β), constructed by the insertion of a cassette containing the early ICP6 promoter regulating the expression of β-galactosidase into the KOS strain of HSV-1 (a gift from David A. Leib, Washington University School of Medicine, St. Louis, MO), was used as the parental strain in this study (9
). HSV-1 (KOS) wild type (KOS wt), without β-galactosidase inserted, was also used in some experiments. The stocks of all viruses used in this study were propagated on Vero cells (American Type Culture Collection [ATCC], Manassas, VA) grown in complete Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS) and antibiotics. The titers of the virus stocks were determined by standard plaque assays on Vero cells. The stocks were stored at −80°C in 0.5-ml aliquots, and a fresh aliquot of virus stock was thawed and diluted for each experiment. The titers of virus stocks used in these studies were 2.20 × 108
β), 2.75 × 108
), 4.04 × 108
), 2.02 × 108
), and 9.3 × 108
PFU/ml (KOS wt).
pMuTNF, a plasmid that contains a 1.1-kb sequence for mouse TNF-α (ATCC), was used in the study. Mouse TNF-α DNA was excised from pMuTNF by EcoR1 restriction digestion and purified. The gene for TNF-α was then cloned into a pCI mammalian expression vector (Promega, Madison, WI). This vector contains the human cytomegalovirus (CMV) major immediate-early gene enhancer/promoter region. The mouse TNF-α DNA was cloned into the pCI vector at the EcoR1 site to generate the expression vector pCI/TNF
-α. In this vector, the mouse TNF-α gene is located downstream of the CMV major immediate-early gene enhancer/promoter region. The plasmid pUIC contains a unique BglII site in the intergenic region between the UL49 and UL50 genes of HSV-1 (9
). The CMV IE promoter::TNF-α cassette was digested with BamHI and BglII and ligated into pUIC at the BglII site to create pUIC/TNF
-α. The pUIC/lacZ
plasmid contains an ICP6 promoter::lacZ
cassette at the unique BglII site in the intergenic region between the UL49 and UL50 genes of HSV-1 (KOS) (9
Generation of recombinant viruses.
The TNF-α recombinant virus HSV-1 (KOSTNF) was generated from HSV-1 (KOS6β), as shown in Fig. . HSV-1 (KOS6β) was grown to a high titer on Vero cells, and viral DNA was extracted from KOS6β virions that had been isolated by centrifugation. pUIC/TNF-α DNA was amplified by transforming Escherichia coli HB101 cells (Promega), and plasmid DNA was extracted and purified by using a S.N.A.P. MidiPrep kit (Invitrogen, Carlsbad, CA). An isolate containing the TNF-α gene was selected, grown in a large culture, and purified by using a S.N.A.P. MidiPrep kit. pUIC/TNF-α plasmid DNA was extracted and purified by using a S.N.A.P MidiPrep kit and digested with FspI (New England Biolabs, Beverly, MA). BHK-21 (hamster kidney) cells (ATCC) were cotransfected with KOS6β and digested plasmid DNA, using a lipophilic transfection agent, Lipofectamine 2000 (Invitrogen). To screen for recombinants, transfected cell supernatants were incubated with complete DMEM containing 5% FBS, antibiotics, 1% agarose, and 250 μg/ml β-galactosidase chromogenic substrate, X-GAL (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Promega), at 37°C in 5% CO2 for several days on cultured Vero cells. Clear plaques (indicative of TNF-α-containing recombinant virus) were selected and plaque purified by three passages through a cell culture before being grown to a high titer. To control for the effects of cloning the TNF-α cassette into the intergenic region between the UL49 and UL50 genes of the pUIC plasmid, a control virus containing a 1,344-bp fragment of pCI plasmid DNA alone, without TNF-α DNA, was also made, using the process described above (Fig. ).
FIG. 1. Construction of HSV-1 recombinants. (A) Diagram of the HSV-1 (KOS6β) genome and of pUIC relative to the locations of the UL49 and UL50 genes in the cloned subgenomic PvuII-PstI fragment containing a unique BglII site in the intergenic region between (more ...)
A rescue recombinant virus was generated from HSV-1 (KOSTNF) by the removal of the TNF-α and reinsertion of the lacZ gene, as shown in Fig. . HSV-1 (KOSTNF) was grown to a high titer on Vero cells, and viral DNA was extracted from KOSTNF virions that had been isolated by centrifugation. pUIC/lacZ DNA (a gift from David A. Leib) was amplified by transforming E. coli HB101 cells, and plasmid DNA was extracted and purified by using a S.N.A.P. Midiprep kit. BHK-21 cells were cotransfected with KOSTNF and digested plasmid DNA, using Lipofectamine 2000. To screen for recombinants, transfected cell supernatants were incubated with complete DMEM containing 5% FBS, antibiotics, 1% agarose, and 250 μg/ml β-galactosidase chromogenic substrate, X-GAL (Promega), at 37°C in 5% CO2 on cultured Vero cells. Blue plaques (indicative of recombinant rescue virus) were selected and plaque purified by three passages through cell cultures prior to preparation of the virus stock.
The viruses used in this study were genotyped by PCR. Primers were designed from publicly available GenBank sequences to ensure the specificity of the primers for the genes of interest. Samples were mixed with 100 pmol of primers specific for mouse TNF-α DNA, (sense) 5′-TCCAGAACATCCTGGAAATAGCTC-3′ and (antisense) 5′-AGAGGCCCACAGTCCAGGTCACTG-3′; lacZ DNA, (sense) 5′-GATGCGCCCATCTACACCAACGTG-3′ and (antisense) 5′-CAGCGCGGATCATCGGTCAGACGA-3′; or the HSV-1 VP16 gene, (sense) 5′-CGGTACCTGCGCGCCAGCGTC-3′ and (antisense) 5′-CAGCGGGAGGTTAAGGTGCTC-3′.
PCR was done, using a PCR reagent system (Invitrogen) and a Biometra T3000 thermocycler (Biometra, Goettingen, Germany). The PCR was performed according to the manufacturer's PCR protocol with 35 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 90 seconds. After 35 cycles, the reaction mixture was maintained at 72°C for an additional 90 seconds. The products were subjected to electrophoresis on a 1% agarose gel and visualized under UV light.
Virus replication and production of TNF-α in cultured cells.
Vero (African green monkey kidney) cells and SH-SY5Y (human brain [neuroblastoma]) cells (ATCC) were infected with HSV-1 (KOSTNF), HSV-1 (KOS6β), HSV-1 (KOSpCI), HSV-1 (KOS6βrescue), or HSV-1 (KOS wt) at a multiplicity of infection of 5/PFU per cell and incubated at 37°C in 5% CO2 for 1 h to allow the viruses to attach. The plates were then washed with phosphate-buffered saline (PBS) to remove any unattached viruses, overlaid with 2 ml DMEM containing 5% FBS and antibiotics, and incubated at 37°C in 5% CO2. At 0, 2, 4, 6, 12, and 24 h after infection, the cell supernatants were collected and stored at −80°C. To determine if TNF-α was produced, the samples were thawed and centrifuged at 300 × g at 4°C for 15 min. The protein concentrations were determined and normalized, using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The supernatants were then assayed for TNF-α using a Quantikine mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN). The results were read by a microplate reader (AT 400; SLT, Hillsborough, NC) set to 450 nm and analyzed using DeltaSoft plate reader software (DeltaSoft, Princeton, NJ). Uninfected-cell supernatant was used as the negative control. The results were plotted graphically (DeltaGraph version 5.0; RockWare, Inc., Golden, CO). For virus titration, samples were freeze-thawed three times and centrifuged. The titers of virus in the supernatants were determined by plaque assays on duplicate cultures of Vero cells.
Biological activity of virally produced TNF-α.
The cytotoxic activity of the recombinant TNF-α was assayed using WEHI-13VAR (mouse fibrosarcoma) cells (ATCC). The cells were grown in RPMI 1640 medium (ATCC) containing 10% FBS and 1% antibiotics-antimycotics (Gibco). The cells were seeded in a 96-microwell plate at 2 × 104 cells per well in 0.1 milliliters of RPMI medium containing 3% FBS and 1 μg/ml actinomycin D (Sigma). Recombinant TNF-α was concentrated from HSV-1 (KOSTNF)-infected Vero cell supernatants, using a Centricon Plus-70 centrifugal filter device (Millipore, Bedford, MA), and quantified using a Quantikine mouse TNF-α ELISA kit. Tenfold dilutions of recombinant TNF-α from HSV-1 (KOSTNF) and recombinant mouse TNF-α (R&D Systems, Inc.) were made in RPMI 1640 medium, and 0.1 ml of the dilutions were added to WEHI cells (in triplicate). After a 6-h incubation, cytotoxicity was determined, using a CytoTox 96 nonradioactive cytotoxicity assay (Promega). Absorbance was recorded at 490 nm, using a microplate reader and DeltaSoft plate reader software.
Adult female BALB/c mice 6 to 8 weeks old (Taconic, Germantown, NY) were used in all in vivo experiments. The mice were housed in accordance with National Institutes of Health guidelines, and all study procedures conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia. The mice were maintained on a 12-h light/dark cycle and were given unrestricted access to food and water. The mice were anesthetized by intramuscular injection of a mixture of 42.9 mg/ml ketamine, 8.57 mg/ml xylazine, and 1.43 mg/ml acepromazine (0.5 to 0.7 ml/kg) before all experimental manipulations.
Mice were anesthetized and inoculated through the AC route. The right eye was proptosed, aqueous humor was removed by paracentesis, and 2 μl of inoculum containing 2 × 104 PFU of HSV-1 (KOS6β), HSV-1 (KOSTNF), HSV-1 (KOS6βrescue), or HSV-1 (KOSpCI) was injected into the AC with a 30-gauge needle attached to a 100-μl microsyringe (Hamilton, Reno, NV). The inocula were prepared by diluting the virus stocks in DMEM with antibiotics.
The mice were deeply anesthetized and perfused transcardially with PBS for approximately 3 min. After perfusion, the brains and both eyes were removed and immediately stored at −80°C.
The brains and eyes from mice infected with HSV-1 (KOS6β), HSV-1 (KOSTNF), HSV-1 (KOS6βrescue), or HSV-1 (KOSpCI) were removed. One millimeter of coronal slices was prepared from the brains, using a brain matrix (ASI Instruments, Inc., Houston, TX), and slices containing the SCN were collected and homogenized in 500 μl of DMEM containing antibiotics. Both eyes from each mouse were homogenized individually in 500 μl of DMEM containing antibiotics. Each homogenate was serially diluted and plated on Vero cells (ATCC) that were 80% confluent. After adsorption of the virus for 1 h at 37°C in a CO2 incubator, the cells were washed to remove any unattached viruses and overlaid with a 1:1 solution of 2× DMEM (containing 10% serum and antibiotics) and 1% low-melt agarose (Life Technologies, Rockville, MD). After 5 days at 37°C, the cells were fixed with 10% buffered formalin and stained with 0.13% crystal violet (Sigma). The plaques were counted, and the titers of virus were calculated and analyzed using DeltaGraph (DeltaPoint, Inc., Monterey, CA).
In vivo TNF-α production.
The uninjected eyes from the mice infected with HSV-1 (KOS6β), HSV-1 (KOSTNF), or HSV-1 (KOSpCI) were removed and homogenized in 500 μl of DMEM containing antibiotics. The samples were then centrifuged at 300 × g at 4°C for 15 min. The protein concentrations were determined and normalized, using Bio-Rad protein assays. The supernatants were then assayed for TNF-α using Quantikine mouse TNF-α ELISA kits. The results were read by a microplate reader (AT 400; SLT) set to 450 nm and analyzed using DeltaSoft plate reader software. The results were plotted graphically, using DeltaGraph version 5.0.
Frozen sections were fixed with 4% paraformaldehyde, washed in PBS, and blocked with normal goat serum (Vector Laboratories) for 30 min. For virus detection, sections were incubated with an anti-HSV-1/fluorescein isothiocyanate (FITC) polyclonal antibody (DAKO, Carpinteria, CA), washed with PBS, and mounted with VectorShield containing DAPI (4′-6′-diamidino-2-phenylindole; Vector Laboratories). Slides were examined using a fluorescence microscope connected to the computer program Spot Advanced (Diagnostic Instruments, Inc., Sterling Heights, MI).
Hematoxylin and eosin staining.
Frozen sections were fixed with acetone, air dried, and placed in hematoxylin for 2 min. The sections were washed in water, placed in 0.14N HCl in 70% ethyl alcohol, washed again in water, and stained with eosin for 30 seconds. The sections were then washed in water, dehydrated in a graded ethanol series, cleared with xylene, coverslipped, and examined microscopically.
Single-cell suspensions were prepared from the eyes of HSV-1 (KOSTNF)-, HSV-1 (KOSpCI)-, and HSV-1 (KOS6β)-infected mice on different days postinfection. Enucleated whole eyes were incubated with collagenase IV for 60 min at 37°C in a CO2 incubator. After being incubated, cells were removed by pressing the digested eye through a 70-μm nylon cell strainer (BD Falcon). The cells were suspended in Hank's balanced salt solution (Cellgro; Mediatech, Herndon, VA), centrifuged at 400 × g at 4°C for 5 min, and resuspended in PBS containing 0.5% FBS, 1 mM EDTA, and 25 mM HEPES. FITC anti-mouse CD11b (integrin αM chain, Mac-1 α chain; Pharmingen) and FITC anti-mouse Gr-1 (Ly-6G and Ly-6C) (RB6-8C5; Pharmingen), antibodies recognizing macrophages and neutrophils, respectively, were used to determine the extent of cellular infiltration. Flow cytometry of stained cell samples was performed (FACSCalibur; Becton, Dickinson, and Co., Franklin Lakes, NJ), and the flow cytometry results were analyzed using CellQuest software (Becton, Dickinson, and Co.).