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Herpes Simplex Virus Latency in Isolated Human Neurons [Herpesviruses/Neuron-Specific Marker/Human Leukocyte Interferon/(E)-5-(2-Bromovinyl)-2'-Deoxyuridine]

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Herpes Simplex Virus Latency in Isolated Human Neurons [Herpesviruses/Neuron-Specific Marker/Human Leukocyte Interferon/(E)-5-(2-Bromovinyl)-2'-Deoxyuridine] Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6217-6221, October 1984 Microbiology Herpes simplex virus latency in isolated human neurons [herpesviruses/neuron-specific marker/human leukocyte interferon/(E)-5-(2-bromovinyl)-2'-deoxyuridine] BRIAN WIGDAHL, CAROL A. SMITH, HELEN M. TRAGLIA, AND FRED RAPP* Department of Microbiology and Cancer Research Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033 Communicated by Gertrude Henle, June 6, 1984 ABSTRACT Herpes simplex virus is most probably main- latency was maintained after inhibitor removal by increasing tained in the ganglion neurons of the peripheral nervous sys- the incubation temperature from 370C to 40.50C, and virus tem of humans in a latent form that can reactivate to produce replication was reactivated by decreasing the temperature recurrent disease. As an approximation of this cell-virus inter- (20, 22). As determined by DNA blot hybridization, the la- action, we have constructed a herpes simplex virus latency in tently infected HEL-F cell and neuron populations con- vitro model system using human fetus sensory neurons as the tained detectable quantities of most, if not all, HSV-1 Hin- host cell. Human fetus neurons were characterized as neuronal dIII, Xba I, and BamHI DNA fragments (21). Furthermore, in origin by the detection of the neuropeptide substance P and there was no detectable alteration in size or molarity of the the neuron-specific plasma membrane A2B5 antigen. Virus la- HSV-1 junction or terminal DNA fragments obtained by tency was established by blocking complete expression of the HindIII, Xba I, or BamHI digestion of DNA isolated from virus genome by treatment of infected human neurons with a latently infected HEL-F cells or neurons (21). The data sug- combination of human leukocyte interferon and (E)-5-(2-bro- gested that the predominant form of the HSV-1 genome in movinyl)-2'-deoxyuridine for 7 days. After removal of inhibi- either latently infected cell population was nonintegrated, tors, virus latency was maintained for at least 9 days. This in linear, and unit-length DNA (21). We now report an in vitro vitro model will provide a system to analyze, in a primary hu- latency model using isolated primary human neurons as the man neuron, the state of the herpes simplex virus genome dur- host cell type. ing establishment and maintenance of experimental latency. MATERIALS AND METHODS Herpes simplex virus (HSV) infection of humans can pro- Cells, Viruses, and Infectious Virus Determination. Mono- duce diseases ranging in severity from mild gingivostomatitis layer cultures of HEL-F cells and primary rabbit kidney to life-threatening encephalitis (1-4). In addition, HSV can (PRK) cells were grown and maintained as previously de- remain latent in sensory and autonomic peripheral ganglia of scribed (16-19). HSV-1 (strain Patton) was grown in HEL-F humans, with subsequent virus reactivation often culminat- cells infected at 0.01 pfu per cell as previously described (16- ing in recurrent epithelial lesions (5, 6). To examine the 19). HSV-1 stocks with titers between 1 and 2 x 109 pfu/ml, mechanisms involved in the establishment and maintenance as assayed on either HEL-F or PRK cells, were stored at of HSV latency and virus reactivation, experimental ap- -70'C. After disruption of cells by freezing and thawing proaches have included analyses of (i) central and peripheral three times, total infectious HSV-1 present in infected hu- nervous system tissue isolated from human cadavers (5, 7, man neuron cultures was quantitated by plaque assay on 8); (ii) in vivo animal models (9, 10); (iii) animal nervous sys- PRK cells as described (16, 17). To quantitate infectious vi- tem explantation models (11); and (iv) in vitro cell culture rus in HSV-1-infected human fetus neurons during combined models (12-15). treatment with BVdUrd and IFN-a, the inoculum was re- Because the peripheral ganglion neuron most probably moved from indicator cells after a 2-hr adsorption period and maintains the HSV genome during virus latency, thereby the infected cells were washed twice with Tris-buffered sa- playing a central role in latent and recurrent infection, we line prior to overlay with 0.5% methylcellulose (20). have constructed several in vitro systems to approximate Isolation and Purification of Human Fetus Neurons. All hu- this cell-virus interaction (16-22). We initially described a man fetus nervous system tissue was isolated from elective, prototype HSV latency system established by treatment of nontherapeutic, suction-aborted fetuses after 6-16 weeks of human embryo lung fibroblast (HEL-F) cells infected with a gestation. Abortion specimens were maintained in Liebovitz low multiplicity of virus [0.1 plaque-forming unit (pfu) per L-15 buffer prior to aseptic microscopic surgical removal of cell] with 1-,B3D-arabinofuranosylcytosine; virus latency was nervous system tissue initiated between 45 min and 2 hr after maintained after inhibitor removal by increased temperature pregnancy termination. The dorsal root ganglia (DRG) were (16-19). To increase the fraction of the latently infected cell removed by making a midline incision through the vertebrae population containing an HSV genome that could be activat- of the entire spinal column followed by surgical removal of ed into a productive replication cycle and consequently fa- individual ganglia from their attached spinal roots. With cilitate molecular analysis of virus latency, a more effective abortion-terminated fetal specimens obtained at approxi- inhibitor combination consisting of (E)-5-(2-bromovinyl)-2'- mately 6-9 weeks gestation, a time prior to extensive devel- deoxyuridine (BVdUrd) and human leukocyte interferon opment of the dorsal root nerve, the DRG were removed by (IFN-a) was used to block complete expression of the virus negative pressure in conjunction with a glass capillary tube. genome after a high multiplicity HSV type 1 (HSV-1) infec- During the isolation and prior to dissociation, the DRG were tion (2.5 pfu per cell) of HEL-F cells (20). In addition, virus maintained in Eagle's basal medium with Earle's salts, gluta- latency was also established in isolated rat fetal neurons af- infections with ter treatment of similar high-multiplicity HSV and IFN-a In both systems, virus Abbreviations: HSV, herpes simplex virus; HSV-1, type 1; combined BVdUrd (20). BVdUrd, (E)-5-(2-bromovinyl)-2'-deoxyuridine; IFN-a, human leu- kocyte interferon; HEL-F, human embryo lung fibroblast; PRK, The publication costs of this article were defrayed in part by page charge primary rabbit kidney; pfu, plaque-forming unit(s); DRG, dorsal payment. This article must therefore be hereby marked "advertisement" root ganglia; CPE, cytopathic effect; IU, international units. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint request should be addressed. 6217 Downloaded by guest on September 25, 2021 6218 Microbiology: Wigdahl et aL Proc. NatL Acad. Sci. USA 81 (1984) mine (0.03%), glucose (0.6%), sodium bicarbonate (0.17%), Laboratories, Burlingame, CA). Incubations with each rea- and 2% fetal calf serum in a 5% CO2 atmosphere at 370C. gent were performed at room temperature for 20 min. The After removal, the DRG were washed with isolation medi- A2B5 and substance P monoclonal antibodies were obtained um, collected by gravitational force, and dissociated as de- from Accurate Chemicals (Westbury, NY) and the HSV-1 scribed (23, 24) with several modifications. The DRG were ICP-8 monoclonal antibody was generously provided by M. resuspended in calcium- and magnesium-free Hanks' buff- Zwieg (National Cancer Institute, Frederick, MD). Immuno- ered saline containing 0.02% collagenase type IIS and fluorescence was observed with a Zeiss photosystem III flu- 0.004% bovine pancreatic DNase I. After a 0.5-hr incubation orescence microscope. at 37TC, porcine pancreatic trypsin, type II, was added to a final concentration of 0.25% and the suspension was incu- RESULTS bated for an additional 0.5 hr. The resultant cell suspension Characterization of Isolated Human Fetus DRG Neurons. was subjected to centrifugation at 230 x g for 10 min and the As previously observed by light microscopic analysis (23- cell pellet was washed and resuspended in maintenance me- 25), cell cultures prepared from human fetus DRG contained dium consisting of Eagle's basal medium containing gluta- predominantly two cell types; very large, flat, fibroblastoid mine (0.03%), glucose (0.6%), sodium bicarbonate (0.17%), cells and round phase-bright cells with long extended pro- chicken embryo extract (1%), nerve growth factor (0.05 cesses. When mitotic inhibitors were not added to the neuro- kkg/ml), 10% fetal calf serum, and 10% heat-inactivated nal cultures, the proliferation of the fibroblastoid cells result- horse serum and supplemented with the mitotic inhibitors, 1- ed in the formation of a confluent cell monolayer within 3-6 ,-D-arabinofuranosylcytosine (10 ,uM), 5-fluoro-2'-deoxyur- days after isolation. However, as described for preparation idine (10 MtM), and uridine (10 /iM). After a single-cell sus- of neurons from rat fetus DRG (15, 22), when the cell cul- pension had been obtained by trituration through a 23-gauge tures were treated with mitotic inhibitors to inhibit cell pro- needle, the cells were plated on collagen-coated glass cover- liferation, essentially pure human fetus DRG neuron cultures slips in medium with mitotic inhibitors to minimize contami- were obtained. As shown by light microscopic analysis (Fig. nation from dividing cell types, predominantly fibroblasts. 1 A and B), mitotic inhibitor-treated cultures used for experi- After an initial 36-hr treatment with mitotic inhibitors, neu- mentation were greater than 99% neurons, consisting of a ronal cultures were alternated at 24-hr intervals between dense matrix of neuritic processes and neuron-like single maintenance medium and maintenance medium containing cells or multiple cell aggregates, with only a few contaminat- mitotic inhibitors.
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