1. Volume 143, Issue 2, Pages 439-447.e1 (August 2012)
Hepatocytes That Express Variants of Cyclophilin A Are Resistant to HCV Infection and Replication 
Thomas von Hahn, Cordelia Schiene–Fischer, Nguyen Dinh Van, Stephanie Pfaender, Behya Karavul, Eike Steinmann, Andrej Potthoff, Christian Strassburg, Nabila Hamdi, Ahmed I. Abdelaziz, Christoph Sarrazin, Tobias Müller, Thomas Berg, Eric Trépo, Heiner Wedemeyer, Michael P. Manns, Thomas Pietschmann, Sandra Ciesek 
Gastroenterology 
Volume 143, Issue 2, Pages e1 (August 2012)
DOI: /j.gastro
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2. Figure 1
Characterization of a novel Huh-7.5 CypAlow cell line. (A) Immunoblot for human CypA and β-actin in Huh-7.5 cells expressing an irrelevant or CypA-specific shRNA (Huh-7.5/CypAlow). (B) Parental Huh-7.5 cells, Huh-7.5 cells expressing an shRNA against CypA (Huh-7.5/CypAlow), and Huh-7.5/CypAlow cells transduced to express an shRNA-resistant CypA wild-type transgene were transfected with a luciferase reporter virus genome (F-luc Jc1) and seeded into replicated tissue culture plates. HCV RNA replication was quantified by measuring luciferase activity after 4, 24, 48, and 72 hours. Mean values and the SD of 3 independent experiments are shown.
Gastroenterology  , e1DOI: ( /j.gastro )
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3. Figure 2
Effect of different PPIA SNPs on the HCV life cycle. (A) Parental Huh-7.5 cells, Huh-7.5/CypAlow cells, and Huh-7.5/CypAlow cells expressing human CypA variants were inoculated with supernatant containing F-luc Jc1 reporter virus. Luciferase activity was assayed 48 hours after infection. Mean values and the SD of 3 independent experiments are shown. P < .05. (B) Lentiviral HCV pseudoparticles with strain H77 (genotype 1a) glycoproteins were used to infect Huh-7.5 shCypA expressing different CypA SNPs. Pseudoparticles with the glycoprotein of the vesicular stomatitis virus (VSV-G) served as positive controls; pseudoparticles without viral glycoproteins obtained after transfection of an empty vector (pcDNA) in lieu of a vector encoding a viral glycoprotein were used to define the background of the assay. A representative of 3 independent experiments is shown. Mean values of quadruplicate measurement and the SD are given. (C and D) Huh-7.5/CypAlow cells expressing different CypA variants were electroporated with (C) F-luc Jc1 RNA transcripts or (D) F-luc JFH1 subgenomic RNA transcripts, and luciferase activity over the following 72 hours was monitored as indicated. (E) To assess the efficiency of viral particle production, the different cells were transfected with Jc1 wild-type RNA transcripts and after 48 hours intracellular and extracellular core was measured by a core enzyme-linked immunosorbent assay. (F) Huh-7.5/CypAlow cells expressing the CypA variant N106I or parental Huh-7.5 cells were transduced either with CypA wild-type or CypA N106I, respectively, at decreasing dilutions. After 48 hours, cells were inoculated with supernatant containing F-luc Jc1 reporter virus. Luciferase activity was assayed 48 hours after infection. *P < .05.
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4. Figure 3
Effects of PPIA SNPs on different HCV genotypes. Huh-7.5/CypAlow cells expressing the different CypA variants were inoculated with chimeric viruses consisting of the structural region and NS2 from genotypes 1a, 1b, 3a, 4a, and 5a and NS3-5B from the genotype 2a isolate JFH-1. HCV replication was assessed in a 50% tissue culture infective dose assay. Cells were fixed and stained for NS5A expression 48 hours after inoculation, and 50% tissue culture infective dose per milliliter was determined.
Gastroenterology  , e1DOI: ( /j.gastro )
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5. Figure 4
Alisporivir retains its antiviral activity in the presence of CypA variants. (A and B) Huh-7.5/CypAlow cells expressing CypA variants were transfected with a luciferase reporter virus genome (F-luc Jc1) and increasing doses of alisporivir were added to the medium 4 hours after transfection. At 48 hours after transfection, HCV replication was measured by luciferase activity. A representative of 3 independent experiments is shown.
Gastroenterology  , e1DOI: ( /j.gastro )
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6. Figure 5
SNPs associated with an HCV refractory phenotype lead destabilize CypA. (A) PPIA messenger RNA in Huh-7.5 or Huh-7.5/CypAlow cells expressing CypA variants was measured by quantitative reverse-transcriptase polymerase chain reaction using oligonucleotides binding within the CypA open reading frame. (B) CypA protein expression in Huh-7.5/CypAlow cells expressing CypA variants was detected by immunoblotting with a monoclonal antibody against CypA. β-actin served as a loading control. (C) Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of cell lysate of E coli M15 cells overexpressing wild-type CypA or CypA variants R55A, D66E, N106I, or G96D, respectively, induced by 1 mmol/L isopropyl β-D-1-thiogalactopyranoside before and after bacterial cell disruption using a French press. Samples were loaded on 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis using sodium dodecyl sulfate sample loading buffer. (D) Detection of CypA over time by immunoblotting in cells treated with the translation inhibitor cycloheximide (100 μg/mL). β-actin served as a loading control. Untreated naïve Huh-7.5 cells served as an internal control. (E) Thermal unfolding of 10 μmol/L CypA wild-type or CypA D66E was observed by circular dichroism, measuring ellipticity at 222 nm from 30°C to 80°C in 10 mmol/L sodium phosphate buffer, pH 7.5. The transition midpoints of CypA wild-type or CypA D66E are 52.7 ± 0.2°C and 47.2 ± 0.1°C, respectively.
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7. Figure 6
Effect of proteasome inhibition on the stability of CypA N106I. Huh-7.5/CypAlow cells expressing the CypA N106I variant were treated with the proteasome inhibitor atazanavir at different concentrations for 6 hours. Then cells were lysed and CypA expression was detected by immunoblotting. Untreated Huh-7.5/CypAlow cells expressing wild-type CypA served as controls.
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8. Supplementary Figure 1
Nonsynonymous SNPs in PPIA. (A) Schematic representation of PPIA with the position of 6 amino acid changes caused by known nonsynonymous SNPs in the coding region (exon 4 and 5). Boxes indicate exons, with gray and black portions indicating the untranslated regions and the open reading frame, respectively. (B) The structure of human cyclophilin A (PDB entry 1RMH) in complex with a tetrapeptide substrate with the positions of the SNP mutations highlighted. Amino acid side chains are shown in stick representation in green, and the tetrapeptide substrate Suc-AAPF-pNa is depicted in blue. (Left) Side view; (right) front view.
Gastroenterology  , e1DOI: ( /j.gastro )
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9. Supplementary Figure 2
Flow cytometric analysis of GFP expression in Huh-7.5/CypAlow cells transduced to express CypA variants using an expression vector (pWPI-GUN) that also encodes a GFP-neomycin-resistance fusion protein from a separate cistron. Measurements were performed at the time when susceptibility to HCVcc was assessed.
Gastroenterology  , e1DOI: ( /j.gastro )
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