1. Volume 141, Issue 3, Pages 1057-1066 (September 2011)
Mouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus Particles 
Gang Long, Marie–Sophie Hiet, Marc P. Windisch, Ji–Young Lee, Volker Lohmann, Ralf Bartenschlager 
Gastroenterology 
Volume 141, Issue 3, Pages (September 2011)
DOI: /j.gastro
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2. 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 μg 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. β-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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. 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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. 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.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Association of apoE with muHCVTCP particles. (A) ApoE-specific Western blot analysis of total proteins from Hep56-sgJFHcl3-CNS2 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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. 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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. 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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions