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GASTROENTEROLOGY 2011;141:1057–1066 BASIC AND TRANSLATIONAL—LIVER Mouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus Particles GANG LONG, MARIE–SOPHIE HIET, MARC P. WINDISCH, JI–YOUNG LEE, VOLKER LOHMANN, and RALF BARTENSCHLAGER See editorial on page 806. BACKGROUND & AIMS: Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. METHODS: A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. RESULTS: Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. CONCLUSIONS: Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection. Keywords: HCV Mouse Model; HCV Assembly; Liver Disease; Virology. H epatitis C virus (HCV) is a leading cause of acute and chronic liver diseases, affecting 130 –170 million people worldwide.1 Up to 80% of infected individuals are unable to clear the virus and have a high risk of developing cirrhosis and hepatocellular carcinoma.2 There is no HCV-specific vaccine available, and the only approved therapy, pegylated interferon-␣ combined with ribavirin, has numerous adverse effects and limited efficacy.3 HCV has a positive-strand RNA genome encoding a polyprotein that is cleaved co- and post-translationally into 10 products.4 Core protein and envelope glycoprotein 1 (E1) and E2 are main constituents of the virus particle, whereas p7 and nonstructural protein 2 (NS2) are auxiliary assembly factors. NS3, NS4A, NS4B, NS5A, and NS5B constitute the viral replicase that catalyzes the amplification of the viral genome. HCV assembly and release is tightly linked to the very low density lipoprotein (VLDL) biosynthesis pathway.5,6 Inhibition of microsomal triglyceride transfer protein or knockdown of apolipoprotein B (apoB) was reported to reduce production of infectious HCV.7,8 Additionally, apoE is a constituent of infectious HCV particles,9 and depletion reduces infectivity titers.10 –12 Although the establishment of a fully permissive culture system has greatly advanced molecular analyses of the viral replication cycle,13–15 there is still an unmet need for an HCV permissive immunocompetent small animal model. A major obstacle to its development is the very narrow host range of the virus that infects only humans and chimpanzees. Although entry of HCV into mouse cells can be achieved by ectopic expression of the human entry factors CD81 and occludin16 or by using an HCV mutant adapted to the use of murine CD81,17 RNA replication is extremely limited.18,19 Only when using a selection approach and subgenomic replicons, mouse hepatoma cell lines such as Hepa1-6, MMH1-1, or AML12 containing stably replicating HCV RNAs can be obtained.20,21 However, given the lack of stable replication of Abbreviations used in this paper: apoB, apolipoprotein B; apoE, apolipoprotein E; GFP, green fluorescent protein; DMEM, Dulbecco’s modified minimal essential medium; (h), human; HA, hemagglutinin; HCV, hepatitis C virus; HCVTCP, trans-complemented HCV particles; IRES, internal ribosome entry site; LDL, low-density lipoprotein; LDLR, LDL receptor; (m), mouse; NS, nonstructural protein; SCARB1, scavenger receptor class B type I; TCID, tissue culture infective dose 50; TCP, transcomplementation particle; VLDL, very low density lipoprotein. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.06.010 BASIC AND TRANSLATIONAL LIVER Department of Infectious Diseases, Molecular Virology, Medical Facility, Heidelberg University, Heidelberg, Germany 1058 LONG ET AL complete HCV genomes in mouse liver cells, it is unknown whether they support assembly and release of infectious HCV particles. Taking advantage of mouse liver cell lines containing a self-replicating HCV replicon and a trans-complementation approach, we demonstrate that mouse hepatic cells are permissive for assembly and release of infectious HCV particles. A key factor is the expression level of apoE. Importantly, human and mouse apoE are equally efficient in supporting HCV particle production, arguing against a species restriction of the late steps of the HCV replication cycle in mouse cells. Materials and Methods Cell Culture and Reagents The cell lines Huh7.5 and Hep56.1D (CLS-Cell Line Service)22 were cultured in Dulbecco’s modified minimal essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 2 mmol/L L-glutamine, nonessential amino acids, 100 U penicillin per milliliter, 100 g streptomycin per milliliter, and 10% fetal calf serum (complete DMEM). Huh7.5 and Hep56.1Dderived cell lines containing a subgenomic replicon and the trans-complementing expression cassette were kept in complete DMEM containing 100 g/mL of G418 (to select for the replicon) and 5 g/mL of blasticidin (to select for expression of core to NS2). AML12-sgJFH1 and MMH1-1-sgJFH1 cells were cultured as previously described.20 Plasmid Construction The plasmids pFK-Jc123 and pSGR-JFH1-H2476L24 have been described recently. Construction of further plasmids is described in Supplementary Materials and Methods. Electroporation and Selection of Stable Replicon Cells BASIC AND TRANSLATIONAL LIVER In vitro transcripts of individual constructs were generated as previously described.25 For electroporation of HCV RNA into cell lines Huh7.5 and Hep56.1D, trypsinized cells were washed twice with phosphate-buffered saline and used for transfection and selection as described in Supplementary Materials and Methods. Characterization of HCV Particles by Iodixanol Density Gradient Analysis and Neutralization Assays Concentrated trans-complemented HCV particles (HCVTCP) were analyzed by density gradient centrifugation as described in the Supplementary Materials and Methods. For neutralization assays, Huh7.5 cells were inoculated at a multiplicity of infection of 0.01 in the presence or absence of a given antibody. Two hours later, cells were washed twice with phosphate-buffered saline and cultured in DMEM complete for 72 hours. Cells were fixed and analyzed by immunohistochemistry as described in the Supplementary Materials and Methods. Virus Purification by Using Flag- or Hemagglutinin-Specific Affinity Gel These methods are described in the Supplementary Materials and Methods. GASTROENTEROLOGY Vol. 141, No. 3 Results Transient and Stable Replication of Genomic HCV RNAs in Mouse Cells The primary goal of our study was to determine HCV assembly competence of mouse cells for which we selected the cell line Hep56.1D that has excellent growth and transfection properties (not shown). In the initial set of experiments, we transiently transfected Hep56.1D cells with the HCV genome Jc1 and quantified virus production. Although considerable amounts of core protein were produced, no infectivity was detected (data not shown). Given the very low replication of HCV in mouse cells,20,21 which might have limited virus production, we attempted to establish mouse cell-derived clones containing selectable Jc1 genomes. Although cell clones could be obtained, no infectivity was detected because of accumulation of deletions affecting the region encoding the structural proteins (not shown). Production of Infectious HCV Particles in Mouse Liver Cells To overcome these technical limitations, we devised an alternative strategy based on trans-complementation of subgenomic HCV replicons with the viral “assembly factors” (core E1, E2, p7, and NS225). A JFH-1derived selectable subgenomic replicon containing the cell culture-adaptive mutation H2476L in NS5B24 was transfected into Hep56.1D cells (Figure 1A). Upon selection with G418, cell clones could be obtained containing HCV RNA of the correct size (Figure 1B). The cell clone with highest HCV RNA amounts (clone 3; designated Hep56sgJFH-cl3) was transduced with a lentiviral expression construct encoding for the assembly factors (Figure 1A). Western blot analysis of this cell clone designated Hep56sgJFHcl3-CNS2 confirmed expression of the assembly factors and the presence of the JFH1 replicon (Figure 1C). Culture supernatants did not contain a statistically significant higher amount of HCV RNA released from cells expressing the assembly proteins as compared with the replicon cells (Figure 1D). However, when using the more discriminating tissue culture infective dose 50 (TCID50) assay to monitor infectivity titers, a detectable amount of infectious particles was found in supernatants of Hep56sgJFHcl3-CNS2 cells but not with replicon control cells (Figure 1E). This result suggested production of infectious HCV particles in Hep56.1D mouse liver cells, albeit with low efficiency. Given the origin of these particles, by analogy to our earlier report,25 they were designated muHCVTCP to indicate the use of a trans-complementation (TCP) approach in mouse (mu) cells. Expression of Human or Mouse apoE in Mouse Cells Promotes Production of Infectious muHCVTCP Particles to the Same Extent In a search for the reasons of this low efficiency, we argued that host factors playing a crucial role in HCV particle production might either be incompatible between HCV ASSEMBLY IN MOUSE LIVER CELLS 1059 Figure 1. Production of infectious HCV particles in mouse cells by using trans-complementation. (A) A schematic representation of the selectable subgenomic JFH1 replicon neo-sgJFH1-H2476L is shown in the top. The neo gene is fused at the 5= end with the 16 5= terminal codons of the core gene. The respective fusion protein is expressed via the HCV IRES residing in the 5= NTR. The replicase genes (NS3 to NS5B) are translated via the IRES of the encephalomyocarditis virus (EI). H2476L refers to the position of the cell culture-adaptive mutation in NS5B. The trans-complementing assembly cassette (core to NS2) expressed via a lentiviral vector is drawn below. The coding region is derived from the Jc1 genome and composed of the core to p7 region of the HCV isolate J6; the NS2 gene is chimeric and composed of the first transmembrane domain of the J6 isolate, whereas the remainder of NS2 (indicated by the dashed line) is derived from the JFH-1 isolate.23 (B) Detection of replicon RNA in Hep56.1D cell clones by Northern blot analysis. For each cell clone, 5 g total RNA was analyzed. Left 2 lanes: Given amounts of in vitro transcripts spiked with total RNA from naïve cells served as size marker and to estimate HCV RNA amounts. Total RNA from naïve Hep56.1D (control cells) was used as negative control. -actin (lower panel) was used as loading control. Positions of replicon RNA (HCV) and 28S ribosomal RNA are indicated in the left. (C) Western blot analysis of Hep56.1D cells and cell lines derived thereof 3 weeks after lentiviral transduction, for proteins specified on the right of each panel. Positions of molecular size markers are indicated on the left. For comparison of apoE amounts contained in 105 Hep56.1D cells, total protein in a homogenate of given numbers of mouse liver cells was analyzed in parallel. Actin was used as loading control. (D) Intra- and extracellular HCV RNA amounts in replicon cells with or without stable expression of core to NS2. Total RNA prepared 48 hours after seeding from cells or culture supernatant of 105 cells was quantified by using quantitative reverse-transcription polymerase chain reaction. (E) Detection of infectivity in culture supernatants of cells specified in the bottom by using a limiting dilution assay. Data in panels D and E represent the mean of 3 independent assays; error bars represent standard deviations from the means. mouse and human or limiting in Hepa56.1D cells. To address the latter possibility, a comparative transcriptome analysis between naïve Hep56.1D cells and 3 independent replicon-containing cell clones was conducted. Most striking was the low expression of apoE in Hep56.1D cells (Figure 1C) that was reduced even further in the 3 replicon cell clones (Supplementary Figure 1). In contrast, in a homogenate prepared from the same number of mouse liver cells, apoE was highly abundant, arguing that low apoE expression is a property of Hep56.1D cells. We therefore concluded that apoE, which is essential for HCV particle production, might limit assembly in Hep56.1D cells but probably not in mouse liver cells. Focusing our analysis on apoE, we studied the impact of different apoE expression levels and possible species BASIC AND TRANSLATIONAL LIVER September 2011 1060 LONG ET AL GASTROENTEROLOGY Vol. 141, No. 3 BASIC AND TRANSLATIONAL LIVER Figure 2. HCV particle production is limited in mouse cells by (m)apoE amounts. (A) Schematic representation of constructs used for expression of authentic or tagged apoE genes, respectively. (B) Western blot analysis of Hep56-sgJFHcl3-CNS2 cells transiently transfected with constructs given in the top Cells were transfected with equal amounts of the expression construct, and, 48 hours later, proteins were analyzed. Molecular sizes are indicated on the left of each blot, and proteins are specified on the right. Actin served as loading control. (C–F) Hep56-sgJFHcl3-CNS2 cells were transfected with constructs specified in the bottom of each panel; 48 hours later, cells and supernatants were harvested and used for determination of (C) released core protein by enzyme-linked immunosorbent assay, (D) released HCV RNA by quantitative reverse-transcription polymerase chain reaction, and (E) intra- and (F) extracellular infectivity by using limiting dilution assay. Mock-transfected cells and cells transfected with the pcDNA-GFP expression construct served as negative controls. Dashed lines in C and D indicate assay backgrounds. Shown are the means of 3 independent experiments; error bars indicate standard deviations from the means. restriction. An amino acid sequence alignment between mouse (m) apoE, which is the only allele in this species, and the predominant human (h) apoE3 gene revealed a similarity of 80.9% (Supplementary Figure 2) and an 8-amino acid deletion in (m)apoE. We therefore constructed apoE expression vectors, with or without a C-terminal hemagglutinin (HA)-affinity tag (Figure 2A), the latter allowing the unambiguous detection of apoE proteins and their quantification. Transient transfection of Hep56-sgJFHcl3-CNS2 cells each with equal amounts of an apoE construct demonstrated comparable expression level of all HA-tagged proteins, thus allowing direct comparisons in assembly assays (Figure 2B). Importantly, (h)apoE3-HA and (m)apoE-HA shared a similar subcellular localization pattern (Supplementary Figure 3), suggesting that the fusion with the HA tag did not affect apoE properties. Finally, core and NS5A amounts were not affected by apoE overexpression (Figure 2B). We next studied the impact of apoE expression on HCV particle production in mouse cells. Hep56-sgJFHcl3CNS2 cells were transfected with the various apoE constructs or the green fluorescent protein (GFP) control or mock-transfected and release of core protein and viral RNA as well as intra- and extracellular infectivity were assessed (Figure 2C–F). Expression of each individual apoE led to a slight increase of core protein release as compared with the control cells independent from the apoE species origin (Figure 2C). In contrast, when we measured RNA amounts in the culture supernatants, we did not detect a difference (Figure 2D). Instead, we found that high amounts of RNA were released from replicon cells independent from the structural proteins (Supplementary Figure 4), which is similar to observations made with replicons in human hepatoma cells.26 However, with the more discriminatory TCID50 assay, we observed an unambiguous difference between Hep56-sgJFHcl3-CNS2 control cells (GFP and mock) and cells transfected with any of the apoE expression constructs (Figure 2E and F). In fact, in all cases of apoE expression, extracellular infectivity titers were enhanced about 100-fold as compared with Hep56-sgJFHcl3-CNS2 expressing only limiting amounts of endogenous (m)apoE. Most importantly, (h)apoE and (m)apoE enhanced production of muHCVTCP particles to the same extent, arguing that apoE is not a determinant of HCV species specificity. We also did not detect a difference between the various (h)apoE alleles, showing that they all support HCV assembly to the same extent. Finally, the comparable infectivity titers attained with authentic and HA-tagged apoE demonstrates full functionality of the latter. We note that production of infectious muHCVTCP particles was also achieved in 2 other Hep56.1D-derived replicon cell clones (numbers 8 and 10; Figure 1B), thus excluding a cell clone-specific effect (data not shown). We also note that Hep56.1D replicon cells expressing very low amounts of apoE do not release (noninfectious) HCV particles (Supplementary Figure 5), arguing that the high release of viral RNA and core from such cells is either nonspecific or reflects the release of naked noninfectious particles or RNA-containing replication complexes.26,27 In conclusion, our results demonstrate that Hep56.1D mouse cells are permissive for HCV assembly and that expression of (m)apoE is a limiting factor. Biophysical Properties of Mouse Cell-Derived Infectious muHCVTCP Particles To study biophysical properties of mouse cell-derived particles and the impact of apoE on association with lipids and lipoproteins, muHCVTCP particles released from Hep56-sgJFHcl3-CNS2 cells after transfection with GFP control or apoE constructs were analyzed by equilibrium density gradient centrifugation (Figure 3). HCV RNA, core protein, and infectivity in each fraction were quantified. Cells transfected with the GFP vector released substantial amounts of viral RNA that sedimented to very low density in agreement with our earlier report on nonspecific RNA HCV ASSEMBLY IN MOUSE LIVER CELLS 1061 release from replicon cells (Figure 3A).26 In contrast, cells transfected with either of the apoE expression construct released RNA-containing structures with peak density of about 1.1 g/mL similar to the one of HCV particles produced in Huh7 cells.5 A high amount of core protein was detected in the supernatant of the Hep56-sgJFHcl3-CNS2 cells transfected with the GFP control plasmid and these core-containing structures had a high density (⬃1.18 g/mL), whereas the peak of Core protein released from apoE-transfected cells was much broader and covered the range from 1.07 to 1.18 g/mL (Figure 3B). Most importantly, culture supernatants of apoE-transfected cells contained high amounts of infectivity with a peak density of ⬃1.1 g/mL similar to HCV produced in human liver cells (Figure 3C).9,25 In contrast, only very low infectivity was found with the GFP control cells, corroborating the limiting amounts of endogenous (m)apoE expressed in Hep56.1D cells. For direct comparison of HCVTCP produced in mouse or human hepatic cells, we developed the same trans-complementation system with Huh7.5 cells (cell line Huh7.5sgJFH-CNS2). Infectivity titers attained with this cell line were well comparable with those of mouse cells demonstrating the high assembly competence of Hep56.1D mouse liver cells (Supplementary Figure 6). Biophysical properties of mouse and human cell-derived HCVTCP were determined by ultracentrifugation as described above. The density profile of viral RNA of both samples was comparable (Figure 3D). Core protein distribution across the gradient was also comparable, although an additional peak was detected at very low density (Figure 3E), which was more pronounced when analyzing infectivity (Figure 3F), arguing that either human cells are more competent in producing lipoproteins or that huHCVTCP have a higher competence to associate with lipids. In summary, these results demonstrate that infectious muHCVTCP particles share similar although not identical biophysical properties with HCV produced in human liver cells. ApoE Is a Component of Mouse Cell-Derived Infectious HCV Particles Because apoE is required for HCV infectivity and assembly and an integral component of infectious HCV particles,9,10 we wondered whether the same holds true for mouse cell-produced particles. MuHCVTCP particles were generated in cells transiently expressing HA-tagged (h)apoE3 or (m)apoE and subjected to HA-specific affinity purification. Only tagged apoEs were detected in the immunocomplexes even though untagged proteins were expressed to comparable level (Figure 4A). Importantly, infectious particles were detected in immunocomplexes obtained with supernatants of HA-apoE transfected cells (Figure 4B). Thus, similar to human hepatocyte-derived HCV, mouse cell-derived HCV particles are tightly associated with apoE residing on the surface of the particles. BASIC AND TRANSLATIONAL LIVER September 2011 1062 LONG ET AL GASTROENTEROLOGY Vol. 141, No. 3 BASIC AND TRANSLATIONAL LIVER Figure 3. Biophysical properties of muHCVTCP particles. (A–C) Hep56-sgJFHcl3-CNS2 cells were transfected with constructs specified in the top. Forty-eight hours later, culture supernatant was collected, concentrated by ultrafiltration, and fractionated by density gradient centrifugation. Ten fractions were harvested from each gradient, and amounts of HCV RNA (A), core protein (B), and viral infectivity (C) contained in each fraction were determined. (D–F) muHCVTCP contained in supernatant of Hep56-sgJFHcl3-CNS2 cells transiently expressing (m)apoE and huHCVTCP released from Huh7.5-sgJFH-CNS2 cells expressing (h)apoE were analyzed by ultracentrifugation. Amounts of HCV RNA (D), core protein (E), and viral infectivity (F) contained in each fraction were determined as above. Representative data of at least 2 independent experiments are shown. Equal Entry Factor Dependency of Mouse and Human Cell-Derived HCV Particles Entry of HCV into human hepatocytes requires multiple cell surface molecules, including CD81,28 scavenger receptor class B type I (SCARB1),29 Claudin 1,30 and Occludin.16 To test whether HCV particles produced in mouse cells utilize these entry molecules to the same extent, neutralization experiments were conducted. Entry of human and mouse cell-derived HCV particles was blocked with comparable efficiency by all 3 tested antibodies, whereas matching control antisera had no effect (Figure 5). We therefore concluded that entry receptor dependency of HCV particles derived from human or mouse cells is comparable. This result argued against mouse cell-specific modifications of HCV particles affecting their entry receptor usage. September 2011 HCV ASSEMBLY IN MOUSE LIVER CELLS 1063 MMH-sgJFH-CNS2. Amounts of HCV RNA in these cell lines were comparable with those in Hep56.1D-derived cells (Figure 6A). Endogenous amount of (m)apoE was below the detection limit, but robust expression was obtained upon transfection with (h)apoE3 or (m)apoE expression constructs (Figure 6B). None of the transduced genes affected HCV RNA replication as determined by NS5A-specific Western blot, and core protein amounts were comparable in all cell clones. Infectious HCV particles were not detectable in either of the cell lines transfected with the GFP control vector (Figure 6C). However, infectivity was consistently detected in the supernatant of MMH1-1- and AML12-derived cells expressing either the human or mouse apoE gene. In agreement with the results obtained with Hep56.1D cells, infectivity titers were not affected by the species origin of the apoE gene. Whereas the titers attained with the MMH1-1 cells were well comparable with those achieved with Hep56.1D cells, infectivity titers achieved with AML12-derived cells were about 10-fold lower, arguing for possibly limiting factors of HCV assembly and release. Nevertheless, the fact that 3 different mouse liver-derived cell lines support production of infectious HCV particles provides compelling evidence that HCV assembly competence is a general property of mouse hepatocytes. Discussion To establish an immunocompetent fully permissive mouse model that is urgently required to study HCVspecific immune responses and pathogenesis, multiple species restrictions need to be overcome. HCV entry into mouse cell lines is possible upon ectopic expression of HCV Assembly Competence Appears to be a General Property of Mouse Liver Cells So far, all our analyses have been based on the mouse hepatoma cell line Hep56.1D. To determine whether assembly competence is a unique property of this cell line or of mouse liver cells in general, we established the analogous trans-complementation system by using 2 other mouse cell lines containing a stably replicating subgenomic HCV replicon: AML12-sgJFH1 and MMH11-sgJFH1.20 Both cell lines were transduced to express core to NS2, resulting in cell lines AML12-sgJFH-CNS2 and BASIC AND TRANSLATIONAL LIVER Figure 4. Association of apoE with muHCVTCP particles. (A) ApoEspecific Western blot analysis of total proteins from Hep56-sgJFHcl3CNS2 cells transfected with (h)apoE3-, (h)apoE3-HA- (m)apoE-, or (m)apoE-HA-expression constructs. Input and immuno-captured proteins are shown in the left and right panels, respectively. Molecular sizes are indicated on the left of the blots, and proteins are specified on the right. (B) Infectivity determination of immunocaptured muHCVTCP particles. Equivalent amounts of infectious particles were subjected to HA affinity capture as described in the Materials and Methods section. Captured particles were eluted with an HA peptide and infectivity titers in input and the corresponding eluates were determined by using limiting dilution assay. Shown are the means of 3 independent experiments; error bars represent standard deviations from the means. Figure 5. Entry factor dependency of muHCVTCP particles. Huh7.5 cells were inoculated with muHCVTCP (released from Hep56-sgJFHcl3-CNS2 cells overexpressing (m)apoE) or huHCVTCP particles (released from Huh7.5-sgJFH-CNS2 cells) for 1 hour in the absence or presence of antibodies specified in the bottom. Cells were washed 3 times with fresh medium and incubated for 72 hours. Infectivity titers were assessed by counting HCV-positive foci detected by immunohistochemical staining. Mock incubation of virus samples served as negative control and was used for data normalization (set to 100%). Shown are the means of 3 independent experiments; error bars represent standard deviations from the means. 1064 LONG ET AL GASTROENTEROLOGY Vol. 141, No. 3 Figure 6. Assembly of muHCVTCP particles in mouse liver cell lines AML12 and MMH1-1 cells. (A) Quantification of intracellular HCV replicon RNA by using quantitative reverse-transcription polymerase chain reaction. HCV RNA copy number in 105 mouse cells specified in the bottom was determined. (B) Expression analysis of HCV proteins and apoEs in MMH1-1 and AML12-derived cell lines by using Western blot. Cells were harvested 48 hours after transfection; mock transfected cells served as negative control. Molecular sizes are indicated on the left, and proteins are specified on the right. (C) Infectivity released into supernatants of cells transfected with apoE expression constructs specified in the bottom was determined using TCID50 assay. Shown are the means of 3 independent experiments; error bars represent standard deviations from the means. BASIC AND TRANSLATIONAL LIVER CD81 and occludin or by using a mouse CD81-adapted HCV genome.16,17 Whereas HCV internal ribosome entry site (IRES) activity in mouse and human cells appears to be comparable,18,19 viral RNA replication is very inefficient and requires selection approaches.20,21 Transient replication that mimics more closely an infection is virtually undetectable, although the ectopic expression of microRNA-122 and knockout of interferon regulatory factor 3 or protein kinase R appears to increase RNA replication.31,32 Thus, HCV replication might be limited both by intrinsic and induced restriction factors but also by low expression and/or mouse/human incompatibility of important proviral factors. Owing to this insufficient RNA replication, it could not be studied whether mouse cells support production of infectious HCV particles. To overcome this limitation, we devised a trans-complementation system with multiple mouse liver cell lines. We demonstrate that mouse cells do support assembly and release of infectious HCV particles as long as sufficient levels of apoE are expressed. In addition, we found that (1) each human apoE isoform and mouse apoE support particle production to the same extent and (2) (m)apoE supports assembly also in human cells (Supplementary Figure 7). Thus, apart from apoE, host factors contributing to HCV particle production in mouse cells are neither limiting in abundance, nor do they critically determine species restriction. However, as exemplified by the different expression levels of apoE in mouse hepatic cell lines and mouse hepatocytes in vivo, we can not exclude the possibility that other host factors affecting HCV assembly might limit particle production in vivo. Accumulating reports show that HCV morphogenesis is tightly associated with lipoproteins and virions are re- leased as unique lipoviro particles.5 A hallmark of this assembly process is its dependence on apoE.9 –12 In this study, we observed the same for mouse cell-derived HCV particles corroborating the similarity of HCV assembly in human and mouse cells. Interestingly, Hep56.1D cells containing the subgenomic JFH-1 replicon expressed much lower amounts of apoE as compared with naïve cells. Moreover, also in the replicon cell clones derived from AML12 and MMH1-1, apoE expression was barely detectable. The underlying reasons are not known, but low apoE expression in mouse replicon cells might be an epiphenomenon perhaps linked to host cell conditions that are more favorable for viral replication. HCVTCP produced in mouse and human cells share similar properties. First, we found that infectivity of human or mouse cell-derived HCVTCP has a broad density range, comparable with HCV produced in Huh-7 cells.9 We note, however, that a fraction of HCVTCP particles produced in human cells appear to be associated with lipids to a higher extent and that these particles have a high specific infectivity. Second, infectivity of HCV particles produced in human or mouse liver cells depends on the same set of entry molecules. Third, assembly of human and mouse cell-derived HCV particles depends on apoE amounts, and both particle types are associated with it. ApoE on lipoprotein particles directly binds to the LDL receptor (LDLR), which is important for clearance of remnant lipoproteins. LDLR is also involved in HCV entry presumably via interaction with apoE on the surface of the virion.33 LDLR binding affinity to apoE is remarkably isoform specific. In vitro, apoE3 and E4 bind with similar affinity to LDLR, whereas apoE2 has ⬃2% of this binding capability.34,35 In line with these results, Hishiki et al reported that HCV particles produced in apoE2-expressing cells were about 75% less infectious than apoE3- or apoE4-containing HCV.36 We found however, that HCV infectivity is not affected by a particular apoE allele indicating that the apoE-LDLR affinity is not decisive for infectivity, at least in Huh7.5 cells. Moreover, very recently Cun et al also reported that human apoE2, E3, and E4 are equally efficient in supporting infectious HCV production in Huh7.5 cells.37 The reason for these discrepancies is not clear, but differences between the used cell lines eventually expressing different levels of LDLR as well as other apoE receptors such as Heparan sulfate proteoglycans, lowdensity lipoprotein receptor-related protein 1 (LRP1), or apoE receptor R2 (LRP8) might account for that. Apart from enabling assembly studies in mouse cells, the extremely low expression levels of apoE in Hep56.1D cells, which mimics a functional knock-down, provides an important tool to dissect the role of individual apolipoproteins for HCV assembly independent from apoE. Moreover, HAtagged apoEs are fully functional thus providing an additional tool to isolate and characterize HCV particles as well as apoE-binding viral and host cell proteins. In summary, we demonstrate that mouse liver cell lines are competent for assembly and release of HCV particles provided that sufficient levels of apoE are expressed. This result closes another gap in our understanding of species restriction of HCV. In this respect, the block of viral RNA replication is most likely the only remaining hurdle that needs to be overcome to establish a fully permissive mouse liver cell-based HCV system. Without doubt, such a system will pave the way for the development of an immunocompetent HCV permissive mouse model. 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GASTROENTEROLOGY Vol. 141, No. 3 Acknowledgments The authors thank Ulrike Herian for excellent technical assistance; Susan Uprichard for providing the replicon cell lines MMH1-1 and AML12; Thomas Baumert for provision of SCARB1- and Claudin 1specific antibodies; Heinrich Wieland for apoE2, 3, 4 constructs; Stephan Urban for providing mouse liver homogenates; Charles M. Rice for Huh7.5 cells and the NS5A-specific antibody 9E10; Takaji Wakita for provision of the original JFH1 clone; and the Nikon Imaging Center at the University of Heidelberg for continuous support and providing access to all necessary equipment. M.P.W.’s present address is Applied Molecular Virology, Institute Pasteur Korea, 696 Sampyung-dong Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea. Conflicts of interest The authors disclose no conflicts. Funding Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB/TRR77, Teilprojekt 1; to R.B. and V.L.; and TRR83, Teilprojekt 13; to R.B.), by a grant from the European Union (ERC grant contract no. 233130), and by the Marie Curie Training Network EI-HCV, contract no. MRTN-CT-2006-035599 EI-HCV. BASIC AND TRANSLATIONAL LIVER