Analysis of Autophagy in HSV-1 Infection of Fibroblast Cells.

<p>A. MEFs were transfected with LC3-GFP. After 24 hr, the cells were uninfected or infected with different HSV-1. At 2 or 24 hpi, the cells were imaged using confocal microscopy. B. Quantification of the number of LC3-GFP punctae per cell in (A); about 30 cells per sample were counted.</p

Analysis of Autophagy in HSV-1 Infection of HeLa Cells.

<p>A. HeLa cells were uninfected (mock), or infected with different strains of HSV-1 (KOS or McKrae). At 2 hpi, the cells were harvested, lysed, and the lysate was immunoblotted for LC3 levels. B. Uninfected or infected HeLa cells were harvested at 6 hpi, lysed, and immunoblotted. C. Immunoblotting of Uninfected or infected HeLa cells at 12 hpi. D. Immunoblotting of Uninfected or infected HeLa cells at 30 hpi. E. Quantification of LC3-II levels in (A-D), normalized to the housekeeping protein GAPDH.</p

Analysis of Autophagy in HSV-1 Infection of Corneal Epithelial Cells.

<p>A. HCE cells were uninfected or infected with HSV-1 at the indicated MOIs. SQSTM1/p62 and LC3 were immunoblotted at 2 hpi. Quantification of band intensities was performed using digital band quantification software (ImageQunatTL; GE). B. Uninfected or infected HCE cells were immunoblotted at 8 hpi.</p

Analysis of Autophagy in HSV-1 Infection of Retinal Ganglion Cells.

<p>A. Experimental layout. B. RGC5 cells were transfected with HcRed-LC3. After 24 hr, the cells were mock-treated, treated with MG132, or infected with HSV-1 for 24 hr. The cells were washed in presence of saponin, and analyzed cytofluorimetrically. C. Quantitation of the relative levels of autophagosomal LC3-II in (B), calculated using integrated MFI of the gated region R6. D. Quantification of autophagosomal LC3-II (from saponin-treated cells), shown as % of total LC3 (non saponin-treated cells), as a function of infection with various MOIs of HSV-1 in RGC5 cells.</p

Syndecan-1 knockdown or overexpression do not affect cell viability.

<p>(A). CHO-K1, CHO-745, and HCE cells were transfected with scrambled (scr) siRNA or syndecan-1 (SDC1) siRNA. 72–96 h after transfection, immunoblots of cell lysates were prepared and probed with anti-SDC1 polyclonal Ab. β-actin protein level was measured as loading control. Representative blots are shown. Protein bands were quantified using ImageQuant TL image analysis software (version: 7). SDC1 protein expression (mean ± 1SD), normalized to that of β-actin, of at least three independent experiments was quantified by calculating the relative intensity of each syndecan-1 band relative to the control scrambled siRNA treated bands, and presented as bar graph. (B) Cells were grown in 6-well plates, mock treated or transfected with human SDC1 plasmid for 48 h. Cell surface level of SDC1 was evaluated by flowcytomety. FITC stained cells were used as background control. Results are representative of two independent experiments (C, D). Cells were grown in 96-well plates, transfected with scrambled siRNA or SDC1 siRNA for 48 h (C), or transfected with control GFP plasmid or human SDC1 plasmid for 24 h (D). Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to scrambled siRNA transfected cells (C), or relative to GFP transfected cells (D), and are mean ± 1SD of at least 2 independent experiments.</p

Comparison of syncytia number and nuclei count after syndecan-1 overexpression in each CHO cell type.

<p>Average number of syncytial cells, as well as, the average number of nuclei per syncytia was counted in CHO-K1 and CHO-745 cells after overexpressing syndecan-1 on target cells or effector cells. Positive controls are target and effector cells expressing normal levels of syndecan-1. Syncytial cells were counted 72 h post mixing. Syncytia were classified as any red cell having two or more nuclei. Number of syncytia was normalized to the number of syncytia detected in the negative control wells where the effector cell population lacks gB. The average is based on results from two independent experiment performed in duplicate (mean ± 1SD).</p

Downregulation of Syndecan-1 results in reduced production of infectious virus.

<p>HCE cells were transfected with either control scrambled siRNA or syndecan-1 siRNA. 72 h post-transfection cells were infected with HSV-1 (KOS) at an MOI of 0.1. At 0, 5, 24, 48, and 72 h post-infection, infectious virus was quantified by a standard plaque assay on HCE cell monolayers. The titers shown are the mean ± 1 SD of a representative experiment of two independent experiments performed in duplicates. <i>SDC1</i>, syndecan-1.</p

Syncytial cell formation in CHO-K1 and CHO-745 after syndecan-1 overexpression.

<p>(A). An illustration of syncytia assay that was exploited to understand the contribution of syndecan-1 during HSV-1 induced cell-to-cell fusion. Effector cell population that expresses HSV-1 fusion glycoproteins, T7 polymerase as well as a RFP-NES construct that restricts the expression of RFP to the cytoplasm is mixed with the target cell population that expresses nectin-1 as a gD receptor, the luciferase reporter gene under the control of T7 promoter as well as a CFP-NLS construct that restricts the level of CFP to the nucleus. (B). Syncytia formation was observed in cells 72 h after the mix of target cells with effector cells. <i>Top panels</i> show representative syncytia formed in CHO-K1 cells after overexpressing syndecan-1 in target, or effector cell. <i>Bottom panels</i> show representative syncytia formed in CHO-745 cells after overexpressing syndecan-1 in target, or effector cells. Positive controls are target cells mixed with effector cells where both populations have the wild-type level of syndecan-1 on the surface. Negative controls are target cells mixed with effector cells missing gB. (Scale bar = 37.5 µm). <i>SDC1</i>, syndecan-1.</p

Syndecan-1 knockdown reduces plaque formation in HCE cells.

<p>(A). Monolayers of HCE cells were transfected with either control plasmid GFP, or with human syndecan-1. 24 h post-transfection cells were infected with serial dilution of HSV-1(KOS) stocks. (B). 50% confluent HCE cells were transfected with either control scrambled siRNA or syndecan-1 specific siRNA. 72 h post-transfection, cells were infected with serial dilution of HSV-1(KOS) stocks. (A, B). 72 h post-infection cells were fixed and stained with crystal violet stain. Infectivity was measured by the number of plaque forming units (PFUs). Number of PFUs was counted at the 10× objective (Zeiss Axiovert 200). Plaques consist of 15 or more nuclei were counted. Results are means ± 1 SD of three independent experiments conducted in duplicate. <i>SDC1</i>, syndecan-1.</p

syndecan-1 ectodomain and cytoplasmic domains are important for inhibiting cell fusion when overexpressed on effector cells.

<p>(A). Syndecan-1 truncation and mutants used in the study are illustrated including the full-length wild type (<i>wt</i>) core protein syndecan-1 (SDC1) that includes an extracellular domain, transmembrane domain (TM), and COOH-terminal cytoplasmic domain. Also illustrated are the construct FcR^ectohS1 that is a chimera comprised of the ectodomain of human IgG Fcγ receptor Ia/CD64 fused to the transmembrane and cytoplasmic domains of human syndecan-1, the construct hS1^pLeu™ that has the transmembrane domain replaced with leucine residues, and a truncation mutant hS1^Δcyto that lacks the 33 C-terminal amino acids. (B) Cells were grown in 96-well plates, transfected with control GFP plasmid, full-length <i>wt</i> human SDC1 plasmid, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto for 24 h. Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to control GFP plasmid transfected cells, and are mean ± 1SD of at least 3 independent experiments. (C). Effector cells for CHO-K1 and CHO-745 cells were transfected with either control GFP plasmid, full-length <i>wt</i> syndecan-1, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto and mixed with the target cells 24 h post-transfection. Fusion was measured 16 h post mixing. Results are presented as mean ± 1 SD of at least 3 independent experiments. As a negative control, target cells were mixed with effector cells lacking HSV-1 gB.</p