rIDE enhances VZV infectivity and increases the stability of virus.

<p>(A) Cell-free ROka-LacZ was pre-incubated with rIDE or BSA at 25°C for the indicated time before infecting MeWo cells. The ratio of the number of blue foci in cells infected with virus treated with rIDE or BSA(control) was determined. The experiment was preformed 11 times and a representative experiment is shown. Vertical lines indicate standard deviations. (B) Pre-treatment of VZV with rIDE at 4°C enhances viral infectivity. Cell-free ROka-GFP virus was incubated with medium, BSA, rIDE or denatured rIDE (rIDE treated with acid followed by base to a final neutral pH) for 60 min at 4°C before infecting cells. (P = 0.001 for rIDE vs. denatured rIDE, Student t test). Vertical lines indicate standard deviations. The experiment was performed 3 times, and a representative result is shown.</p

Accumulation of a VZV mutant deleted for the IDE binding domain of gE at the cell surface and at the cell-cell junction.

<p>Melanoma cells were infected with VZV ROka or ROka68D32-71, fixed, and incubated with monoclonal antibody to VZV gE followed by FluoroNanogold-conjugated anti-mouse antibody and visualized by transmission electron microscopy. (A) Representative virions at cell-cell junctions are indicated with arrows. Magnification 12000×. The insets in panel A provide higher-power images of selected virions. (B) Representative virions on the cell surface are indicated with arrows. Magnification 5000×. (C) Quantification of gE-positive virions on the cell surface and at cell-cell junctions. Multiple cells from the experiment in panel A were coded and observed under a Hitachi H7500 transmission electron microscope. gE positive viral particles at cell surface and/or junctions were counted.</p

VZV mutants deleted for the IDE binding domain of gE are impaired for syncytia formation.

<p>(A) Melanoma cells were infected with VZV ROka or ROka68D32-71 and stained with DAPI (top panels) or mouse monoclonal antibody to gE followed by anti-mouse-Alexa-488 and visualized by immunofluorescence microscopy. Magnification 40X. (B) Melanoma cells were infected with VZV ROka-GFP or ROka68D32-71-GFP and visualized directly by fluorescence microscopy. Magnification 40X. (C) Quantification of the number of nuclei in syncytia per nucleus was performed using confocal microscopy (Leica SP2, Leica Microsystems, Exton, PA). Sequential Z-sections of DAPI stained infected cells were acquired for 3D reconstruction of representative cells with Imaris software (version 6.2, Bitplane AG, Zurich, Switzerland). The numbers of nuclei were automatically determined using spot function in Imaris software and manually corrected for errors by independent investigators in Biological Imaging, Research Technologies Branch, NIH. Vertical lines show standard deviations. n represents the number of syncytia analyzed in which nuclei were counted by a microscopist who was unaware of the expected outcome of the experiment.</p

rIDE is expressed in insect cells, degrades insulin, and binds to VZV gE.

<p>(A) Structure of HA-tagged rIDE and its promoter in recombinant baculovirus. (B) rIDE was purified, eluted under native conditions, and stained with Coomassie Blue. Soluble rIDE is expressed as a 110 kDa protein in insect cells. (C) HPLC profiles of insulin degradation fragments observed after co-incubation of radiolabeled insulin with rIDE expressed in insect cells, endogenous IDE extracted from rat liver (top panel), or recombinant IDE (6His/Flag-IDE) expressed in eukaryotic cells (bottom panel). (D) Insulin-degrading enzymatic activities of IDE proteins. (E) ELISA assay for binding of rIDE to gEt, gEt lacking the IDE binding domain (gEtΔ32-71), gEt defective in binding to VZV gI (gEtΔ163-208), and a negative control protein (P7.5, vaccinia 7.5 protein). The horizontal dotted line indicates the level of nonspecific binding based on the control protein.</p

VZV mutant lacking the IDE binding domain of gE shows reduced fusogenicity by membrane fusion assay.

<p>(A) Melanoma cells expressing T7 polymerase were infected with VZV ROka, ROka68D32-71, and the cells were incubated with HeLa cells containing the β–galactosidase gene driven by the T7 promoter or a control GFP plasmid for 20 hr. The cells were lysed, incubated with chlorophenol red-β-D-galactopyranoside, and β–galactosidase activity was measured using a spectrophotometer at OD570 nm. The experiment was also performed at 16 hr and 24 hr and similar results were obtained. Vertical lines indicate standard deviations. (B) Human melanoma cells expressing T7 polymerase were infected with the same MOI of ROka-GFP or ROka68D32-71-GFP, and cells were co-incubated with HeLa cells containing the β–galactosidase gene driven by the T7 promoter for 16 hrs. The cells were lysed, incubated with chlorophenol red-β-D-galactopyranoside, and β–galactosidase activity was measured using a spectrophotometer at OD570 nm. The experiment was performed twice with similar results. (P<0.001 for ROka-GFP vs. ROka68D32-71-GFP, Student t test). Vertical lines indicate standard deviations.</p

Treatment of VZV with rIDE does not increase virus binding.

<p>POka (parental Oka VZV) or mock infected MRC-5 cells were metabolically labeled with ³H-thymidine at 0.25 mCi/ml for 36 hr beginning at 8 hr post-infection, and cell-free virus was prepared by sonication as described above. The virus was then pre-incubated with rIDE or control proteins at 37°C for 30 min before addition to human melanoma cells on ice for 80 min in the presence of 100 µg/ml of heparin (to block binding through cell surface heparin sulfate). After extensive washing with cold PBS, the cells were lysed and radioactivity bound to cells was measured. The number of radioactive counts per minute for mock-infected MRC-5 cells was subtracted from the counts per minute for POka-infected cells (P = 0.1 for rIDE vs. BSA or for rIDE vs. BZLF2, Student t test). The data shown are based on two independent experiments.</p

rIDE modifies gEt and induces a conformational change in gEt.

<p>(A) Biotin labeled gEt-Fc protein (arrow) was incubated with rIDE at the indicated temperature for 30 min. After electrophoresis and transfer onto a nitrocellulose membrane, proteins were visualized with streptavidin-conjugated-horse radish peroxidase. (B) Biotin-labeled gEt-His protein (arrow) was incubated with buffer, control protein BZLF2 or P7.5, rIDE (produced in baculovirus) at 37°C for 30 min, followed by urea for 18 hr and thermolysin for 30 sec. The proteins were separated by electropheresis, transferred to a nitrocellulose membrane and proteins were detected using streptavidin-conjugated horse radish peroxidase. (C) Biotin-labeled gEt-His protein (arrow) was incubated with rIDE or control proteins at 37°C for 30 min, followed by incubation with 4 µg/ml of endoproteinase Asp-N (Roche Applied Science, Indianapolis, IN) at 37°C for 45 sec. The digestion was then terminated by adding 0.5 M EDTA and samples were boiled in SDS-PAGE gel loading buffer with 2.5% 2-mercaptoethanol and separated by electrophoresis. Protein fragments were detected by streptavidin conjugated-horse radish peroxidase. (D) Binding of catalytically inactive IDE mutant protein IDE-E111Q to gEt fails to induce a conformational change in gE. Biotin-labeled gEt-His protein (arrow) was incubated with rIDE (produced by baculovirus), IDE-E111Q (produced in bacteria), or IDE-w. t. (produced in bacteria), or negative control proteins as indicated at 37°C for 30 min, followed by thermolysin and processed as described in panel B. (E) Coomassie Blue stained SDS-PAGE gel showing the amount of IDE-E111Q and IDE-w.t. proteins used for pulse proteolysis assay in panel D.</p

Deletion of IDE binding domain on gE (amino acids 32–71) does not affect myristylation of gE.

<p>ROka or ROka68D32-71 infected cells were radiolabeled with ³H-myristic acid and immunoprecipitated with anti-gE antibody.</p

IDE mutant protein, IDE-E111Q binds to gEt.

<p>gEt but not control protein p7.5, pulled down both IDE-E111Q protein and wild-type IDE (arrow).</p

Distribution of latently infected neurons after cross-serotype infection of LAT-expressing transgenic mice.

<p>Twenty-one or twenty-eight days after ocular inoculation sections of latently infected trigeminal ganglia were assayed by combined FISH/IF. The percentage of LAT-positive (specific for infected HSV type) neurons that co-labeled with monoclonal antibodies A5 or KH10 is presented. The raw data are also presented (dual-labeled neurons/number of LAT-positive neurons evaluated).</p