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Molecular Vision 1999; 5:36 <http://www.molvis.org/molvis/v5/p36> © Molecular Vision Received 1 November 1999 | Accepted 7 December 1999 | Published 20 December 1999 Iontophoresis of lysophosphatidic acid into rabbit cornea induces HSV-1 reactivation: Evidence that neuronal signaling changes after infection Rex E. Martin,1 Jeannette M. Loutsch,2 Hildegardo H. Garza, Jr.,3 Daniel J. Boedeker,1 James M. Hill2 1Department of Cell Biology and Oklahoma Center for Neurosciences, University of Oklahoma College of Medicine, Oklahoma City, OK; 2LSU Eye Center and Neuroscience Center, Louisiana State University Medical Center School of Medicine, New Orleans, LA; 3Department of Biology, Texas A & M University, Kingsville, TX Purpose: Lysophosphatidic acid induces neurite retraction; it is also present in tears and aqueous humor. We determined whether lysophosphatidic acid induces HSV-1 reactivation in latently infected rabbits and whether the nerve growth associated protein GAP-43 undergoes posttranslational modification during the course of HSV-1 infection. Methods: Rabbits were infected with HSV-1 and acute infection was documented by slit lamp examination. Corneas of latently infected rabbits were treated with lysophosphatidic acid or lysophosphatidylserine (structurally similar but lack- ing biological potency). For application to the cornea, these compounds were impregnated into collagen shields, applied as topical drops, or iontophoresed. In another experiment, corneas of latently infected rabbits were either untreated or treated iontophoretically with lysophosphatidic acid, lysophosphatidylserine, or saline. Ocular swabs detected shedding of infectious virus. Western blot and immunoprecipitation identified GAP-43 in corneal extracts and densitometry of silver-stained isoelectric focusing gels measured changes in GAP-43 isoform abundance. Results: Iontophoresis of lysophosphatidic acid induced HSV-1 shedding more frequently than lysophosphatidylserine or saline. Viral shedding induced by collagen shield and topical drop administration was low and not significantly different for lysophosphatidic acid and lysophosphatidylserine. Five discrete GAP-43 isoforms predominated in the IEF gels. Most abundant were the pI 4.7 band in uninfected cornea and the pI 5.05 band in latently-infected cornea. Compared to latently-infected cornea, there was no significant change in isoform abundance 1 h after lysophosphatidic acid iontophore- sis, but 24 and 72 h later, the pI 5.05 band was diminished. Conclusions: Lysophosphatidic acid can induce HSV-1 reactivation and changes in GAP-43 pI suggest that posttransla- tional modifications, possibly related to phosphorylation and ADP-ribosylation, are occurring during HSV-1 latency and after LPA is iontophoretically applied to the cornea. How lysophosphatidic acid-induced signaling, HSV-1 reactivation, and GAP-43 pI are related remains to be determined. Most clinically significant ocular herpes simplex virus When phosphorylated in the appropriate site by protein ki- type-1 (HSV-1) infections result from the reactivation of la- nase C, GAP-43 interacts with the cytoskeleton to potentiate tent virus [1]. Systemic stresses or physical traumas that dis- actin polymerization and neurite outgrowth [12]. GAP-43 rupt the corneal nerves induce HSV-1 reactivation [2]. Our expression is induced by nerve growth factor [13,14]. This intent is to learn how to impair or prevent HSV-1 reactivation finding, plus the fact that systemic nerve growth factor deple- by characterizing the biochemical changes that occur in neu- tion induces HSV-1 reactivation [15], suggests that neuronal rons during infection. As a marker of these biochemical systems that potentiate neurite elongation are involved in the changes, we have chosen to study a nerve growth associated establishment and maintenance of HSV-1 latency as well as protein called GAP-43 [3] and we have shown that the con- the switch from the latent to the lytic phase. To test this con- centration of GAP-43 increases in corneal nerves during HSV- cept, we hypothesized that if the latent HSV-1 in corneal nerves 1 latency [4,5]. A specific role for GAP-43 in HSV-1 infec- can be reactivated by physical trauma or systemic stress, then tion has not been established. substances that perturb neurite homeostasis in in vitro culture GAP-43 participates in the remodeling and regeneration could also induce latent HSV-1 to reactivate in vivo. of synaptic terminals; it is also implicated in the constant re- Lysophosphatidic acid (LPA) was selected to perturb neu- modeling of nerve endings that occurs in the densely inner- ronal homeostasis because it is present in fluids that bathe the vated cornea [3-7]. GAP-43 can be palmitoylated or ADP- cornea and it induces neurites to retract in culture [16,17]. ribosylated and has at least four phosphorylation sites [8-11]. LPA is a negatively charged, naturally occurring, receptor bind- ing, lysophospholipid that is synthesized from phosphatidic acid by phospholipase A2 [18-20]. In non-pathologic condi- Correspondence to: Rex E. Martin, Ph.D., Room 517, 940 Stanton tions, LPA is present at concentrations of 2-20 µM in serum, 1 Young Boulevard, Department of Cell Biology and Oklahoma Cen- µM in lacrimal gland fluid, and 0.2 µM in aqueous humor ter for Neurosciences, University of Oklahoma College of Medicine, [16,21]. In cryogenically-injured cornea, the LPA concentra- Oklahoma City, OK, 73104; Phone: (405) 271-2377, ext 229; FAX: tion increases 3-6 fold in aqueous humor but not in tear fluid (405) 271-3548; email: [email protected] Molecular Vision 1999; 5:36 <http://www.molvis.org/molvis/v5/p36> © Molecular Vision [16]. The details of signaling events that relate to LPA-recep- lysophosphatidylserine, applied to the corneal surface, and left tor binding and to posttranslational changes in GAP-43 are in place for 8 h. Iontophoretic drug delivery was performed unknown. A direct connection between LPA and GAP-43 sig- once daily for 3 consecutive days as previously described [24]. naling is also unknown, but a compelling connection is sug- In a second experiment, LPA, lysophosphatidylserine, or sa- gested because LPA stimulates neurite retraction and GAP-43 line was iontophoresed once daily in 10 eyes each for 3 con- supports neurite elongation [3,13,14,17]. Moreover, the LPA secutive days as in experiment 1; eight eyes were monitored receptor functions through membrane-bound and soluble G as untreated controls. proteins [18,20] and GAP-43 is associated with G proteins Corneal Proteins: Following previously described pro- [22,23], and ADP-ribosylated [9,10]. cedures [5], corneas were dissected and immediately frozen We delivered LPA and another negatively charged in liquid nitrogen. The frozen tissue from individual corneas lysophospholipid, lysophosphatidylserine, to the corneas. To were ground to a powder in a liquid nitrogen-cooled mortar determine the optimal way to deliver these compounds, topi- and pestle. The powder from each cornea was resuspended in cal drops, collagen shields, or iontophoresis were tested. We 15 volumes of homogenization buffer, which consisted of 20 then determined frequency of HSV-1 reactivation after each mM Tris-HCl (pH 7.5) containing 320 mM sucrose, 10 mM procedure. We observed that the pI of GAP-43 changed dur- ethylene glycol-bis [β-aminoethylether], 2 mM ing HSV-1 infection and after LPA treatment. ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol, 0.05% leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. METHODS The suspension was centrifuged at 2000 x g for 10 min at 4 °C Virus and Rabbits: HSV-1 strain McKrae was propagated on to remove the collagenous stromal matrix. The pellet was primary rabbit kidney cells and titered on African Green Mon- resuspended with sonication in five volumes of ice-cold ho- key cells (CV-1). The virus was aliquoted and stored at -70 mogenization buffer and centrifuged to remove the collagen °C. New Zealand White rabbits (2-3 kg) were inoculated bi- and maximize extraction of non-collagenous proteins. Pro- laterally by applying 2 x 105 plaque forming units of HSV-1 tein concentrations were determined using Bio-Rad (Rich- in a 25-µl suspension into the cul-de-sac of each eye. The eye mond, CA) protein assay reagent and bovine serum albumin was closed and massaged for 30 seconds. The corneas were as a standard. All corneal protein procedures were done at 4 not scarified. Slit-lamp examination and ocular swabbing were °C. done 3 days after inoculation to identify epithelial lesions and GAP-43 Identification and Densitometry: Three differ- to verify a productive infection, respectively. HSV-1 was pre- ent approaches were used to identify discrete GAP-43 isoforms sumed to be latent when slit-lamp examination indicated no in rabbit corneal extracts (Figure 1). Two of these approaches corneal defects and tear film swabs contained no infectious used pI 4-6 horizontal IEF gels and an anti-GAP-43 mono- virus [24]. clonal antibody that binds GAP-43 irrespective of its phos- The reactivation studies were performed 5-6 weeks after phorylation state [13]. In the third approach, proteins migrat- virus inoculation. All eyes underwent membranectomies 1 ing at 43 kDa in sodium dodecyl sulfate (SDS)-polyacryla- week prior to treatment. A sample of the tear film from each mide gels were electroeluted from the gel, concentrated, and eye of each rabbit was collected on Dacron swabs once daily then vertically electrophoresed in precast pI 3-7 IEF gels for 7 days after the start of treatment. The swabs were placed (Novex, San Diego, CA). All procedures were done at room in tubes containing monolayers of primary rabbit kidney cells temperature. and the cells were monitored for the development of cyto- Figure 1, IP/IEF, shows the first approach taken to iden- pathic effect indicative of infectious virus. Rabbits used in tify GAP-43 in IEF gels. GAP-43 was immunoprecipitated studies of reactivation were sacrificed seven days after initia- with staphylococcal protein G-coated agarose beads (Hyclone tion of the first treatment.