Viral Activation of Heparanase Drives Pathogenesis of Herpes Simplex Virus-1  Alex M. Agelidis, Satvik R. Hadigal, Dinesh Jaishankar, Deepak Shukla  Cell Reports  Volume 20, Issue 2, Pages 439-450 (July 2017) DOI: 10.1016/j.celrep.2017.06.041 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Overexpression of Constitutively Active HPSE Worsens Disease in Corneal HSV-1 Infection (A) Schematic showing GS3-HPSE expression variant, in which the linker of the inactive 65-kDa form is replaced with a triple repeat of Gly-Ser to produce a constitutively active HPSE. Protein expression is confirmed in the HCE cell line with anti-HPSE PT-16673. EV, empty vector. (B) Representative micrographs of mice 7 days after HSV-1 (McKrae; 106 PFUs) corneal infection. Scale bar, 1 mm. (C) Clinical scores based on corneal opacity of mice in (B). (D) Representative micrographs of corneal surface of mice 14 days after HSV-1 (KOS; 106 PFUs) corneal infection. Scale bar, 1 mm. (E–G) Analysis of ipsilateral draining (submandibular) lymph nodes of mice 7 days after HSV-1 (McKrae) infection. (E) Representative flow cytometry data showing surface staining of F4/80 versus Gr-1, CD3 versus CD45, and CD4 versus CD8. Percentages of parent gates are labeled on each plot. (F) Total numbers of cells in each mouse lymph node. (G) Numbers of each cell type present in the draining lymph node. Asterisks denote a significant difference as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figures S1, S2, and S6. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Active HPSE Delays Corneal Wound Healing In Vivo and In Vitro (A) Representative micrographs of in vivo corneal wound healing assay in murine corneas previously transfected with GS3-HPSE or empty vector, in the absence of infection. At specified time points post-application of the circular wound, fluorescein was applied to highlight tissue damage (arrowheads), and corneas were imaged. Scale bar, 1 mm. (B) Mean ± SEM of extent of wound healing in (A). (C) Representative micrographs of in vitro wound healing assay showing closure of HCE cellular defect over specified times after scratching. Before wound application, cells were transfected with empty vector, WT HPSE (not depicted), or GS3-HPSE. Scale bar, 50 μm. (D) Quantification of extent of wound healing in (C). Pixel distances between wound fronts in each panel were measured, and percentages of initial wound widths are plotted. (E and F) The same procedure described in (C) and (D) was performed with the exception that HCE cells were infected with HSV-1 (KOS; MOI = 0.1) at the time of wound application. Scale bar, 50 μm. All results are presented as mean ± SEM of three independent experiments (n = 3). Asterisks denote a significant difference as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figure S3. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 HPSE Inhibits Type I Interferon Signaling (A) Transcript copy numbers relative to GAPDH from HCE cells transfected with specified HPSE variants and HSV-1 infected. (B) Luciferase assay results from HCE cells co-transfected with HPSE variants and pIFNb-luc and infected for specified time points. (C) Transcript copy numbers relative to GAPDH obtained as described in (A). (D) Representative western blot analysis of whole-cell lysates of HCE cells transfected with specified HPSE variants in the absence or presence of HSV-1. (E) Densitometry analysis of (D). (F) Transcript copy numbers relative to GAPDH from wild-type and HPSE KO MEFs in the absence or presence of HSV-1 for specified time points. (G) Representative western blot analysis of whole-cell lysates of wild-type and HPSE KO MEFs in the absence or presence of HSV-1 for specified time points. (H) Densitometry analysis of (G). All results are presented as mean ± SEM of three independent experiments (n = 3). Asterisks denote a significant difference compared to empty vector (EV) or WT MEF where applicable, for each time point, as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figures S3 and S6. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 HPSE Translocates to Nucleus upon Infection and Drives Pro-inflammatory Factor Production and Nuclear Translocation of NF-κB (A) Transcript copy numbers of HCE cells transfected with specified HPSE variants or transfected and HSV-1 infected for 24 hr. (B and C) Representative immunofluorescence micrographs of HCE cells mock treated or HSV-1 infected for 36 hr. (B) HPSE (red) and (C) HS (green) are shown with respect to DAPI stain of nucleus (blue). Scale bars, 10 μm. (D) Representative western blot analysis of cytoplasmic and nuclear extracts of HCE cells transfected with empty vector or GS3-HPSE. GAPDH and histone H3 reflect cytoplasmic and nuclear content, respectively. (E) Densitometry analysis expressed as fold change compared to EV mock samples, mean ± SEM of three independent experiments (n = 3). (F) Representative immunofluorescence micrographs of HCE cells transfected with empty vector or GS3-HPSE and HSV-1 infected for 24 hr. Nuclear translocation (arrowheads) of NF-κB p65 (green) is shown relative to DAPI (pseudocolored red). Scale bars, 10 μm. All results are presented as mean ± SEM of three independent experiments (n = 3). Asterisks denote a significant difference compared to empty vector (EV) for each time point, as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Viral Upregulation of Cellular HPSE Can Be Driven by HSV-1 ICP34.5 (A) Copy number of HPSE transcripts relative to GAPDH in HCE cells infected with HSV-1 (strain 17) parental strain or 17Δγ34.5 mutant virus lacking ICP34.5 expression or mock treated. (B) Luciferase induction in HCE cells transfected with HPSE-luc plasmid and then infected for 24 hr with HSV-1 (strain 17) parental strain or 17Δγ34.5 mutant virus or mock treated. (C) Luciferase induction in HCE cells co-transfected with HPSE-luc plasmid and empty vector (EV) or γ134.5 plasmid and then assayed at specified time points. (D) Representative immunofluorescence micrographs of porcine corneas infected for 24 hr with HSV-1 (strain 17) parental strain or 17Δγ34.5 mutant virus and then stained for HSV-1 (green), HPSE (red), and DAPI (blue). Antibody staining controls are presented in Figure S4. Scale bar, 100 μm. All plotted results are presented as mean ± SEM of three independent experiments (n = 3). Asterisks denote a significant difference as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figure S4. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Pharmacological Inhibition of HPSE by OGT 2115 Blocks Progression of HSV-1 Infection (A) Transcript fold expression relative to DMSO mock, normalized to GAPDH, in HCE cells mock treated or infected with HSV-1 (KOS) for 24 hr. (B) Representative fluorescence micrographs of HCE cells infected with GFP-HSV-1 (K26-GFP; MOI = 0.1) in the presence of DMSO or OGT 2115. Scale bar, 50 μm. (C) Representative western blot of cytoplasmic and nuclear extracts of HCE cells mock treated or infected for 24 hr with HSV-1 (KOS) in the presence of DMSO or OGT 2115. GAPDH and histone H3 reflect cytoplasmic and nuclear content, respectively. (D) Representative plaque assay and quantification of virus released into supernatants of HCE cells infected with HSV-1 (KOS; MOI = 0.1) in the presence of DMSO or OGT 2115 at the specified concentrations. Lactate dehydrogenase (LDH) cytotoxicity assay results are shown at right. (E) Representative fluorescence micrographs of ex vivo porcine corneas infected with GFP-HSV-1 (17-GFP) in the presence of DMSO or OGT 2115; quantification of fluorescence at right. Scale bar, 200 μm. Tx, treatment. All results are presented as mean ± SEM of three independent experiments (n = 3). Asterisks denote a significant difference as determined by Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figures S5 and S6. Cell Reports 2017 20, 439-450DOI: (10.1016/j.celrep.2017.06.041) Copyright © 2017 The Author(s) Terms and Conditions