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1 Supplementary information Exosomes mediate the cell-to-cell transmission of interferon alpha-induced antiviral activity Jianhua Li, Kuancheng Liu, Yang Liu, Yan Xu, Fei Zhang, Huijuan Yang, Jiangxia Liu, Tingting Pan, Jieliang Chen, Min Wu, Xiaohui Zhou, and Zhenghong Yuan * * Corresponding author: Zhenghong Yuan, zhyuan@shaphc.org, Telephone: , Fax:

2 Supplementary Figure 1 Supplementary Figure 1. LNPCs contribute to the antiviral activity of IFN-α against HBV (a) Schematic of the transwell co-culture model with macrophages, LSECs, or HepG2 cells in the top well and HepG cells in the bottom well. A porous (0.4-μm) membrane allows transfer of exosomes but precludes direct cell contact. (b) Southern blot analysis of secreted enveloped viral DNA (top) and intracellular viral replicative DNA (bottom) from HepG cells co-cultured with THP-1-derived macrophages or LSECs at the indicated ratio in a Transwell co-culture system with or without stimulation with 1,000 U/ml IFN-α for 48 h. (c) Fluorescence confocal microscopy images of the indicated cells that were stimulated with or without 1000 U/ml IFN-α for 12 h and then stained by a rabbit 2

3 anti-stat1 antibody and an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (green) and DAPI. The nuclei were counter-stained with DAPI (blue). (d) Luciferase assay of ISRE promoter activity in the indicated cells that were transduced with lentiviral vectors expressing scrambled or STAT1 shrna, followed by treatement with or without 1,000 U/ml IFN-α for 36 h. (e) Luciferase assay of the indicated cells from mice that were hydrodynamically injected with CMV promoter- or liver cell type-specific promoter-driven luciferase expression plasmids (10 μg each), together with 1 μg of prl-tk (internal control) for 48 h. (f) Fluorescence confocal microscopy images of macrophages stained with a fluorescent anti-f4/80 antibody in the livers of mice that were or were not administrated BMDMs 48 h after treatment with or without liposomal clodronate for an additional 72 h. The data are the mean ± standard error of the mean (SEM) from two independent experiments (d, e). For the image panels, representative images from one (e) or two (b, c) independent experiments are presented. **p < 0.01 (Student s t-test). The numbers below panels represent relative intensities. MΦ, macrophage; RC, HBV relaxed circular DNA; SS, HBV single-stranded DNA. 3

4 Supplementary Figure 2 Supplementary Figure 2. Exosome biogenesis is required for the cell-to-cell transfer of IFN-α-induced anti-hbv activity in vitro (a) Bradford assay of the total amount of proteins (top) and immunoblot analysis of the expression of exosomal proteins CD63 and LAMP2 (bottom) in purified exosomes from the cell culture supernatants of LSECs that were treated with the indicated amounts of the exosome release inhibitors GW4869 or spiroepoxide or with control vehicle (DMSO) for 48 h. (b) Southern blot analysis of the secreted enveloped viral DNA (top) and intracellular viral replicative DNA (bottom) from HepG cells that were co-cultured with LSECs and treated with an exosome release inhibitor as in a or with an inhibitor of shedding vesicle release (imipramine, 10 μm) or of apoptotic body release (DEVD, 60 μm). The HepG cells were then exposed to 1,000 U/ml IFN-α for 48 h. 4

5 (c) Bradford assay of the total amount of protein in purified exosomes (top) and immunoblot analysis of nsmase2 and β-actin (loading control) expression in cell lysates and of CD63 and LAMP2 expression in purified exosomes (bottom) from LSECs that were transduced with scrambled or nsmase2 shrna and with or without sirna-resistant nsmase2 cdna. (d) Southern blot analysis of HBV replication and production in HepG cells co-cultured with LSECs that were transduced with lentiviral vectors as in c, followed by treatment with or without 1,000 U/ml IFN-α for 48 h. (e) Evaluation of the amount of exosomes from LSECs that were transduced with scrambled or Rab27a shrna and with or without sirna-resistant Rab27a cdna, as described in c. (f) Evaluation of HBV replication and production in HepG cells co-cultured with LSECs that were transduced with lentiviral vectors as described in e, followed by treatment with or without 1,000 U/ml IFN-α for 48 h. The data are the mean ± SEM from two (c, e) or three (a) independent experiments. For the image panels, representative images from two (c-f) or three (a, b) independent experiments are presented. *p < 0.05, **p < 0.01 (Student s t-test). 5

6 Supplementary Figure 3 Supplementary Figure 3. Exosomes are mediator of the transfer of IFN-α-induced antiviral response in vitro (a) Southern blot analysis of the secreted enveloped viral DNA (top) and intracellular viral replicative DNA (middle) from HepG cells cultured with IFN-α-treated or untreated, LSEC-derived cell culture supernatants that had been incubated with biotinylated antibodies against CD63 or a control IgG and immunodepleted using streptavidin beads. An immunoblot analysis of the CD63 immunoprecipitate for the exosomal protein LAMP2 is also included (bottom). (b) Electron microscopy of the purified exosomes from IFN-α-treated or untreated LSECs. The scale bar represents 100 nm. (c) Comparison of exosome and non-exosome markers in exosomes (5 μg/well) prepared from IFN-α-treated or untreated LSECs and in the corresponding whole-cell 6

7 lysates (15 μg/well, intracell) by immunoblot analysis using a range of antibodies, as indicated. (d) Analysis of density of exosomes prepared from IFN-α-treated or untreated LSECs by probing for exosomal proteins (LAMP2 and β-actin) in fractions from a sucrose gradient ultracentrifugation. (e) Northern blot analysis of viral RNAs (top) and Southern blot analysis of the intracellular viral replicative DNA (middle) and secreted enveloped viral DNA (bottom) from HepG cells that were treated with an increasing concentration of exosomes (μg/ml) from IFN-α-stimulated LSECs for 48 h. (f) Antiviral activity of the cell culture supernatants of IFN-α-treated LSECs and the indicated pellets and final supernatant (top and middle) from the sequential centrifugation of equivalent cell culture supernatants of IFN-α-treated LSECs at 300 g for 10 min, 2,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min. An immunoblot analysis of aliquots for the exosomal protein LAMP2 is also included (bottom). Representative images from two (a, d, f) or three (b, c, e) independent experiments are presented. 3.5 kb, HBV pregenomic RNA; 2.4 kb, HBV pre-s1/s RNA; 2.1 kb, HBV pre-s2/s RNA; RC, HBV relaxed circular DNA; SS, HBV single-stranded DNA. 7

8 Supplementary Figure 4 Supplementary Figure 4. Effects of exosomes from LNPCs on HCV replication (a, b) Immunoblot analysis of expression of viral proteins NS3, NS5A and core (a) and qrt-pcr analysis of viral RNA levels (b) in Huh7 HCV subgenomic replicon cells that were treated with 5 μg/ml exosomes from IFN-α-treated or untreated macrophages and LSECs for 48 h. (c, d) Luciferase assay of the cell culture supernatants (c) and immunoblot analysis of expression of viral proteins NS3 and core in cell lysates (d) from Huh7.5 cells that were infected with a cell culture infectious virus (HCVcc) expressing a secreted Gaussia luciferase reporter, followed by treatment with exosomes, as described in a, b. (e, f) The cell apoptosis (e) and cell viability (f) of the Huh7 cells treated with exosomes, as described in a, b, were determined by measuring the PARP-1 cleavage and by the CCK-8 cell viability assay, respectively. The data are the mean ± SEM from two independent experiments (b, c, f). For the 8

9 image panels, representative images from two independent experiments are presented (a, d, e). **p < 0.01 (Student s t-test). 9

10 Supplementary Figure 5. Supplementary Figure 5. IFN-α treatment changes the cargo contents of exosomes (a) Immunoblot analysis of APOBEC3G, PKR, and MyD88 expression in whole-cell lysates (top, 15 μg/well) and purified exosomes (bottom, 5 μg/well) from LSECs that were exposed to 1,000 U/ml IFN-α for 48 h. (b) qrt-pcr analysis of the indicated mrnas and mirnas in purified exosomes from LSECs that were exposed to 1,000 U/ml IFN-α for 48 h. The results are presented as fold change (2 -ΔΔCt ) relative to the results for the exosomes from unexposed cells. (c) Immunoblot analysis the indicated proteins and Northern blot analysis of the 10

11 indicated RNAs in fractions from a sucrose gradient ultracentrifugation of IFN-α-treated or untreated, LSEC-derived exosome preparations. (d, e) qrt-pcr analysis of the indicated mrnas and mirnas in LSECs (d) and macrophages (e) that were exposed to 1,000 U/ml IFN-α for 12 or 36 h. The results are presented as the fold change (2 -ΔΔCt ) relative to the results for the unexposed cells. (f) Immunoblot analysis of the expression of APOBEC3G (top) and PCR analysis of HBV DNA at a denaturation temperature of 94 C or 88 C (bottom) in HepG cells that were incubated with increasing amounts of exosomes from IFN-α-stimulated LSECs for 24 h. (g) Immunoblot analysis of the translation products of the indicated exosomal mrnas in HepG cells that were transfected with increasing amounts of exosomal RNA from IFN-α-stimulated LSECs for 24 h. (h) Luciferase activity of the indicated mirna seed-site reporters in HepG cells that were transfected with 0.2 μg each of the reporter plasmids and 20 ng prl-tk for 12 h and then incubated with increasing concentrations of exosomes from IFN-α-stimulated LSECs for an additional 48 h. The data are the mean ± SEM from two independent experiments (b, d, e, h). For the image panels, representative images from two independent experiments are presented (a, c, f, g). *p < 0.05, **p < 0.01 (Student s t-test). 11

12 Supplementary Figure 6. Supplementary Figure 6. Differentially expressed exosomal molecules are responsible for the exosome-mediated antiviral activity of IFN-α (a) Immunoblot analysis of APOBEC3G expression in exosomes (top) and whole-cell lysates (bottom) from LSECs that were transduced with lentiviral vectors to induce the expression of scrambled or APOBEC3G shrna. (b) Southern blot analysis of the secreted enveloped viral DNA (top) and intracellular viral replicative DNA (bottom) from HepG cells that were incubated with 5 μg/ml IFN-α-stimulated, LSEC-derived exosomes with or without APOBEC3G knockdown for 48 h. (c, d) Southern blot analysis of HBV replication and production in HepG cells that were treated with 10 μg/ml cycloheximide for 2 h (c) or transfected with the indicated mirna inhibitors at 50 nm, either alone or in combination, for 12 h (d) 12

13 before incubation with exosomes (5 μg/ml) from IFN-α-stimulated or unstimulated LSECs for an additional 24 h (c) or 48 h (d). (e) Immunoblot analysis of the intended proteins in HepG2 cells that were transfected with 4 μg of individual expression vectors for the indicated mrnas and 2 of μg phbv1.3 using an anti-myc antibody. The arrowheads indicate the position of the intended proteins. For the image panels, representative images from two (a, e) or three (b-d) independent experiments are presented. 13

14 Supplementary Figure 7. Supplementary Figure 7. IFN-α induces the cell-to-cell transfer of antiviral molecules (a) Fluorescence confocal microscopy images of HepG cells that were incubated with 5 μg/ml PKH67- or SYTO RNASelect-labeled exosomes from IFN-α-stimulated or unstimulated LSECs or with the same volume of PKH67- or SYTO RNASelect-labeled PBS, which served as a negative control, for 2 h. (b) Fluorescence confocal microscopy images of HepG cells that were treated with 5 μg/ml PKH67- or SYTO RNASelect labeled exosomes (pre-incubated with 2 μg/ml Annexin V or BSA for 2 h) from IFN-α-stimulated LSECs for 2 h. (c) Southern blot analysis of the secreted enveloped viral DNA (top) and intracellular 14

15 viral replicative DNA (bottom) from HepG cells that were treated with exosomes (5 μg/ml; pre-incubated with 2 μg/ml annexin V or BSA for 2 h) from IFN-α-stimulated or unstimulated LSECs for 48 h. (d) Immunoblot analysis of the expression of APOBEC3G in HepG cells (cultured alone or co-cultured with LSECs) and LSECs that were incubated with or without 1,000 U/ml IFN-α for 48 h in the presence or absence of 10 μm GW4869 or 2 μg/ml annexin V. (e) qrt-pcr analysis of the expression of the indicated mrnas in HepG cells and LSECs treated as described in d. The results are presented as the fold change (2 -ΔΔCt ) relative to the results for the HepG cells that were cultured alone. (f) The luciferase activity of the mirna-638 seed-site reporter in HepG cells and LSECs that were transfected with these reporters for 12 h and then treated as described in d. (g) Immunoblot analysis of APOBEC3G expression detected by an anti-gfp antibody in the purified exosomes and corresponding cell lysates of BMDMs that were transduced with a lentivirus expressing EGFP-APOBEC3G alone or in combination with a lentivirus expressing scrambled or nsmase2 shrna, followed by stimulation with or without 1,000 U of mouse IFN-α for 48 h. The data are the mean ± SEM from two independent experiments (e, f). For the image panels, representative images from two independent experiments are presented (a-d). *p < 0.05, **p < 0.01 (Student s t-test). 15

16 Supplementary Figure 8 Supplementary Figure 8. Inhibiting exosome release by Rab27a shrna weakens the antiviral activity of endogenously produced IFN-α in vivo (a, b) Determination of ALT levels (a) in the serum and MHV-A59 titers (b) in the liver and brain tissues from mice that were hydrodynamically injected with a mouse Rab27a shrna expression construct for 24 h before an injection with 5 pfu of MHV-A59 alone or in combination with IFN-α/ -neutralizing antibodies or control antibodies for an additional 48 h. (c) Bradford assay of the total amount of protein in purified exosomes (top) and immunoblot analysis of nsmase2 and β-actin expression in the tissue lysates and of LAMP2 expression in the purified exosomes (bottom) from the livers of mice that were infected with a lentivirus expressing either scrambled or mouse Rab27a shrna 16

17 (10 6 viruses/mice) by intranasal inhalation for 72 h. (d) qpcr analysis of adenovirus genome copies in the lung tissues from mice that were infected with a lentivirus expressing either the scrambled or mouse Rab27a shrna (10 6 viruses/mice) by intranasal inhalation for 24 h, followed by an intravenous injection with IFN-α/ -neutralizing antibodies or control antibodies and an intranasal infection with 10 9 pfu of MHV-A59 for an additional 72 h. The data are the mean ± SEM from two independent experiments (a-d). **p < 0.01 (Student s t-test). For the image panels, representative images from two independent experiments are presented (c). 17

18 Supplementary Figure 9 Supplementary Figure 9. The mechanistic model of exosome-mediated antiviral activity of IFN-α IFN-α induces the transfer of antiviral molecules from LNPCs to HBV-replicating hepatocytes via exosomes, which bypasses the viral inhibition of IFN-induced antiviral gene expression and results in the inhibition of HBV replication. 18

19 Supplementary Table 1. Differentially expressed mrnas and mirna in the exosomes from IFN-α-stimulated and unstimulated LSECs by microarray Table is too large to be displayed with the supplementary material and can be downloaded as an excel spreadsheet. 19

20 Supplementary Table mrnas and 3 mirnas selected for the confirmation of microarray data and antiviral activity analysis Gene symbol Gene title Genbank or mirbase accession no. Fold change P-value IFI6 Interferon alpha-inducible protein 6 NM_ ALDH3A2 TNFAIP8L2 Homo sapiens aldehyde dehydrogenase 3 family, member A2 Homo sapiens tumor necrosis factor, alpha-induced protein 8-like 2 NM_ NM_ CALHM1 Homo sapiens calcium homeostasis modulator 1 NM_ AGBL4 Homo sapiens ATP/GTP binding protein-like 4 NM_ TRAF4 Homo sapiens TNF receptor-associated factor 4 NM_ FABP5 Homo sapiens fatty acid binding protein 5 NM_ DDIT3 Homo sapiens DNA-damage-inducible transcript 3 NM_ USP20 Homo sapiens ubiquitin specific peptidase 20 NM_ IFITM1 Homo sapiens interferon induced transmembrane protein 1 NM_ GTPBP2 Homo sapiens GTP binding protein 2 NM_ hsa-mir-638 MI hsa-mir-4284 MI hsa-mir-1260 MI

21 Supplementary Table 3. Antibodies used in this study Antibody name Catalogue number Origin anti-nsmase2 sc anti-eea1 sc anti-lamp2 sc SantaCruz anti-pkr sc-707 anti-myc sc-40 anti-cd63 sc-5275 or CBL553 SantaCruz or Millipore anti-apobec3g sc or AP SantaCruz or Proteintech anti-tsg AP anti-rab27a AP anti-alix AP anti-ifi AP anti-tnfaip8l AP Proteintech anti-ddit AP anti-ifitm AP anti-alb AP anti-cytochrome C 4280S anti-stat Cell Signaling anti-phospho-stat1 (Tyr701) 9167 Technology anti-hsp anti-grp S anti-β-actin A2228 anti-myd88 SAB anti-tubulin T5168 Sigma-Aldrich anti-gfp G6539 anti-f4/80 ab6640 Abcam 21

SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURES Supplementary Figure 1. (A) Left, western blot analysis of ISGylated proteins in Jurkat T cells treated with 1000U ml -1 IFN for 16h (IFN) or left untreated (CONT); right, western