Cells and viruses
Vero cells and L7 cells  were propagated in Dulbecco's Modified Eagle's medium (DMEM) containing 0.15% HCO3
- supplemented with 5% fetal bovine serum (FBS), penicillin G (100 U/ml), and streptomycin (100 mg/ml), hereafter referred to as "complete DMEM." Wild-type HSV-1 strains KOS, KOS-GFP, and McKrae were propagated in Vero cells cultured in complete DMEM. The ICP0- virus n212 [53, 54] contains a 14 bp insertion (CTAGACTAGTCTAG) in codon 212 of the ICP0 gene of HSV-1 strain KOS, which inserts stop codons into all three open-reading frames of the ICP0-encoding DNA strand (Fig. 4). The n212 (ICP0-) virus was propagated and titered in ICP0-complementing L7 cells . The ICP4- virus n12 contains a 16 bp insertion (GGCTAGTTAACTAGCC) in codon 262 of the ICP4 gene of HSV-1 strain KOS, which inserts stop codons into all three open-reading frames of the ICP4-encoding DNA strand. The n12 (ICP4-) virus was propagated and titered in E5 cells  (generously provided by Priscilla Schaffer, Harvard University).
KOS-GFP is a recombinant virus derived from HSV-1 strain KOS that expresses green fluorescent protein (GFP) from a CMV promoter-GFP gene cassette inserted in the intergenic region at the 3' ends of the UL26 and UL27 genes, which converge from opposite strands of DNA . To avoid disrupting the 3' untranslated region of either the UL26 or UL27 gene, the intergenic region (Kpn I – Fsp I; 52,733 – 53,150) was duplicated and the CMV-GFP cassette is inserted between these duplicated sequences. No known open-reading frames are disrupted by this 2.0 kbp insertion into the HSV-1 genome.
The ICP0- virus 0--GFP was constructed from a chimeric ICP0-GFP gene , in which the GFP open-reading frame was inserted into a Xho I restriction site in codon 105 of the ICP0 gene (Fig. 4). This mutation was transferred into HSV-1 strain KOS by homologous recombination. Southern blot analysis demonstrated that 0--GFP possessed the desired mutation in both copies of the RL region (Fig. 4). Northern blot analysis confirmed that 0--GFP expresses an ICP0 mRNA that migrates at ~3.1 kb as opposed to the wild-type 2.4 kb ICP0 mRNA transcribed from KOS. 0--GFP is phenotypically identical to the well-established ICP0- null virus n212 [53, 54], based on the following observations: 1. Both n212 and 0--GFP are repressed with identical kinetics in rag2
-/- mice; 2. Both n212 and 0--GFP are virulent in stat1
-/- mice; 3. Both n212 and 0--GFP are hypersensitive to IFN-α/β in vitro ; 4. Only 1% of n212 and 0--GFP virions form plaques on Vero cells [54, 58]; 5. ICP0, but not ICP4 or VP16, provided in trans from adenovirus vectors allow n212 and 0--GFP to efficiently form plaques on Vero cells , and the efficiency of plaque formation is the same as observed on ICP0-complementing L7 cells [14, 31, 52].
HSV-1 infection of mice
Female mice of the strains specified in Table 1 were inoculated with HSV-1 at 6- to 10-weeks of age and were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals. BALB/c mice, BALB/c scid mice, and IFN-γ receptor null (ifngr
-/-) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Strain 129 mice, rag2
-/- mice, stat1
-/- mice, and rag2
-/- mice were obtained from Taconic Farms (Germantown, NY). PML-/- mice were obtained from the National Cancer Institute (Frederick, MD). IFN-α/β receptor-knockout (ifnar
-/-) mice and ifnar
-/- mice were obtained from B & K Universal Ltd. (East Yorkshire, United Kingdom). Rag2
-/- mice were generously provided by Nicole Meissner and Allen Harmsen (Montana State University, Bozeman). Prior to viral inoculation, mice were anesthetized by i.p. administration of xylazine (7 mg/kg) and ketamine (100 mg/kg). Mice were inoculated by scarifying the cornea with a 26-gauge needle and by placing 4 μl complete DMEM containing 2 × 105 pfu of virus on each eye.
Viral titers in the ocular tear film of mice were determined at times after inoculation by swabbing the ocular surface of both eyes with a cotton-tipped applicator, and transferring the tip into 0.4 ml complete DMEM. Viral titers were determined by a 96-well plate plaque assay on the appropriate cell line cultured in complete DMEM containing 0.5% methlycellulose (i.e., Vero cells for wild-type HSV-1, L7 cells for ICP0- mutants, and E5 cells for ICP4- mutants).
Titers of infectious virus in homogenates of whole eyes, trigeminal ganglia (TG), or hindbrain were determined by homogenizing tissues in 0.5 ml complete DMEM with a Pro 200 homogenizer (Pro Scientific, Oxford, CT), removing cell debris via centrifugation, and titering 10-fold serial dilutions of clarified supernatant on 24-well plates containing Vero or L7 cells. For all plaque assays, virus-infected cells were cultured in complete DMEM containing 0.5% methlycellulose for two to three days before staining with 20% methanol and 0.5% crystal violet.
GFP fluorescence in eyes, TG, and brains of mice infected with KOS-GFP was visualized on a Nikon TE2000 inverted fluorescent microscope (Nikon Instruments, Lewisville, TX) using a Jenoptik ProgRes C10+ Digital Camera (JenOptik Laser, Jena, Germany). Images were collected at 2× or 4× magnification using identical exposure conditions within a given comparison group, and composite images of the brain were created by stitching together photographs that covered the ventral surface of the brain using the graphics editor, Paint Shop Pro (Jasc Software, Eden Prairie, MN). GFP fluorescence in the eyes of living mice was obtained by placing anaesthetized mice on a petri dish on the stage of the microscope.
Analysis of HSV-1 replication in vitro
Cultures of Vero cells were established in 12-well plates at a density of 2 × 105 cells per well and were cultured in complete DMEM. Cells were treated 24 hours later by replacing the culture medium with complete DMEM containing no IFN or 200 U/ml IFN-β (PBL Biomedical Laboratories, Piscataway, NJ). Sixteen hours later, Vero cells were inoculated with 2.5 pfu per cell of wild-type HSV-1 strain KOS, 0--GFP, or the ICP4- virus n12. After allowing 45 minutes for adsorption, the inoculum was replaced with complete DMEM containing no IFN or 200 U/ml IFN-β. Cultures were harvested 4.5, 9.0, 13.5, or 18.0 hours after inoculation by transfer to a -80°C freezer. Upon thawing, viral titers were determined by plaque assay on appropriate indicator cells.
Dual labeling of trigeminal ganglion sections for LAT RNA and viral proteins
Mice were euthanized and perfused with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. The six TG from each group of mice were immersed in 4% paraformaldehyde for 1 hour, equilibrated with 30% sucrose, embedded in Tissue-Tek® O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and frozen in liquid nitrogen. Each block of tissue containing the six TG from one group of mice was cut into ~160 sections of 7 μM thickness. Slides were processed to label 1. LAT+ neurons with rhodamine, and 2. HSV antigen+ neurons with fluorescenin, as follows. LAT riboprobes, specific for bases 119629–119975 of HSV-1, were prepared at 37°C with digoxigenin RNA Labeling Mix (Roche, Mannheim, Germany). LAT riboprobe synthesis reactions were treated with DNase I and filtered through a Sephadex G-50 column prior to use. Tissue sections were incubated in a pre-hybridization buffer, and then hybridized to LAT-specific riboprobes by an overnight 45°C incubation. Tissue was washed with 2× SSC, treated with 20 μg/ml of RNase A in 2× SSC at 37°C, followed by serial washes in 0.5× SSC and 0.1× SSC. Tissue sections were equilibrated with 0.1 M PBS, and were sequentially incubated with 1. rhodamine-labeled anti-digoxigenin Fab fragments, 2. 3% normal rabbit serum, and 3. fluorescein-labeled rabbit polyclonal anti-HSV antibodies (DAKO Cytomation, Carpinteria, CA) diluted 1:100 in 1% normal rabbit serum. Fluorescent-labeled tissue sections were then washed with 0.1 M PBS, and mounted under cover slips.
Analysis of HSV-1 reactivation in trigeminal ganglion explants
Latently infected mice were sacrificed on days 38 and 39 p.i., TG were aseptically removed, and each TG was placed in one well of a 24-well plate containing 1 ml of complete DMEM. Once TG were harvested for an entire 24-well plate, the TG were heat stressed by transfer to a 43°C, 5% CO2 incubator for 3 hours. After heat stress, explants were transferred to a 37°C, 5% CO2 incubator. Twenty-four hours later, TG and cell culture medium were transferred to 24-well plates seeded 6 h earlier with 5 × 104 L7 cells per well in a volume of 0.5 ml of complete DMEM. On days 6 and 10 after explantation, TG were transferred to a 24-well plate containing freshly seeded L7 cells. After 14 days in culture, TG explants were homogenized in 500 μl of complete DMEM with a Pro 200 homogenizer (Pro Scientific) and TG homogenates were transferred to a -80°C freezer. Freeze-thawed TG homogenates were centrifuged to remove tissue debris, and 200 μl of clarified supernatant was used to inoculated each well of a 12-well plate of L7 cells (1 × 105 cells per well). After allowing 45 minutes for viral adsorption, the viral inoculum was aspirated, the cell monolayer was rinsed with 1 ml complete DMEM, and the rinse solution was replaced with 1 ml complete DMEM. Monolayers of L7 cells treated with TG homogenates were observed for six days for the development of viral cytopathic effects.
Measurement of HSV-1 DNA load in latently infected mouse trigeminal ganglion
The left and right TG from each mouse were placed into a single 1.5 ml microfuge tube, and transferred to -80°C until the time of DNA extraction. DNA was isolated by a standard phenol: chloroform extraction procedure , and the number of HSV-1 genomes per TG was determined by a competitive PCR assay, which is described as follows.
The oligonucleotide primers used in the competitive PCR assay, VP16-a (5'-GGACTCGTATTCCAGCTTCAC-3') and VP16-b (5'-CGTCCTCGCCGTCTAAGTG-3'), amplified a 260-bp fragment of the HSV-1 VP16 gene. To provide an internal control for each PCR assay, a VP16 competitor template was generated by the method of Siebert and Larrick . In brief, an irrelevant sequence from pUC18 was amplified with the primers VP16 mimic-a (5'-GGACTCGTATTCCAGCTTCACGGAGGACCGAAGGAG-3') and VP16 Mimic-b (5'-CGTCCTCGCCGTCTAAGTGCCAGTGCTGCAATGA), which amplify a 361-bp PCR product whose 5' ends are identical in sequence to the VP16-a and VP16-b primers. The VP16 competitor was cloned into the pCR II vector (Invitrogen Corp., Carlsbad, CA), and the resulting plasmid was used as a competitor template in the competitive PCR assay. DNA for the standard curve was obtained by diluting the plasmid pCRII: VP16 into a solution of TE buffer containing 33 ng/μl of salmon sperm DNA as carrier DNA. The most concentrated standard contained 45 fg of plasmid DNA per μl (10,000 copies per μl), and 13-serial twofold dilutions were made using TE buffer and salmon sperm DNA as the diluent.
PCR assays were conducted as follows: A solution containing 1× Taq buffer, 50 μM of each dNTP, 0.25 μM of each VP16 primer, 5% glycerol, and ~1500 copies of VP16 competitor template per 50 μl reaction was prepared. Forty-two microliters of this master mix were placed in 0.65 ml tubes and overlaid with mineral oil, and 100 ng of TG DNA (3 μl), DNA standards, or negative control DNA sample was added to each tube. The tubes were heated to 90°C in a thermal cycler, and 2.5 U of Taq polymerase diluted in 5 μl of Taq buffer was added to each sample. PCR samples were incubated for 35 cycles of 94°C for 1 minute 15 seconds, 59.5°C for 1 minute 15 seconds, and 72°C for 40 seconds.
Measurement of VP16 gene and competitor PCR product yields was performed by a modification of the dot blot procedure of Hill et al. . From each amplified PCR sample, 15 μl was diluted in 500 μl of a 0.4 M NaOH solution, transferred to a 0.6 ml microfuge tube, heated to 90°C for 10 minutes, snap-cooled on ice, and blotted on Zeta Probe GT nylon membrane (BioRad Laboratories, Hercules, CA) in an 8 × 12 dotblot pattern using a Convertible™ vacuum filtration manifold (Whatman-Biometra, Gröningen, Germany). Two identical dotblots of each set of PCR samples were produced and crosslinked with 0.2 J/cm2 in a UV crosslinker (Spectronics Corporation, Westbury, NY). One dotblot was hybridized to a radiolabeled oligonucleotide specific for VP16 (5'-GTCGTCGTCCGGGAGATCGAGCAGGCCCTC-3'), and the second duplicate dotblot was hybridized to a radiolabeled oligonucleotide specific for the competitor PCR product (5'-CGCTCGTCGTTTGGTATGGCTTCATTCAGC-3'). Both oligonucleotide probes were end-labeled with [α-32P] dATP using terminal deoxynucleotidyl transferase (Promega Corporation, Madison, WI). Each probe was allowed 16 h to hybridize to a membrane at 42°C in a solution containing 20 ng/ml labeled probe, 7% SDS, 120 mM NaH2PO4, and 250 mM NaCl. Excess probe was removed from membranes by sequential rinses in 0.1× standard saline citrate (SSC) containing 0.1% SDS and membranes were exposed to phosphor screens, which were scanned on a Cyclone PhosphorImager (Perkin Elmer Life Sciences, Boston, MA). The amount of radolabeleled probe hybridized to each dotblot was determined using OptiQuant v4.0 software (Perkin Elmer Life Sciences).
A two-fold dilution series of VP16 plasmid DNA defined the relationship between the copy number of VP16 genes in each PCR (x) and the logarithm of the ratio of VP16 to competitor PCR product yields (y) amplified from each DNA sample. The standard curve was described by the equation x = arctanh (y), but for convenience Microsoft Excel's trendline-fitting feature was used to rapidly define a third order polynomial equation (x = ay3 + by2 + cy + d) that approximates the sigmoid shape described by this equation (Fig. 8B). To estimate the copy number of viral genomes per TG, the number of HSV-1 genomes per PCR was multiplied times 150. This derivation is based on the fact that an average of 15 μg of DNA is extracted from each TG pair using an extraction procedure that recovers 50% of the total DNA. Thus, ~15 μg of DNA was present in a single TG, and the input of 0.1 μg of TG DNA per PCR contains ~1/150th (0.1 μg out of 15 μg) of the total number of HSV-1 genomes present in a single TG.
Analysis of numerical data was performed with the software packages Microsoft Excel and Modstat (Modern Microcomputers, Mechanicsville, VA). All viral titers were transformed by adding a value of 1 to the number of plaque-forming units detected per sample, such that all data could be analyzed on a logarithmic scale. The significance of differences between multiple groups was evaluated by one-way analysis of variance followed by a post hoc t-test. The significance of differences between strain 129 mice, rag2
-/- mice, stat1
-/- mice, versus rag2
-/- mice was evaluated by two-way analysis of variance. The goodness of fit of the standard curve used in the competitive PCR assay was determined by regression analysis using the method of least squares.