–S5). In shNT-transduced cells, HCV infection led to the typical membranous web formation with single-, double- and multi-membrane vesicles in close proximity to LDs (cyan and yellow arrowheads). LDs were either isolated or close to ER cisterna (black arrowheads). Interestingly, in HCV-infected PLIN2-deficient cells, LDs as well as the vesicular structures induced by HCV were often found in tight association with double-membrane structures that formed almost closed sacs connected to ER membranes (red arrowheads). These LD- associated membrane sacs were almost never observed in cells expressing PLIN2. The close association of LDs with double- membrane sacs also occurred in uninfected PLIN2-knockdown cells, but without the vesicular structures induced by infection. The double-membrane sacs contained one or more LDs with tight contact sites between the organelles. For some areas, the inner membrane of these sacs was fused with the phospholipid monolayer of LDs inside the sacs (white arrowheads). Quantification of the LD phenotype revealed that more than 50% of LDs were tightly associated with double-membrane sacs in both HCV-infected cells and uninfected controls (Fig. 4B,C). To analyze the 3D structure of the double-membrane sacs, we performed electron tomography (ET). HCV-infected cells expressing PLIN2 shRNAs were identified by epifluorescence and fixed and processed for ET. As illustrated in the colored 3D reconstruction, LDs (yellow) and the vesicular HCV flux, LDs are only slightly affected, indicating compensation through replication structures (cyan) were tightly enclosed by double enhanced recruitment of PLIN3. membranes (red) that were in continuity with ER cisterna (Fig. 5A; Movies 1–3). Membrane sacs devoid of the vesicular structures LDs and HCV-induced vesicular replication organelles are displayed an even closer apposition to LDs in uninfected cells. surrounded by double membranes in cells lacking PLIN2 To further corroborate the origin of the membrane sacs, we Next, we examined PLIN2-knockdown cells by electron microscopy performed correlative light and electron microscopy (CLEM) of (EM) to analyze LDs and their surrounding membrane structures. cells stained with fluorescent ER-Tracker or LysoTracker, or

Tight interactions of LDs with ER membranes have been reported in expressing GFP–LC3B (microtubule associated protein 1 light Journal of Cell Science

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Fig. 3. PLIN2 knockdown harbor slightly larger LDs. (A) Experimental procedure. Cells transduced with RFP– NLS–IPS to enable identification of HCV- infected cells by translocation of RFP from mitochondria to the nucleus were transduced with lentiviral shRNA-Puro constructs and infected with HCV (Jc1wt). (B) The shPLIN2-Puro construct carrying a puromycin-resistance gene was validated by western blotting. (C) After fixation, cells were stained with BODIPY to visualize LDs and z-stacks were recorded on a spinning disk confocal microscope. Shown are representative images and 3D reconstruction of LDs of HCV-infected (marked by a +) and uninfected (marked by a −) shPLIN2- and shNT-transduced cells. Scale bar: 10 µm. Shown are LD volumes, the number of LDs, and the total LD volume per cell determined in 28–34 cells from three independent experiments (LD volumes: median±95% c.i., ***P<0.001, Mann–Whitney U-test; number of LDs and total LD volume per cell: mean±s.e.m., *P<0.05, Welch’s t-test).

chain 3B) as a marker for autophagosomes. Samples were analyzed particle production (Boulant et al., 2007; Herker et al., 2010; by live-cell spinning disk confocal microscopy and directly fixed Liefhebber et al., 2014; Menzel et al., 2012; Miyanari et al., 2007). and processed for EM. ER signals colocalized with and were in Vice versa, cells infected with mutant HCV viruses defective in close apposition to LDs in PLIN2-knockdown and control cells virion morphogenesis often accumulate core at LDs (Gentzsch et al., (Fig. 5B). We then focused on LDs surrounded by double- 2013; Zayas et al., 2016). To investigate whether LD localization of membrane sacs identified in the electron micrographs. Z-stacks of viral proteins is affected by PLIN2 deficiency, we first analyzed fluorescence light microscopy images of these LDs revealed cup- levels of core protein in LD fractions from HCV-infected cells. shaped ER structures at LD edges (red arrowheads) and close-by ER While we did not observe any changes in the cell homogenate, we cisterna (black arrowheads). LDs were in close contact with ER detected a striking ∼75% decrease of levels of core in LD fractions cisterna in cells expressing PLIN2 (black arrowheads). from PLIN2-knockdown cells (Fig. 6A). In HCV Jc1-infected cells In contrast, we did not observe colocalization of membrane- core localizes to LDs and is readily used for virion morphogenesis surrounded LDs with either LysoTracker or GFP–LC3 (Fig. 5B). As (Shavinskaya et al., 2007), limiting the amount of core at LDs. GFP–LC3 might not be representative of endogenous LC3, we Therefore, we ectopically expressed core and investigated the additionally investigated LD isolations. We did not detect enrichment localization in PLIN2-deficient cells. Core was readily detected in of endogenous LC3 in LD fractions of PLIN2-depleted orcontrol cells LD fractions from control cells but almost undetectable in LD (Fig. 5C). Therefore, the double-membrane sacs enclosing LDs and fractions of PLIN2-knockdown cells (Fig. 6B). Intriguingly there is a HCV replication organelles are likely derived from ER cisterna that clear correlation between core and PLIN2 levels in LD fractions, stay attached to LDs in cells lacking PLIN2. while PLIN3 and PLIN2 show an inverse correlation (Fig. S6A,B). These results reveal a severe trafficking defect of core protein to LDs HCV protein trafficking to LDs is impaired in cells depleted in the absence of PLIN2 expression. When we probed LDs from of PLIN2 HCV-infected cells for NS5A, we found a similar decrease of protein HCV core and NS5A proteins localize to LDs to initiate capsid levels in LD fractions from PLIN2-knockdown cells (Fig. 6A). assembly and virion morphogenesis (Paul et al., 2014). Hindering To further validate the trafficking defect of the viral proteins, we trafficking of core or NS5A to LDs either by mutation or by performed immunofluorescence studies of HCV-infected cells and inhibition of critical host factors results in decreased infectious probed for core and NS5A localization. We quantified LD Journal of Cell Science

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Fig. 4. LDs are enclosed by double-membrane sacs in cells lacking PLIN2. (A) Cells containing EGFP–NLS–IPS HCV reporter were transduced with shRNA constructs, infected with HCV (Jc1wt) for 3 days, analyzed by fluorescence microscopy and processed for EM. Depicted are merged fluorescence images, electron micrographs of target cells, and models of LDs and neighboring membrane structures. Boxes indicate the approximate areas of the focus areas that are shown in higher magnification. *, LDs. Colored arrowheads: black, ER membranes; red, double-membrane sac; white, LD membrane contacts; cyan, single-membrane vesicles; yellow, double-membrane vesicles; RC, HCV RNA replication complex; Nu, nucleus; Mi, mitochondria. Scale bars: 10 µm. For more electron micrographs see Figs S2–S5. (B,C) Quantification of LD phenotypes in two independent experiments covering 43 cells. Shown is the percentage of LDs surrounded by double-membrane sacs per cell (B) (mean±s.e.m., *P<0.05, **P<0.01, Welch’s t-test) as well as the fraction of and free membrane-surrounded LDs per condition (C). Journal of Cell Science

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Fig. 5. LDs and HCV-induced vesicular structures are in ER-derived double-membrane sacs in PLIN2-deficient cells. (A) Tomograms of HCV-infected and uninfected PLIN2-knockdown cells. 3D volumes are depicted to illustrate LDs (yellow), double-membrane sacs (red) and vesicular structures inducedby HCV infection (cyan). Scale bars: 200 nm. Movies 1–3 show a progression of the tomograms. (B) CLEM of PLIN2-knockdown and control cells at 5 dpt. Cells were stained with ER-Tracker and LysoTracker fluorescence image z-stacks were acquired in living cells on a spinning disk confocal microscope prior to fixation and staining for EM. GFP–LC3 expression constructs were transfected 2 days prior to analysis. Target LDs were identified in EM and the magnified corresponding fluorescent stacks are shown. *, LDs. Colored arrowheads: red, double-membrane sac; orange, lysosome; green, autophagosome. Scale bars: 10 μm. (C) Western blot analysis of LD fractions isolated from PLIN2-knockdown and control cells. Asterisk marks an unspecific band. localization by determining the Manders’ overlap coefficients, found slight differences between control and PLIN2-knockdown which indicate the amount of signal of one channel that overlaps cells. The normalized signal intensity profiles revealed that core with signal from the other channel. While core mainly localizes to more tightly surrounded LDs in control cells than in PLIN2- LDs, NS5A displays both LD localization and a punctate ER and deficient cells (Fig. 6D). To analyze whether LD-surrounding Golgi staining pattern. Intriguingly, NS5A displayed significantly membranes sacs are lost during our LD isolation procedure, we reduced colocalization with LDs in cells lacking PLIN2 (Fig. 6C). analyzed LD fractions by EM and did not observe differences In contrast, although we observed less core at LDs in cell between control and PLIN2-deficient cells, indicating that the fractionation experiments, we did not observe significantly less membrane sacs are stripped off during isolation (Fig. S6C). These colocalization between core and LDs by microscopy (Fig. 6C). results indicate that core is localizing to the double-membrane sacs

However, when we analyzed core signal intensity around LDs, we that surround LDs in cells lacking PLIN2 while NS5A is not. The Journal of Cell Science

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Fig. 6. See next page for legend. difference between core and NS5A might be due to the differences for trafficking to LDs, we probed DGAT1 localization, activity, and in how core and NS5A interact with membranes and the much interaction with the viral proteins in PLIN2-deficient and stronger LD affinity of HCV core protein. Of note, the resolution control cells (Fig. S7). Of note, core and DGAT1 interact at the limit of confocal microscopy does not allow distinguishing between ER prior to core trafficking to LDs. Accordingly, we did not detect a the localization at the LD surface or the localization to surrounding defect in core–DGAT1 or NS5A–DGAT1 interaction in co- membrane sacs. As core and NS5A both depend on DGAT1 activity immunoprecipitation experiments. DGAT1 localization is also Journal of Cell Science

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Fig. 6. PLIN2 knockdown blocks trafficking of core and NS5A to LDs and We then probed the interaction of the viral envelope protein E2 formation of infectious intracellular particles. (A) Western blot analysis of with ApoE, which occurs after core multimerization and LD fractions of HCV-infected cells transduced with lentiviral shRNAs. Band envelopment, as those steps are not impaired in ApoE-deficient intensities were quantified using Fiji (mean±s.e.m., n=4). **P<0.01 (one sample Student’s t-test). (B) LD fractions of Huh7.5 cells first transduced with cells (Lee et al., 2014). We performed co-immunoprecipitation HCV core expression constructs followed by lentiviral shRNAs were analyzed experiments using a full-length HCV Jc1 construct expressing Flag– by western blotting (mean±s.e.m., n=5). **P<0.01 (one sample Student’s E2 and probed for co-precipitation of ApoE (Fig. 6G). Flag–E2 t-test). (C) shRNA-transduced cells were infected with HCV (Jc1wt) for 3 days interacted with ApoE in the presence or absence of PLIN2, albeit and analyzed by immunofluorescence microscopy. Scale bar: 10 µm. less efficiently. In uninfected cells, ApoE mainly localizes to the ’ Manders colocalization coefficients between the viral proteins (VP) core or Golgi. We previously observed annexin A3-mediated redistribution NS5A (M1) and LDs (M2) were quantified in 50 cells using Coloc2 in Fiji (mean ±s.e.m.). **P<0.01 (Welch’s t-test). (D) Intensity profile of core (black) centered of ApoE to non-Golgi compartments in HCV-infected cells (Rösch around the peak of the LD signals (yellow) and normalized to the full width at et al., 2016). When we investigated if ApoE in PLIN2-knockdown half maximum (FWHM). (E,F) Cells transduced with shNT and shPLIN2 cells changes its localization pattern in response to HCV infection, lentiviral particles were electroporated with HCV Jc1 RNA (Jc1NS5AB-EGFP) and we noted that ApoE staining was weaker in PLIN2-knockdown analyzed for core multimerization and envelopment. Shown are representative compared to control cells. Nevertheless, we detected less blots. (E) Cell lysates were analyzed by 2D Blue native followed by SDS-PAGE colocalization between ApoE and GM130 (GOLGA2), a marker and western blotting. Shown is the distribution of core between high molecular for the Golgi compartment, in cells infected with HCV as compared mass (HMM) and low molecular mass (LMM) complexes. (F) Western blot analysis of cells lysed by freeze–thaw cycles and either left untreated, treated to uninfected cells, independently of PLIN2 expression (Fig. 6H). with proteinase K (PK), or permeabilized with Triton X-100 prior to proteolytic To investigate whether multimerized and enveloped core together treatment. (G) Co-immunoprecipitation analysis of shRNA-transduced cells with an intact E2–ApoE interaction correlates with infectiousness, electroporated with Jc1NS5AB-EGFPor Jc1Flag-E2-NS5AB-EGFP. (H) RFP–NLS–IPS we determined the intracellular infectivity of PLIN2-knockdown HCV reporter cells transduced with shRNA-Puro constructs and infected with and control cells transfected with HCV RNA encoding a Gaussia HCV were used for immunofluorescence microscopy analysis of ApoE and luciferase reporter (Jc1p7-Gluc-2A-NS2). PLIN2-knockdown cells GM130. Shown is the single RFP channel and merged images for ApoE and GM130. HCV-infected cells are marked by a + and uninfected cells by a −. produced intracellular particles that were less infectious as Scale bar: 10 µm. Manders’ colocalization coefficients between ApoE (M1) compared to controls (Fig. 6I). In addition, virions from PLIN2- and GM130 (M2) were quantified using Coloc2 in Fiji. >52 cells were analyzed depeleted cells had a lower specific infectivity (Fig. 6I). The specific in three independent experiments (mean±s.e.m.). *P<0.05, **P<0.01, infectivity of HCV lipoviroparticles depends on their density ***P<0.001 (Welch’s t-test). (I) shRNA-transduced cells were electroporated (Bartenschlager et al., 2011). Thus, we determined the density with HCV RNA encoding secreted Gaussia luciferase between p7 and distribution of intracellular and extracellular lipoviroparticles by NS2 (Jc1p7-GLuc-2A-NS2). Intracellular infectious particles were liberated by iodixanol density gradient centrifugations followed by TCID50 freeze–thaw cycles and infectivity was determined by TCID50 assays and normalized to the protein content of the lysate (mean±s.e.m., n=3). (J) Cell titration. The density peak of intracellular infectious particles shifted lysates were loaded on iodixanol gradients to determine the density profiles towards higher densities in PLIN2-deficient cells, indicating less of infectious and core-containing particles (mean±s.e.m., n=3). The arrow lipidation (Fig. 6J). In addition, the density profile of core- marks the lower amount of low-density core-containing particles isolated from containing particles isolated from PLIN2-knockdown cells PLIN2-knockdown. (K) Determination of the density profiles of infectious showed a lower amount of particles at densities below 1.10 g/ml extracellular HCV particles by TCID50 assays of the individual density fractions (Fig. 6J). Strikingly, very-low-density HCV particles (<1.05 g/ml) (mean±s.e.m., n=3). were almost completely absent from supernatants of PLIN2- deficient cells (Fig. 6K). Overall, the distribution profile independent of PLIN2 expression. We additionally probed DGAT1 resembled the density profile of HCV particles produced in cells and DGAT2 activity in a cell-based assay by using specific lacking ApoE (Lee et al., 2014); thus, we next investigated inhibitors, and found that the relative activity of DGAT1 and apolipoproteins. DGAT2 was similar in control and in PLIN2 -knockdown cells. Therefore, it is likely that the membrane sacs surrounding LDs in PLIN2-knockdown cells secrete less ApoE-containing PLIN2-deficient cells restrict access for the viral proteins. lipoproteins independently of HCV infection We next investigated whether the morphological changes around Neutral lipids stored in LDs are a major source for lipidation of LDs and the restricted LD access for core affect its multimerization lipoproteins. We speculated that the altered LD protein composition and envelopment. First, we performed 2D Blue native PAGE to and structure might affect lipoprotein metabolism, and could detect complexes of core ranging from low molecular mass (LMM) explain the reduced ApoE staining pattern and HCV particle to high molecular mass (HMM) of PLIN2-knockdown and control density and infectivity. Lipoprotein secretion starts with co- cells transfected with HCV RNA as described previously (Gentzsch translational lipidation of ApoB and formation of luminal LDs, as et al., 2013; Rösch et al., 2016). Core multimerization was intact in well as translation of ApoE into the ER lumen (Lehner et al., 2012). cells lacking PLIN2 (Fig. 6E). Using the same experimental set-up, The formation of mature very-low-density lipoproteins (VLDLs) we analyzed envelopment of core: membrane-protected proteins are likely occurs during secretion via the Golgi. Huh7-derived cell lines resistant to proteinase treatment, while cytosolic unprotected display a defect in VLDL metabolism; they produce VLDLs that are proteins are readily degraded. Core protein was partially resistant less lipidated than normal VLDLs and secrete ApoB and ApoE against proteinase K treatment in contrast to NS5A, which served as independently (Meex et al., 2011; Schöbel et al., 2018). a positive control for protease digestion. When cells were treated Interestingly, the formation of infectious HCV particles in Huh7- with Triton X-100 prior to proteolytic digestion to disrupt all derived cells depends on ApoE but not ApoB (Jiang and Luo, membranes, core protein was completely degraded. Comparing 2009). To examine the expression and secretion levels of shPLIN2-transduced with control cells did not reveal differences in apolipoproteins, we performed western blot analysis of cell core envelopment (Fig. 6F). Therefore, core protein can multimerize lysates and supernatants. Strikingly, ApoE, but not ApoB levels, and is protected by membranes even in the absence of prior LD depended on the expression of PLIN2 both in cell lysates and, were localization. even more pronounced, in culture supernatants (Fig. 7A). We Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 detected reduced protein levels of ApoE in supernatants of cells secretion. In contrast to HCV particles, secreted ApoE was of depleted of PLIN2, correlating with PLIN2-knockdown levels similar density in cell lysates and in culture supernatants of PLIN2- induced by different shRNA constructs (Fig. S6D). HCV-infected knockdown and control cells as determined by density gradient cells displayed similar changes in expression and secretion of ApoE experiments (Fig. 7F). To investigate whether the reduced upon PLIN2 depletion (Fig. 7B). Of note, ApoB was barely availability of ApoE explains the HCV phenotype, we performed detectable in supernatants of HCV-infected cells, a phenotype that rescue experiments by overexpressing ApoE. Through lentiviral has been described before (Domitrovich et al., 2005; Mancone et al., delivery, we achieved strong expression and secretion of ApoE in 2012; Tsai et al., 2017b). The reduction of ApoE levels was not caused both PLIN2-deficient and control cells (Fig. 7G). We then by changes in mRNA expression (Fig. 7C). Therefore, we analyzed inoculated these cells with HCV Jc1NS5AB-EGFP and followed viral protein degradation with specific inhibitors; leupeptin for lysosomal spreading. PLIN2-knockdown reduced HCV spreading in both pathways and MG132 for proteasomal pathways. Interestingly, ApoE-overexpressing and control cells to a similar extent (Fig. 7H). inhibition of lysosomal but not proteasomal protein degradation Therefore, PLIN2 expression is required for secretion of ApoE- restored ApoE levels in cells with reduced PLIN2 expression, both containing lipoproteins and for morphogenesis of infectious HCV in control and in HCV-infected cells (Fig. 7D,E). However, while lipoviroparticles. However, HCV morphogenesis cannot be rescued treatment with leupeptin and with an inhibitor of autophagy, by ApoE overexpression, indicating that the lack of core and NS5A 3-methyladenine (3-MA), restored intracellular ApoE levels, we trafficking to LDs and insufficient lipidation are the main reasons could not rescue the amount of secreted ApoE (Fig. S6E), indicating for the impaired production of infectious HCV progeny in PLIN2- that ApoE that is destined for degradation cannot be re-routed for deficient cells.

Fig. 7. PLIN2-knockdown reduces secretion of ApoE-containing lipoproteins. (A) ApoE and ApoB levels in cells and supernatants of shRNA-transduced cells were analyzed by western blotting. Band intensities were quantified and normalized to controls (mean±s.e.m., nCell=4, nSup=3). *P<0.05, ***P<0.001 (one sample Student’s t-test). (B) Representative western blots of HCV-infected cells and supernatants. (C) APOE and APOB mRNA expression levels (mean±s.e.m., n=5). (D) Western blotting of intracellular ApoE levels after inhibition of lysosomal (100 µg/ml leupeptin for 6 h) or proteasomal (2 µg/ml MG132 for 16 h) protein degradation. Band intensities were quantified and normalized to controls (mean±s.e.m., nLeupeptin=5, nMG132=3). *P<0.05 (one sample Student’s t-test). (E) Representative western blots of ApoE degradation in HCV-infected cells. (F) Cell lysates and supernatants were loaded on iodixanol gradients to determine the density profiles of intracellular and secreted ApoE (mean±s.e.m., n=3). (G) Western blot analysis of ApoE overexpression in shRNA-transduced cells.

(H) HCV Jc1NS5AB-EGFP spreading kinetics in shRNA-transduced cells overexpressing ApoE (mean±s.e.m., n=4, *P<0.05, **P<0.01, Student’s t-test). Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042

DISCUSSION more tightly surrounded LDs in control cells than in PLIN2- During the replication cycle of HCV, the viral proteins core and deficient cells. These results indicate that core localizes to the NS5A traffic onto the LD surface, a process essential for successful double-membrane sacs that surround LDs in cells lacking PLIN2 viral progeny production (Boulant et al., 2007; Miyanari et al., while NS5A does not. This mislocalization of core and NS5A 2007). Active TG synthesis is vital for this translocation as correlated with reduced levels of intracellular infectious viral inhibition of TG biosynthesis results in an HCV assembly block particles. Interestingly, lack of LD localization did not interfere with (Camus et al., 2013; Herker et al., 2010; Liefhebber et al., 2014). multimerization and envelopment of core, processes that have been Proteins present at the LD surface have been previously described as shown to occur in the absence of virion production (Ai et al., 2009). HCV host factors with, for example, PLIN3 and Rab18 required for In addition, the envelope glycoprotein E2 still interacted with ApoE. both successful HCV RNA replication and assembly of virions We previously failed to detect an interaction between E2 and ApoE (Ploen et al., 2013a,b; Salloum et al., 2013; Vogt et al., 2013). Here, in cells only expressing the structural proteins (Rösch et al., 2016) we investigated the role of PLIN2, the most abundant protein that even though other groups have reported it (Lee et al., 2014) coats LDs in hepatocytes. We describe that PLIN2 expression is indicating that E2 and ApoE can indeed interact without virion required for the production of infectious HCV particles (see model assembly. Taken together, multimerization and membrane in Fig. 8). protection of core, as well as the interaction of E2 with ApoE and Previous publications investigated PLIN2 in HCV infection and its mislocalization to non-Golgi compartments all occur in the had inconsistent findings: knockdown of PLIN2 using siRNAs had absence of core trafficking to LDs in PLIN2-deficient cells, but do either no or variable effects on HCV infection and on LD not result in the formation of intracellular and secreted infectious morphology (Branche et al., 2016; Zhang et al., 2016). In virions. In contrast, core localization at LDs clearly correlates with contrast, when overexpressed, PLIN2 enhanced virus infection, by intracellular infectivity. upregulating the expression of the HCV entry factor occludin HCV particles mature during assembly or secretion by (OCLN), and increased average LD volumes. The reduction of incorporating lipids and apolipoproteins, most importantly ApoE PLIN2 mRNA and protein levels by siRNAs was only moderate. (Chang et al., 2007; Gastaminza et al., 2008). PLIN2-knockdown Our results using the successful and strong shRNA-mediated cells produced intracellular particles that were less infectious with knockdown clearly point to a pro-viral role for PLIN2 in HCV decreased specific infectivity and lower amounts of very-low- replication. HCV spreading correlated very well with knockdown density lipoviroparticles. This fits with experiments in ApoE- levels induced by different shRNAs targeting PLIN2. In line with deficient cells, which supported HCV capsid formation and the function of LDs in particle production, we found that PLIN2 is envelopment but where maturation into lipoviroparticles did not mainly required for morphogenesis of progeny virions. However, occur (Lee et al., 2014), indicating defective lipidation and/or the sequence and timing of morphogenesis is still unknown. incorporation of ApoE in PLIN2-knockdown cells. Indeed, we PLIN2 deficiency impaired trafficking of the viral proteins core detected a decrease of intracellular and extracellular ApoE levels and NS5A to LDs. Core expressed either in HCV-infected cells or as after PLIN2 knockdown both in uninfected and HCV-infected cells. a single viral protein was nearly undetectable in LD fractions despite Inhibiting lysosomal but not proteasomal protein degradation similar protein levels in cell homogenates. In line with this, less restored intracellular but not secreted ApoE levels. In line with NS5A was present in LD fractions of PLIN2-knockdown cells. This this, lysosomal inhibitors increased ApoE levels in HepG2 cells and was the case even though all other probed LD-associated host macrophages (Deng et al., 1995; Ye et al., 1993). A recent report proteins displayed increased LD localization in PLIN2-knockdown even suggested that HCV replication itself causes enhanced cells. But although NS5A displayed significantly reduced autophagic degradation of ApoE (Kim and Ou, 2018). Of note, colocalization with LDs in cells lacking PLIN2, we did not the strongest effect on ApoE degradation was seen with subgenomic observe a lower degree of colocalization of core with LDs. replicons that do not produce virions. It is also well known that Nevertheless, the analysis of signal intensities revealed that core Huh7-derived cell lines secrete ApoE and ApoB independently (Meex et al., 2011; Schöbel et al., 2018; Takacs et al., 2017). Consistent with earlier reports, we detected less ApoB after infection with HCV (Domitrovich et al., 2005; Mancone et al., 2012; Tsai et al., 2017b), but in our hands ApoE remained stable during infection. However, mechanistically the effects of PLIN2 depletion on ApoE-containing lipoproteins and on HCV morphogenesis clearly differ: while HCV lipoviroparticles are of higher density in PLIN2-deficient cells there is no change in the density of ApoE-containing lipoproteins. And while ApoE levels are higher after inhibition of lysosomal degradation, total HCV core levels are unaffected by PLIN2 knockdown and inhibitor treatment. In addition, the defect in HCV morphogenesis cannot be rescued by ApoE overexpression, indicating that PLIN2 expression is required to allow core and NS5A trafficking to LDs and for the formation of functional low-density HCV particles prior to ApoE incorporation. Fluorescence microscopy analysis of LDs in PLIN2-deficient Fig. 8. Model of consequences of PLIN2 deficiency that impact HCV and cells revealed a slight increase in average LD volumes. These results lipoprotein maturation and egress. HCV core and NS5A proteins localize to LDs to initiate HCV assembly. In PLIN2 knockdown cells, ultrastructural corroborate findings in oleate-loaded cells showing that only alterations of LDs correlate with impaired trafficking of the viral proteins and simultaneous PLIN2 and PLIN3 knockdown severely changes LD infectious HCV and lipoprotein production. See text for details. RC, replication morphology (Bell et al., 2008; Sztalryd et al., 2006). In EM and ET complex; Mi, mitochondria; LVP, lipoviroparticle. analyses of PLIN2-depleted cells, we noticed LDs that are Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 surrounded by double-membrane sacs that are likely ER-derived, as RFP-NLS-IPS (Jones et al., 2010) were as described previously. For EGFP- indicated by CLEM. Interestingly, overexpression of Rab18 causes NLS-IPS, the EGFP was cloned into RFP-NLS-IPS via XbaI and BsrGI (the dissociation of PLIN2 from LDs concomitant with tight interactions XbaI site was destroyed). PLIN2 and NT shRNAs were cloned into pSicoR- MS1 (mCherry) and pSicoR-Puro (target sequences: shNT 5′-GCGCGA- of LDs with ER membranes (Ozeki et al., 2005). These membranes ′ ′ were termed LD-associated membranes (LAMs) and occur after TAGCGCTAATAATT-3 , shPLIN2#1 5 -GCTAGAGCCGCAAATTGC- A-3′, shPLIN2#2 5′-GGTTCAGAAGCCAAGTTATTA-3′, shPLIN2#3 5′- depletion of PLIN2 from LDs in 3T3 cells. The membrane sacs we CAGAAGCTAGAGCCGCAAATT-3′, shPLIN2#4 5′-TGGTTCAGAA- observed are likely LAMs and, in addition to LDs, enclose vesicular GCCAAGTTATT-3′, shPLIN2#5 5′-CAGCCATCAACTCAGATTGTT- structures induced by HCV infection. 3′, shPLIN2#6 5′-TGAAGGATTTGATCTGGTT-3′). PLIN2WT and PLI- The near-complete PLIN2-deficiency induced by the shRNAs N2MT were cloned into pSicoR-MS1 replacing mCherry by overlap exten- had no major effects on overall TG and LD content, similar to what sion PCR using pCMV6-XL4 PLIN2 (Origene) as a template (primers: has been described before (Bell et al., 2008). In western blot PLIN2fw 5′-CTGTGACCGGCGCCTACGATGGCATCCGTTGCAGTT- ′ ′ analysis of isolated LDs, we detected recruitment of PLIN3 to LDs 3 ,PLIN2MTrev 5 -AGGTATTGGCAACTGCAATCTGTGGTTCCAGC- ′ ′ ′ ′ in PLIN2-knockdown cells. PLIN3 likely compensates for the lack 3 ,PLIN2MTfw 5 -GCAGTTGCCAATACCTATGCCT-3 ,PLIN2rev 5 -T- ′ of PLIN2 to shield LDs from degradation. Less PLIN2 at LDs also AGGTCCCTCGACGAATTTTAATGAGTTTTATGC-3 ). ApoE was clon- caused an increased LD localization of the lipase ATGL and ed into pSicoR-MS1 replacing mCherry with ApoE3 using pCMV4-ApoE3 (Addgene plasmid #87086; Hudry et al., 2013) as a template (primers: Ap- elevated LD-associated lipase activity. Previous publications have oE 5′-CGGCGCCTACGCTAGCATGAAGGTTCTGTGGGCT-3′,Apo- investigated the role of PLIN2 and PLIN3 proteins in the regulation fw Erev 5′-GTCCCTCGACGAATTCTCAGTGATTGTCGCTGGGCAC-3′). of ectopic fat deposition after loading of hepatoma cells with fatty acids (Bell et al., 2008). PLIN3 localizes to LDs under conditions of Cell lines, culture conditions, and viability assays LD biogenesis, and increased levels of PLIN3 at LDs were observed HEK293T cells were obtained from the American Type Culture Collection in PLIN2-deficient cells after oleate loading. Our results indicate and Huh7.5 cells from Charles M. Rice (Rockefeller University, NY) were that, under steady-state conditions, the regular turnover of LDs is grown under standard cell culture conditions in high-glucose DMEM sufficient to recruit PLIN3 to LDs. In contrast to our study, Bell et al. supplemented with 10% FBS (Biochrom Superior), 1% GlutaMax (Gibco), (2008) detected recruitment of the lipase complex and increased and 1% penicillin-streptomycin (Sigma). All cell lines were authenticated by – lipase activity, as measured by determining the amount of FFAs in STR fingerprinting and were tested for mycoplasma every 3 6 months. Cell the medium after oleate loading, only when both PLIN2 and PLIN3 viability was analyzed with CellTiter 96 AQueous One Solution Reagent (Promega). were depleted simultaneously. In the case of single PLIN2 deficiency, liberated fatty acids are likely directly shuttled towards β Antibodies and reagents -oxidation. When challenged with excess amounts of fatty acids, All antibodies and reagents were obtained commercially and used as the capacity to esterify fatty acids to TGs was reduced in PLIN2- indicated: PLIN2 (ab52355), ApoE (ab52607), ApoB (ab31992), ATGL deficient cells, but degradation via β-oxidation was elevated. These (ab2138S), CGI-58/ABDH5 (ab73551), PLIN3 (ab47639) [all Abcam, – – findings corroborate recent data for oleate-loaded cultured PLIN2 / 1:1000 for western blotting (WB), 1:100 for immunofluorescence (IF)], myotubes revealing reduced TG levels are mostly attributable to PLIN2 (610102 Progen, 1:250 WB), tubulin (T6074, Sigma, 1:2000 WB), increased ATGL activity and fatty acid oxidation rates (Feng et al., HCV core (clone C7-50, sc-57800), GM130 (sc-16268), DGAT1 (H-255, 2017). sc-32661) (all Santa Cruz Biotechnology, 1:250 WB, 1:25 IF), HCV NS5A How does PLIN2 deficiency interfere with ApoE metabolism? (HCM-131-5, IBT, 1:500 WB, 1:100 IF), Flag (F7425, Sigma, 1:1000 WB), Biochemical studies in PLIN2−/− mice indicated elevated luminal LC3B (D11, Cell Signaling, 1:1000 WB), Flag agarose (A2220 Sigma), TG levels (Chang et al., 2006). In addition, inhibition of ApoB Alexa 488-, Alexa 594-, and Alexa 647-conjugated secondary antibodies [all donkey, IgG (H+L), Life Technologies 1:1000–1:1500 IF], HRP-labeled degradation triggers tight association of ApoB-containing ER secondary antibodies (Jackson Laboratories, 1:10,000 WB), BODIPY493/ membranes that form crescents around LDs (Ohsaki et al., 2008). 503 (D-3922), BODIPY 655/676 (B-3932) ER-Tracker Green (E34251), Remarkably, PLIN2 localized to ApoB-opposing sites of LDs and LysoTracker Green (L7526) (all Thermo Fisher), DGAT1 inhibitor (PF- overexpression of PLIN2 abolished, whereas its downregulation 04620110) and DGAT2 inhibitor (PF-06424439) (Sigma). Chemicals were augmented, the accumulation of ApoB and membranes around LDs. purchased from AppliChem, Sigma, and Merck, if not noted otherwise. A defect in ER membrane and LD interaction concomitant with membrane alterations around LDs could cause the ApoE phenotype Production of lentiviral particles in PLIN2-deficient cells, where ApoE is degraded. Future studies Lentiviral particles were produced in HEK293T cells as described will need to address the molecular mechanisms underlying ApoE previously (Naldini et al., 1996; Rösch et al., 2016). Viral titers were destabilization in PLIN2-depleted cells. determined by transduction of Huh7.5 cells. In summary, our study demonstrates that PLIN2 expression is HCV infection assays required for proper LD architecture that is needed for trafficking of in vitro viral proteins to LDs and for formation of functional low-density HCV viral stocks were prepared by electroporation of -transcribed HCV RNA into Huh7.5 cells as described (Herker et al., 2010; Rösch et al., HCV particles prior to ApoE incorporation. 2016). The infectivity (TCID50) was assessed by serial limiting dilution on Huh7.5 cells stably expressing the HCV reporter RFP-NLS-IPS (Jones et al., 2010; Rösch et al., 2016) or using the Gaussia luciferase reporter strain (Jc1p7- MATERIALS AND METHODS Gluc-2A-NS2). HCV infection or HCV RNA transfection rates were measured Plasmids and HCV constructs with the Fortessa flow cytometer (BD Bioscience) and analyzed using FlowJo HCV Jc1 reporter constructs encoding fluorescent proteins or firefly (Treestar). Replication of luciferase reporters was determined using luciferase between a duplicated NS5A–NS5B cleavage site, and Flag- Luciferase assay systems (Promega) and a Tecan plate reader. tagged E2 or envelope-deleted versions (Jc1FLAG-E2, Jc1NS5AB-EGFP and Jc1FLAG-E2-NS5AB-EGFP, Jc1ΔE1E2 Jc1NS5AB-Fluc), as well as the secreted Gaussia luciferase reporter (Jc1p7-GLuc-2A-NS2) were as described previously Fluorescence microscopy (Eggert et al., 2014; Schöbel et al., 2018; Webster et al., 2013). The lentiviral For LD quantification, cells seeded on µ-dishes (Ibidi) were fixed with 2% constructs expressing the JFH1 core and NS5A (Rösch et al., 2016), and paraformaldehyde prior to staining with BODIPY493/503 to visualize the Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042

LDs on a custom-made spinning disk confocal microscope (Nikon) 100 mM KCl, 1 mM EDTA, 20 µg/ml leupeptin, 2 µg/ml antipain, and 1 µg/ equipped with a CFI Apo TIRF 100×1.49 NA objective, a dual-camera ml pepstatin) and centrifuged at 36,000 g for 2 h in an SW41 rotor Yokogawa W2 spinning disk confocal scan head and Andor iXON 888 (Beckmann). The LD fraction was harvested using a blunt bended cannula cameras. An Andor Borealis System ensured illumination flatness of 405, and further analyzed by SDS-PAGE, silver staining or western blotting, or 488, 561 and 647 nm lasers. Images were deconvolved with 75 iterations were used to determine LD-associated lipase activity as described and a high noise level with AutoQuant X2 (Media Cybernetics) were used previously (Camus et al., 2014; Schweiger et al., 2014). TG for 3D reconstruction and volumetric analysis with the 3D objects counter concentrations in the isolated LDs were measured using the Infinity function of Fiji (Bolte and Cordelieres,̀ 2006; Schindelin et al., 2012) and Triglyceride Kit (Thermo Fisher). 0.4–1 mM of TGs were used for self- 3D reconstruction with Imaris (Bitplane). Objects below the resolution of digestion with 1% BSA (fatty acid free, Sigma) at 37°C with continuous the microscope and the acquisition settings (0.028 µm3) were excluded. shacking for 1 h. To solubilize the released FFAs, Triton X-100 was added Living cells were incubated with ER-Tracker or LysoTracker and BODIPY to final concentration of 1%, incubated for 10 min at room temperature and, 655/676 for CLEM. For colocalization studies, cells seeded on coverslips following centrifugation (20,000 g for 30 min), the FFA concentration of the were fixed, permeabilized with 0.1% Triton X-100, incubated in blocking underlying solution was determined with a NEFA kit (WAKO chemicals). solution (5% BSA, 1% fish skin gelatin, 50 mM Tris in PBS) and stained The amount of released FFA reflects the LD-associated lipase activity. with the antibodies in blocking solution. The target cells were visualized either with the spinning disk confocal microscope or with an Ti2-A1R-HD Western blot and co-immunoprecipitation analysis plus laser-scanning confocal microscope (Nikon) using a galvano scanner Cells were lysed in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl pH 7.6, x2 with a LU-NV series laser unit [405 nm, 488 nm, 561 nm and 647 nm; 1% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, protease inhibitor built-in acousto-optic tunable filter (AOTF)] and equipped with a CFI cocktail (Sigma)] for 1 h. To determine ApoE and ApoB secretion, cells PlanApo 60×1.40 oil objective. For colocalization analysis, we used the seeded in a six-well plate were cultured in serum-free medium (Opti-MEM) coloc2 plugin of Fiji to determine the Manders’ colocalization coefficients overnight and the supernatant was directly used for western blot analysis. (Schindelin et al., 2012). Z-stacks of 3D images were deconvolved with the Proteins were transferred onto a nitrocellulose membrane (Amersham). For NIS-Elements AR software (Nikon, blind 3D deconvolution, noisy level chemiluminescent detection, we used Lumi-Light western blotting substrate with 30 iterations and 0 spherical aberration). To determine intensity profiles (Roche), SuperSignal West Femto (Thermo Fisher), and ECL hyperfilm around LDs, intensities and distances of signals were measured using NIS- (Amersham). Band intensities were quantified using the densitometric Elements AR software (Nikon). Intensity profiles were centered around the quantification function of Fiji (Schindelin et al., 2012). peak of the LD signals and normalized to the full width at half maximum For immunoprecipitation analysis, cells were lysed in NP-40 lysis buffer (FWHM) signal intensity of LD signals in RStudio (RStudio Team, 2015). [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Sigma)] for 1 h on ice and, for DGAT1 immunoprecipitation, Transmission electron microscopy, electron tomography, passed 10 times through a G23 needle. Clarified lysates were incubated with correlative light and electron microscopy and negative FLAG M2 affinity gel (Sigma) for 2 h at 4°C to capture Flag-tagged staining of LDs proteins. Beads were then washed five times with ice-cold NP-40 lysis Target cells grown in gridded µ-dishes (Ibidi) were visualized via live-cell buffer and analyzed by western blotting. epifluorescence or spinning disk confocal microscopy (Nikon) and fixed following a modified OTO fixation method (Hofmann et al., 2018; 2D Blue native PAGE Seligman et al., 1966). Cells were fixed with EM-grade 2.5% glutaraldehyde 2D Blue native PAGE was performed as described previously (Gentzsch and 1% osmium tetroxide in PBS for 1–16 h, washed with PBS and then et al., 2013; Rösch et al., 2016). 106 shRNA-transduced Jc1NS5AB-EGFP- treated with 0.1% thiocarbohydrazide for 30 min, followed by staining with electroporated cells were lysed in 80 µl native PAGE buffer (0.75 M 1% reduced osmium tetroxide in 1.5% potassium hexacyanoferrate for aminocaproic acid, 50 mM Tris-Bis, pH 7.0) supplemented with 15 µl 10% 15 min. Samples were treated with 1% gallic acid for 30 min, dehydrated n-dodecyl-β-D-maltopyranoside for 30 min on ice. Post-nuclear with ethanol by progressive lowering of temperature, infiltrated with low supernatants were supplemented with 20 µg/ml leupeptin, 2 µg/ml viscosity epoxy resin (Epon 812), and polymerized at 60°C overnight. The antipain, and 1 µg/ml pepstatin, mixed with 10 µl 5% Coomassie Brilliant embedded targeted cells were tracked back, trimmed and sectioned at 50– Blue G and the same volume of 2× non-reducing sample buffer (62.5 mM 60 nm for transmission electron microscopy (TEM) and 350–450 nm for Tris-HCl, pH 6.8, 25% glycerol, 0.1% Bromophenol Blue). 40 µl of each ET. LDs isolated as described below were subjected to negative staining as sample was loaded on a 4–20% polyacrylamide precast gradient gel described previously (Pogan et al., 2018). TEM samples were visualized on (BioRad). After separation, the gel was incubated in 2× SDS sample buffer a FEI Tecnai G20 Twin electron microscope at 80 kV. Micrographs were (150 mM Tris/HCl, pH 6.8, 1.2% SDS, 30% glycerol, 0.002% acquired using a Veleta-2K×2K side-mounted TEM CCD camera Bromophenol Blue, 15% β-mercaptoethanol) for 1 h. Gel stripes were (Olympus). Representative TEM images were analyzed and assembled placed horizontally on a 15% polyacrylamide SDS gel and core complexes using ImageJ. Tomograms were acquired using a bottom-mounted 4K CCD were analyzed by western blotting. camera (Eagle 4K, FEI) with tilt series from −65° to +65° (1° increments). 3D datasets were aligned and reconstructed with Inspect 3D Xpress software Proteinase K digestion assay (FEI) using the sequential iterative reconstruction with 20 iterations. Envelopment of the core protein was analyzed as described previously Rendering and visualization was performed with Imaris (Bitplane). For (Gentzsch et al., 2013; Rösch et al., 2016). shRNA-transduced cells were CLEM, spinning disk confocal images were deconvolved with NIS- electroporated with Jc1NS5AB-EGFP RNA, lysed by freeze–thaw cycles, and Elements AR (Nikon, blind 3D deconvolution, noisy level with 25 iterations treated with 100 µg/ml proteinase K in the absence or presence of 1% Triton and 0 spherical aberration). Fluorescence and TEM images were aligned by X-100 and analyzed by western blotting. hand in Adobe Photoshop. Iodixanol gradient centrifugation Determination of LD-associated proteins and lipase activity To determine HCV particle and ApoE density through centrifugation LDs for western blotting and in vitro self-digestion were isolated as experiments, we used linear 6–56% iodixanol (Progen) gradients as described previously (Camus et al., 2014; Rösch et al., 2017; Schweiger described previously (Nielsen et al., 2006; Rösch et al., 2016; Schöbel et al., 2014). Briefly, cells were detached in PBS and lysed in hypotonic et al., 2018). Jc1p7-Gluc-2A-NS2 RNA-transfected cells were harvested by sucrose buffer (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, pH 7.2, and trypsinization at 6 days post transduction (dpt), resuspended in 1 ml TNE lysis 20 µg/ml leupeptin, 2 µg/ml antipain, and 1 µg/ml pepstatin) using a buffer (10 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA), and lysed Dounce homogenizer. Post-nuclear supernatants were overlaid with an through multiple freeze–thaw cycles. Cell debris was removed by isotonic potassium phosphate buffer (0.1 M potassium phosphate pH 7.4, centrifugation (300 g for 5 min). Clarified culture supernatant was Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs217042. doi:10.1242/jcs.217042 concentrated with polyethylene glycol. Cell lysate, unconcentrated (ApoE) or References Ai, L.-S., Lee, Y.-W. and Chen, S. S.-L. (2009). Characterization of hepatitis C virus concentrated supernatant (TCID50, core) was loaded on top of a gradient and centrifuged in an SW41 rotor (Beckman) at 204,095 g for 18 h. From top to core protein multimerization and membrane envelopment: revelation of a cascade of core-membrane interactions. J. Virol. 83, 9923-9939. bottom, 500 µl fractions were harvested and used to determine the infectivity, Andre, P., Komurian-Pradel, F., Deforges, S., Perret, M., Berland, J. L., core and ApoE protein levels (BioCat HCV Core Antigen and MabTech AB Sodoyer, M., Pol, S., Brechot, C., Paranhos-Baccala, G. and Lotteau, V. ApoE ELISA), and the density using a refractometer (DR 201-95, Krüss). (2002). 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Hepatitis C virus increases occludin (Macherey Nagel). cDNA was synthesized using Superscript III reverse expression via the upregulation of adipose differentiation-related protein. PLoS transcriptase (Invitrogen) with random hexamer primers (Qiagen) and ONE 11, e0146000. RNaseOut (Thermo Fisher). cDNA was subjected to quantitative PCR Brasaemle, D. L., Barber, T., Wolins, N. E., Serrero, G., Blanchette-Mackie, E. J. (qPCR) using the Maxima SYBR Green master mix (Thermo Fisher) on a and Londos, C. (1997). Adipose differentiation-related protein is an ubiquitously 7900HT Fast Real-time PCR System (Applied Biosystems). qPCR primers expressed lipid storage droplet-associated protein. J. Lipid Res. 38, 2249-2263. were selected from the Harvard primer bank (Wang et al., 2012). Bulankina, A. V., Deggerich, A., Wenzel, D., Mutenda, K., Wittmann, J. G., Rudolph, M. G., Burger, K. N. J. and Höning, S. (2009). TIP47 functions in the biogenesis of lipid droplets. J. Cell Biol. 185, 641-655. Statistical analysis Camus, G., Herker, E., Modi, A. A., Haas, J. T., Ramage, H. R., Farese, R. V., Jr. For statistical analysis, we used R (R Core Team, 2015), RStudio (RStudio and Ott, M. (2013). Diacylglycerol acyltransferase-1 localizes hepatitis C virus Team, 2015) and Prism (GraphPad). Statistical analysis was performed NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid using unpaired two-tailed Welch’s t-test, Mann–Whitney U-test, and, in case core. J. Biol. Chem. 288, 9915-9923. Camus, G., Schweiger, M., Herker, E., Harris, C., Kondratowicz, A. S., Tsou, C.- of normalized data, a one sample t-test, as indicated in the figure legends. n L., Farese, R. V., Jr., Herath, K., Previs, S. F., Roddy, T. P. et al. (2014). The Samples size ( ) represents independent experiments. hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene Acknowledgements identification 58 (CGI-58) and lipid droplets. J. Biol. Chem. 289, 35770-35780. We thank Ralf Bartenschlager (University of Heidelberg, Germany) for Jc1 Chang, B. H.-J., Li, L., Paul, A., Taniguchi, S., Nannegari, V., Heird, W. C. and constructs, Charles M. Rice (Rockefeller University, NY) for Huh7.5 cells and RFP– Chan, L. (2006). Protection against fatty liver but normal adipogenesis in mice NLS–IPS, Takaji Wakita (National Institute of Infectious Diseases, Japan) for JFH1, lacking adipose differentiation-related protein. Mol. Cell. Biol. 26, 1063-1076. Brian Webster and Warner C. Greene (Gladstone Institutes, CA) for the HCVcc Chang, K.-S., Jiang, J., Cai, Z. and Luo, G. (2007). Human apolipoprotein e is reporter constructs, Matt Spindler (Gladstone Institutes) for pSicoR-MS1, and Boris required for infectivity and production of hepatitis C virus in cell culture. J. Virol. 81, Fehse for Lego-iCer2 (University Clinic Hamburg Eppendorf, Germany). We thank 13783-13793. Rudolph Reimer and Carola Schneider from the Core facility Microscopy & Image Deng, J., Rudick, V. and Dory, L. (1995). Lysosomal degradation and sorting of Analysis for support. apolipoprotein E in macrophages. J. Lipid Res. 36, 2129-2140. Domitrovich, A. M., Felmlee, D. J. and Siddiqui, A. (2005). Hepatitis C virus nonstructural proteins inhibit apolipoprotein B100 secretion. J. Biol. Chem. 280, Competing interests 39802-39808. The authors declare no competing or financial interests. Eggert, D., Rösch, K., Reimer, R. and Herker, E. (2014). Visualization and analysis of hepatitis C virus structural proteins at lipid droplets by super-resolution Author contributions microscopy. PLoS ONE 9, e102511. Conceptualization: S.L., E.H.; Methodology: S.L., V.N.-D., E.H.; Investigation: S.L., Feng, Y. Z., Lund, J., Li, Y., Knabenes, I. K., Bakke, S. S., Kase, E. T., Lee, Y. K., C.G., V.N.-.D.; Writing - original draft: S.L., E.H.; Writing - review & editing: S.L., Kimmel, A. R., Thoresen, G. H., Rustan, A. C. et al. (2017). Loss of perilipin 2 in V.N.-D., E.H.; Visualization: S.L., V.N.-D., E.H.; Supervision: E.H.; Funding cultured myotubes enhances lipolysis and redirects the metabolic energy balance acquisition: E.H. from glucose oxidation towards fatty acid oxidation. J. Lipid Res. 58, 2147-2161. Fujimoto, T. and Parton, R. G. (2011). Not just fat: the structure and function of the Funding lipid droplet. Cold Spring Harbor Perspect. Biol. 3, a004838. This work was in part supported by funds from the Deutsche Fujimoto, Y., Itabe, H., Sakai, J., Makita, M., Noda, J., Mori, M., Higashi, Y., Kojima, S. and Takano, T. (2004). Identification of major proteins in the lipid Forschungsgemeinschaft (HE 6889/2). The Heinrich Pette Institute, Leibniz Institute droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. for Experimental Virology is supported by the Free and Hanseatic City of Hamburg Biochim. Biophys. Acta 1644, 47-59. and the Federal Ministry of Health (Bundesministerium für Gesundheit). The funders Gastaminza, P., Cheng, G., Wieland, S., Zhong, J., Liao, W. and Chisari, F. V. had no role in study design, data collection and analysis, decision to publish, or (2008). Cellular determinants of hepatitis C virus assembly, maturation, preparation of the manuscript. degradation, and secretion. J. Virol. 82, 2120-2129. Gentzsch, J., Brohm, C., Steinmann, E., Friesland, M., Menzel, N., Vieyres, G., Supplementary information Perin, P. M., Frentzen, A., Kaderali, L. and Pietschmann, T. (2013). 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