Alpha-herpesvirus infection induces the formation of nuclear actin filaments.

Virus and cells. The swine kidney epithelial cell line (PK15) was purchased from the American Type Culture Collection (CCL-22). All non-neuronal cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% of fetal bovine serum and 1% penicillin/streptomycin. All PRV stocks were produced in the PK15 cell line. PRV stocks used in this report include PRV Becker, a virulent isolate [ 64 ] and PRV-GS443, a recombinant expressing GFP fused to the VP26 capsid protein [ 31 ]. Infection of mouse SMG. Each 6–8-wk-old C57 B6 mouse was anesthetized with 100 μl of a freshly prepared, sterile filtered solution of ketamine (100 ± 10 mg/kg)/xylazine (10 ± 1 mg/kg) by intraperitoneal (IP) injection. The neck of the mouse, from the base of the chin to just above the ribcage, was shaved using a platinum razor blade. The shaved area was prepared for surgery in the laminar flow hood using aseptic technique by applying disinfectant scrub and swabbing the area with 70% isopropyl alcohol. The mice were at a surgical plane of anesthesia prior to incising the neck region to expose the salivary glands. An approximately 1.5-cm incision was made with a sterile scalpel blade on a scalpel handle, with the skin grasped using forceps in order to ensure a shallow incision. The animal was monitored during the entire surgical procedure; if at any time the animal is no longer in a surgical plane of anesthesia (e.g., increased respiratory rate, movement), then we injected the animal with additional ketamine/xylazine or ketamine alone (up to 10–20 mg/kg) to induce a deeper plane of anesthesia. Four separate 2-l injections of PRV inoculum (diluted in sterile PBS) was injected into the submandibular glands. The incision was closed with 6–0 silk sutures. The mouse was administered an IP injection in the scapular region of 2.0 mg/kg of buprenorphine for prophylaxis against post-surgical pain. At 48 hpi, the mouse was euthanized by CO 2 inhalation and fixed with 4% paraformaldehyde by cardiac perfusion. The salivary glands were surgically removed and the SMG dissected out on Sylgard plates. This experimental protocol related to animal use has been approved by the Institutional Animal Care and Use Committee of the Princeton University Research Board under protocol number 1539-AR1 in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care and those in the Animal Welfare Act (Public Law 99–198). Block-face serial section scanning electron microscopy. SMG were stained in a manner similar to what is described for TEM. Post-staining, the infected ganglia were embedded in Epon resin (EM Sciences). The embedded samples were mounted on an aluminum rivet and trimmed following the procedure given in Denk and Horstman, 2004 [ 25 ]. All data shown were taken on an environmental SBFSEM with a field-emission electron gun (QuantaFEG 200, FEI, Eindhoven, The Netherlands) at a gas (H 2 0) pressure of 23 P, and an electron energy of 3.0 keV. The mounted, trimmed samples were placed on the SEM microtome and sequential images from the block-face were acquired in between cut cycles. The images were taken at a digital resolution of 26 nm/pixel. The data were analyzed using ImageJ 1.32j software (National Institutes of Health). The reslicing of image stacks (see Figure 1 e) was done using the ImageJ Volume Viewer plugin, which interpolates the z-axis data so that the digital resolution matches that of the lateral direction. Neuron culture. Detailed protocols for dissecting and culturing neurons are found in Ch'ng et al. [ 65 ]. Briefly, sympathetic neurons from the SCG were dissected from E15.5 to E16.5 pregnant Sprague-Dawley rats (Hilltop Labs Incorporated, Pennsylvania, United States) and incubated in 250 μg/ml of trypsin (Worthington Biochemicals, Lakewood, New Jersey, United States) for 10 min. 1 mg/ml of trypsin inhibitor (Sigma-Aldrich, St. Louis, Missouri, United States) was added to neutralize the trypsin for 3 min and then removed and replaced with neuron culture medium. Prior to plating, the ganglia were triturated into dissociated neurons using a fire-polished Pasteur pipette and then plated onto glass cover slips in a 35-mm plastic tissue culture dish coated with 500 μg/ml of poly-DL-ornithine (Sigma-Aldrich) diluted in borate buffer and 10 μg/ml of natural mouse laminin (Invitrogen, Carlsbad, California, United States). The neuron culture medium consists of Dulbecco's modified Eagle medium (Invitrogen) and Ham's F12 (Invitrogen) in a 1:1 ratio. The serum-free medium was supplemented with 10 mg/ml of bovine serum albumin (Sigma-Aldrich), 4.6 mg/ml glucose (J. T. Baker), 100 μg/ml of holotransferrin (Sigma-Aldrich), 16 μg/ml of putrescine (Sigma-Aldrich), 10 μg/ml of insulin (Sigma-Aldrich), 2 mM of L-glutamine (Invitrogen); 50 μg/ml or units of penicillin and streptomycin (Invitrogen), 30 nM of selenium (Sigma-Aldrich); 20 nM of progesterone (Sigma-Aldrich) and 100 ng/ml of nerve growth factor 2.5S (Invitrogen). After 2 d post-plating, the neuronal cultures are treated with 1 μM of an antimitotic drug called cytosine β-D-arabinofuranoside (Sigma-Aldrich) to eliminate any non-neuronal cells. The neuron culture medium was replaced every 3 d and cultures were kept in a humidified, CO2 regulated 37 °C incubator. This experimental protocol related to animal use has been approved by The Institutional Animal Care and Use Committee of the Princeton University Research Board under protocol number 1453-AR2 in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care and those in the Animal Welfare Act (Public Law 99–198). Viral infections. Protocols for viral infection of neurons have been described by Ch'ng et al. [ 65 ]. All PRV infections of neuron cultures were carried out under high multiplicities of infection (MOI) unless otherwise stated. Briefly, neurons were cultured on glass cover slips in 35-mm dishes for approximately 2 wk prior to any experiment. The viral inoculum was diluted in 2% fetal bovine serum in Dulbecco's modified Eagle (GIBCO, San Diego, California, United States) and overlaid on the neuronal culture for 1 h in a humidified 37 °C incubator. After 1 h, the viral inoculum was removed and replaced with neuron medium. Infections usually lasted for 15 h (unless otherwise stated) before the samples were fixed and processed for staining and immunofluorescence. The production of infectious virus over time in SCGs has been characterized [ 30 , 66 ]. Antibodies and stains. Antibodies used in this study include mouse monoclonal against lamin-associated protein 2 (LAP2) (BD Biosciences, Palo Alto, California, United States; used 1:500), anti-VP5 (major capsid protein) antibody and anti-GM130 antibody (BD Transduction laboratories; used 1:250). Myosin Va antibody against neuronal rat myosin V was generously provided by Paul Bridgman (Washington University, St. Louis, Missouri, United States) and used at 1:2000. Myosin II antibody against neuronal rat myosin II (Covance) was used at 1:500. Actin was detected by Alexa 568-phalloidin (Molecular Probes, Eugene, Oregon, United States) used at 1:40. All secondary Alexa fluorophores were purchased from Molecular Probes and used at 1:500 dilution. Fluorescence, immunofluorescence, and drug treatments. All fluorescence experiments carried out were performed as follows. Dissociated neurons on glass cover slips were incubated in phosphate-buffered saline containing 3% bovine serum albumin and 0.5% triton for 10 min before the addition of primary antibodies for 1 h. After 1 h, the primary antibodies were removed and the sample was washed three times with phosphate-buffered saline containing 3% bovine serum albumin (and 0.5% saponin when noted in text). Next, secondary antibodies were added to the sample and incubated for 1 h. After 1 h, the secondary antibodies were removed and the sample was washed three times with phosphate-buffered saline containing 3% bovine serum albumin (and 0.5% saponin when noted in text). To stain for filamentous actin, Alexa-568-phalloidin was added to the cover slip at a concentration of 6.6 μg/ml. The cover slip was mounted on a glass slide using Aqua polymount (Polysciences, Warrington, Pennsylvania, United States) and allowed to dry for 24 h prior to imaging. To depolymerize actin, latrunculinA (latA; Molecular Probes) was dissolved in DMSO and added directly to the media at 3 hpi at a final concentration of 5 μM. Jasplakinolide (jasp; EMD Biosciences, Darmstadt, Germany) was dissolved in DMSO and was added directly to the media at 3 hpi at a final concentration of 100 nM. Phosphonoacetic acid (PAA; Sigma-Aldrich) was dissolved in DMSO and added directly to the media at 1 hpi at a final concentration of 400 μM. Cycloheximide was used at 100 μg/ml (10 mg/ml stock dissolved in PBS). Cells were pre-treated with cycloheximide in neuronal media for 1 h and then infected with viral inoculum containing cycloheximide. After 1 h, the viral inoculum was removed and replaced with neuron medium containing cycloheximide. For UV-inactivation, we exposed aliquots of PRV443L strain to UV light using a UV Stratalinker 1800 (Stratagene, La Jolla, California, United States). We used a dosage of UV that reduced the titer by approximately 1,000-fold. Samples were imaged with a Perkin-Elmer (Wellesley, California, United States) RS3 spinning disk confocal microscope side-mounted on a TE200-S Nikon Eclipse microscope (Tokyo, Japan) with an Argon/Krypton laser producing excitation lines of 488, 568, and 647 nms. Optical sections were acquired in 0.1, 0.25, or 0.5 μm steps, as stated. 2-D projections of confocal stacks and channel merges were created by ImageJ 1.32j software (National Institutes of Health). All figures were assembled in Adobe Photoshop 7.0.1. Alterations to image brightness and contrast were conducted in a linear manner and were applied equally to controls, except where otherwise noted. Supplemental video was assembled in ImageJ and converted to QuickTime format. TEM. Whole SCG were cultured for 2 wk on Aclar (EM Sciences) in a manner similar to what is described above for glass cover slips (except without dissociation). Neurons were infected at a high MOI and treated with latA as described above. After 15 hpi, the plates were washed twice with phosphate-buffered saline, fixed with 2% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 4 h, and post-fixed with 1% osmium tetroxide in sodium veronal buffer for 1 h on ice. Samples were then rinsed with sodium veronal buffer four times and incubated with 0.25% toluidine blue in 0.2 M cacodylate buffer (pH 7.2) for 1 h; the staining solution was then removed with four rinses of sodium veronal buffer (pH 7.2), followed by four rinses with 0.05 M sodium maleate buffer (pH 5.1). Overnight incubation with 2% uranyl acetate in 0.05 M sodium maleate buffer was done in the dark followed by four rinses with 0.05 M sodium maleate buffer (pH 5.1). The fixed samples were then dehydrated with ethyl alcohol, embedded in Epon resin (EM Sciences) and cut into 70-nm sections using a Reichert Ultracut E ultramicrotome.

Supporting Information Figure S1 Profile Plots of the GFP-VP26 (Green) and Anti-Myosin II (Blue) Signal Intensities along a Straight Line Intersecting GFP-VP26 Foci through the Nucleus Inset shows the trajectory of the straight line on a merged image of the nucleus. Signal intensity profile plots were obtained using ImageJ and corrected for background noise by subtracting the average intensity of the approximate nuclear area from the profile data. Curve points below zero correspond approximately with points outside the nucleus. Correlation coefficients between GFP-VP26 and myosin II for plots shown: (A) −0.11 and (B) −0.18. (9.9 MB TIF) Click here for additional data file. Video S1 QuickTime Video of a SBFSEM Stack of 65 Serial Sections from an Infected Cell, Sectioned at 50 nm The volume shown is at the inner edge of the nucleus, with the nuclear envelope at the right-hand side of the image. This video is a cropped substack from Video S2 . (3.1 MB MOV) Click here for additional data file. Video S2 QuickTime Video of a SBFSEM Stack Comprising 100 Serial Sections from an Infected Cell, Sectioned at 50 nm Lower half of the cell was not obtained during image acquisition. (944 KB MOV) Click here for additional data file. Video S3 QuickTime Video of a SBFSEM Stack Comprising 150 Serial Sections from an Uninfected Cell, Sectioned at 50 nm (1.4 MB MOV) Click here for additional data file.