Volume 67, Issue 3, Pages 480-489 (September 2017) Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity  Dorothea Bankwitz, Mandy Doepke, Kathrin Hueging, Romy Weller, Janina Bruening, Patrick Behrendt, Ji-Young Lee, Florian W.R. Vondran, Michael P. Manns, Ralf Bartenschlager, Thomas Pietschmann  Journal of Hepatology  Volume 67, Issue 3, Pages 480-489 (September 2017) DOI: 10.1016/j.jhep.2017.04.010 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2017 67, 480-489DOI: (10. 1016/j. jhep. 2017. 04 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 1 ApoE conditioned medium of Huh-7.5 cells enhances HCV infection. (A–D) Jc1 and Jc1/ΔHVR1 renilla luciferase reporter viruses were incubated for 20min at 37°C with serial dilutions of culture fluids from Huh-7.5 ApoE-overexpressing (ApoE sup) or -silenced cells (Ctrl sup) before infection of Huh-7.5 cells. The cells were harvested 72h after infection and luciferase reporter activity was measured. In panel A and B as well as in C and D the same experiments are shown either relative to the reciprocal dilution factor of ApoE conditioned medium (A and C) or relative to the ApoE concentration in ApoE conditioned medium (B and D). The light blue area indicates typical serum ApoE concentrations. Infection in the absence of ApoE sup or ctrl sup is expressed as hundred percent. Three independent experiments are shown. Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Secreted ApoE mediates HCV infection enhancement. (A and B) ApoE was depleted from culture fluids of ApoE-overexpressing (ApoE sup) or ApoE-silenced cells (Ctrl sup) by two consecutive immune-precipitations with a monoclonal anti-ApoE antibody (Progen) and two sequential immune-precipitations with polyclonal anti-ApoE antibodies (Calbiochem). (A) HCV was incubated for 20min at 37°C with ApoE depleted supernatant prior to infection and cells were harvested 72h after infection to measure luciferase reporter activity. (B) ApoE depleted supernatant was analysed for ApoE, ApoC1 and ApoB by ELISA; apolipoprotein concentration in the input supernatant is expressed as a hundred percent. Mean values of at least three independent experiments are shown by blue bars and black dots represent the results of each individual experiment. Comparison of input and ApoE depleted supernatant was performed using one-way ANOVA followed by Dunns multiple comparison correction (**p<0.01). Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Secreted ApoE enhances infectivity of HCV from primary cells and across all genotypes independent of particle density. (A) Chimeric renilla luciferase reporter viruses were incubated for 20min at 37°C with ApoE enriched supernatant (20µg/ml ApoE) or equal volume of control supernatant (∼0.2µg/ml ApoE) prior to inoculation of Huh-7.5 cells. Infectivity was determined 72h later by luciferase assays. Mean values of four independent experiments are shown by light blue bars; black dots represent the results of each individual experiment. (B) Primary human hepatocytes were inoculated with Jc1, Jc1/ΔHVR1 and SA13 (GT5a) renilla luciferase reporter viruses for 4h. Cells were washed with PBS 3 times to remove input virus. Supernatant containing released HCV was collected 24h after infection and incubated for 20min at 37°C with ApoE enriched supernatant (20µg/ml ApoE) or control supernatant (∼0.2µg/ml ApoE) before inoculation of naive Huh-7.5 cells. Infectivity was determined 72h later by luciferase assays. Mean values of at least three independent experiments are shown by light blue bars and black dots represent the results of each individual experiment. (C) Individual virus samples were harvested 48h and 72h after transfection of Huh-7.5.1 cells and were separated using an iodixanol step gradients. Eleven fractions were harvested from the bottom and infectivity in the presence of secreted ApoE (ApoE sup) or control supernatant (Ctrl sup) was determined for each fraction. Percent total infectivity was calculated by relating the amount of infectious virus detected in each fraction to the collected total amount of infectious virus treated with control supernatant (Ctrl sup) and plotted against density of the respective fraction measured by refractometry. Three independent experiments are shown. (D) For each fraction shown in panel C infection-enhancing effect was calculated by relating viral infectivity in the presence of secreted ApoE to infectivity of viruses treated with control supernatant and is expressed as x-fold infectivity. Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Secreted ApoE is incorporated into HCV particles and serum HCV is decorated with more ApoE than cell culture-derived HCV. (A) Cell culture-derived HCV (HCVcc) were incubated with supernatant enriched for HA-ApoE (20µg/ml HA-ApoE) or untagged ApoE (20µg/ml ApoE) as control for 20min at 37°C and were then mixed with anti-HA coupled protein G agarose beads. After 2h at 4°C, co-precipitated HCV core protein was eluted with 400µl PBS/1% Triton and quantified using core ELISA. The amount of core protein associated with anti-HA coupled protein G beads is plotted. Mean values of three independent experiments are shown by bars; black dots represent the results of each individual experiment. Comparison of viruses incubated with HA-ApoE and ApoE was performed using two-way ANOVA followed by Holm-Sidak multiple comparison correction (***p<0.001). (B) Sh-ApoE and sh-NT cell lines were electroporated with Jc1 RNA. After 48h, supernatants were harvested. Samples containing equal amounts of HCV RNA copies (8×105) were used for immuno-capture with ApoE antibody-coated protein G-Sepharose beads for 2.5h at room temperature in the presence or absence of 0.5µg of recombinant human ApoE2 full length protein. Co-precipitated HCV RNA was quantified by qRT-PCR. Immunoprecipitation is expressed as x-fold and calculated by relating immunoprecipitation in the presence of ApoE to untreated Jc1. (C and D) HCV patient sera were treated and analysed as descripted in Fig.4A. Dark blue bars in (C) represent serum ApoE levels (µg/ml). (B–D) Mean values of at least two independent experiments are shown by blue bars; black dots represent the results of each individual. (E) To analyse the degree of ApoE decoration, Jc1 or sera from HCV-infected patients and serum from a healthy donor were captured by E2-specific antibodies (AR4A, CBH5, HC11) and captured core and ApoE was eluted with 50µl PBS/1% Triton and quantified using ApoE ELISA and core ELISA. One representative experiment is shown (n=3). Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Extracellular ApoE enhances HCV entry. Huh-7.5 cells were inoculated with indicated renilla luciferase reporter viruses. 20µg/ml ApoE or equal volume of control supernatant (∼0.2µg/ml ApoE) was added to the cells only before inoculation (white), only during inoculation (light blue), or directly after inoculation (dark blue) as schematically depicted at the top. Infectivity was determined 72h later by luciferase assays. Infectivity is expressed as x-fold and calculated by relating viral infectivity in the presence of secreted ApoE to infectivity of viruses treated with control supernatant. Mean values of three independent experiments are shown by bars and black dots represent the results of each individual experiment. (This figure appears in colour on the web.) Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Enhancement of HCV infectivity by secreted ApoE is abrogated by HPSG-targeting agents independent from SR-B1. (A) SR-BI receptor on Huh-7.5 cells was blocked with anti-SR-BI antibody C167 or with a SR-BI specific small molecule (ITX5061). These cells were infected with renilla luciferase reporter viruses pre-incubated with ApoE enriched supernatant (ApoE sup) or control supernatant (Ctrl sup). (B) Lunet N#3 hCD81 SR-BI knockout cells were created with CRISPR/Cas9 technology and incubated with Jc1 renilla luciferase virus pre-incubated with either ApoE sup or Ctrl sup. (C) Recombinant renilla luciferase reporter viruses were first incubated with ApoE sup or Ctrl sup and then with heparin for 30min at 37°C before Huh-7.5 cells were infected. Huh-7.5 cells were incubated with heparinase III for 1h at 37°C and subsequently incubated with renilla luciferase reporter viruses mixed with ApoE sup or Ctrl sup and incubated for 20min. (A–C) After 72h the cells were harvested for luciferase measurement. Mean values of at least three independent experiments are shown by bars, black dots represent the results of each individual experiment. Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Secreted ApoE protects from neutralizing antibodies. (A) Neutralization with E2-specific antibodies: Renilla luciferase reporter viruses (JcR2a, JcR2a/ΔHVR1 and SA13/5a/R2a) were incubated with ApoE enriched supernatant (20µg/ml ApoE) or control supernatant (∼0.2µg/ml ApoE) for 20min at 37°C. Subsequently, viruses were incubated for 1h at 37°C with serial dilutions of antibodies specific for E1 (IGH526) or E2 (CBH7, HC11, AP33, AR4A) prior to infection of Huh-7.5 cells. 72h after infection luciferase assay was performed. Mean values of three independent experiments including the standard deviations are shown. (B) Equal numbers of given viruses were incubated with ApoE enriched supernatant (20µg/ml ApoE) or control supernatant (∼0.2µg/ml ApoE) for 20min at 37°C. Subsequently, viruses were incubated with 1µg of the specified antibodies. Immune complexes were precipitated using protein A/G-coated beads, and associated HCV core protein was quantified by ELISA. Mean values of at least two independent experiments including the standard deviations are shown. Journal of Hepatology 2017 67, 480-489DOI: (10.1016/j.jhep.2017.04.010) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions