JOURNAL OF VIROLOGY, Sept. 1996, p. 6524–6528 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 70, No. 9
In Vitro Establishment of Lytic and Nonproductive Infection by Herpes Simplex Virus Type 1 in Three-Dimensional Keratinocyte Culture ¨ NEN,1,2* HANNAMARI MIKOLA,2,3 MARJA NYKA ¨ NEN,2 STINA SYRJA
AND
VEIJO HUKKANEN2,3
Department of Oral Pathology, Institute of Dentistry,1 MediCity Research Laboratory, Faculty of Medicine,2 and Department of Virology, Faculty of Medicine,3 University of Turku, Finland Received 11 December 1995/Accepted 2 April 1996
The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation. ments of cultured cells may not accurately reflect the events leading to establishment of HSV latency in the sensory neurons of animals and humans (19). In the present report, we describe a novel approach to the study of HSV infection at the tissue level. We have used a three-dimensional epithelial culture system (1) which promotes the differentiation of human keratinocytes. These raft cultures have been recently used in studies of other viruses, particularly human papillomavirus (14). In HSV-infected organotypic raft cultures, we have observed cytopathic changes identical to those found in the squamous epithelium in vivo, including the formation of typical intraepithelial vesicles and multinucleation. Moreover, depending on the time point of the HSV infection relative to the confluence of the cell layers, a nonproductive form of HSV infection develops in the culture. Culture of HaCat cells and normal skin fibroblasts. The HaCat cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% nonessential amino acids, 2 mM L-glutamine, 50 mg of streptomycin per ml, 100 U of penicillin per ml, and 10% fetal calf serum. To establish the fibroblast culture, a biopsy from uninfected skin was transported to the laboratory in DMEM containing 100 mg of streptomycin per ml, 200 U of penicillin per ml, and 0.5 mg of Fungizone (Gibco Laboratories, Grand Island, N.Y.) per ml. The connective tissue was cut into small fragments of 1 mm3, and explant cultures were started according to standard techniques. The fibroblasts were grown in supplemented DMEM. Skin fibroblasts derived from the explants were from their fifth passage. Preparation of reorganized collagen gel. Vitrogen 100 collagen (Celtrix Pharmaceuticals, Inc., Santa Clara, Calif.) was mixed with 103 DMEM and neutralized by 0.1 M NaOH to pH 7.4 6 0.2. Fibroblasts were suspended in the collagen solution at a cell density of 280,000 cells per 0.7 ml, and this suspension was plated in 16-mm-diameter tissue culture dishes
Productive infection with herpes simplex virus (HSV) is characterized by coordinately regulated and sequentially ordered expression of three classes of genes: the a, b, and g genes (9, 10). The cells lytically infected with HSV undergo a series of structural alterations, with cell death as the final outcome (18). Lytic infection with HSV involves an early shutoff of the host cell metabolism, attributed to the g class protein vhs (virion-host shutoff) (6, 12), and a secondary shutoff, appearing late during the infection and requiring another viral protein, ICP27 (5, 7). These shutoff functions contribute to the fate of the infected cell. The typical changes observed in productively HSV-infected cells include chromatin margination, disaggregation of the nucleolus, and modification of the cellular membranes (18). The cytopathic effects observed at the tissue level (i.e., in the infected epithelium) are a reticular degeneration and ballooning of the cells as well as the appearance of large irregular or multinucleated giant cells. Latency is the hallmark of herpesvirus infections (18). HSV establishes latent infection in sensory neurons (19). In the sensory ganglion, the latent HSV genome resides in the nucleus of the neuron, expressing only one of its genes, the gene for latency-associated RNA (LAT) (22, 25). The LAT RNA is predominantly localized in the neuronal nucleus, and it consists of several overlapping RNA species with different termini (4). Although LAT does not apparently code for a protein, it is a widely used marker in the demonstration of latently infected cells (19, 21). A variety of in vitro models have been developed for the study of HSV latency in different cell types (3, 13, 16, 17, 23). However, the restriction of HSV replication by different treat* Corresponding author. Mailing address: MediCity Research Laboratory, Faculty of Medicine, Tykisto ¨katu 6, 20520 Turku, Finland. Phone: 358 21 3338349. Fax: 358 21 3338399. Electronic mail address:
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(Costar, Cambridge, Mass.). The collagen-fibroblast suspension was allowed to gel at 378C for 1 h, after which the dishes were filled with fresh Green’s medium. The medium consisted of DMEM-Ham F12 (3:1), 10% fetal calf serum, 4 mM glutamine, 5 mg of insulin (Sigma, St. Louis, Mo.) per ml, 0.18 mM adenine (Sigma), 0.4 mg of hydrocortisone (Sigma) per ml, 0.1 nM cholera toxin (Sigma), and 5 ng of epidermal growth factor (Boehringer Mannheim, Germany) per ml and was maintained at 378C in an atmosphere of 5% CO2 in 90% relative humidity. Medium was changed three times a week for 1 week. Epidermal cell culture. HaCat cells, passage 15 (200,000 cells per well), were gently added onto the surface of the fibroblast-collagen gels. The Green’s medium was changed every second day. After 3 days, when the cells had reached confluence, the cultures were lifted into the air-liquid interface with a stainless steel grid. Epithelial cells were then allowed to stratify for 7 days. One day before the lifting, cultures (with a confluence of 80%) were infected with HSV type 1 (HSV-1). Additional cultures were infected with HSV-1 30 min after lifting, while the other uninfected cultures served as negative controls. HSV-1. The F strain of HSV-1 was obtained from the American Type Culture Collection (ATCC, Rockville, Md.) and propagated in human foreskin fibroblasts. The infected cultures were centrifuged at 48C in a Sorvall Technospin R centrifuge at 2,700 3 g for 10 min. The pellet was resuspended in a small volume of phosphate-buffered saline, frozen and thawed three times, and sonicated for 3 min at 40% duty, continuous cycle. The infectious titer of the virus preparation was determined by standard plaque assay. Virus was stored at 2708C at a concentration of 5 3 106 PFU/ml. For each well, 5 PFU were used. Fixation and preparation of the tissue cultures. All tissue cultures were collected at day 18 (1 week after infection) and divided into two parts. One part was fixed in buffered 10% formalin for 24 h, while the other was snap frozen and kept at 2708C for further PCR analysis. The formalin-fixed material was embedded in paraffin and cut into 5-mm sections onto organosilane-coated slides for hematoxylin and eosin (HE) staining and for DNA and RNA in situ hybridizations (ISHs). ISH for HSV-1 DNA. As a probe for HSV-1, a biotinylated commercial probe was used (Enzo Diagnostics, N.Y.). The probe is a mixture of two clones of HSV DNA sequences in the BamHI site of pBR322, with insert sizes of 16.0 kb and 8.0 kb. ISH was performed as previously described (24). Shortly after deparaffinization and deproteinization with proteinase K (Boehringer) (0.3 mg/ml) for 15 min at 378C, the sections were simultaneously denatured with hybridization mixture containing 2 mg of biotinylated probe per ml in 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50% formamide, 0.4 mg of herring sperm DNA per ml, and 10% dextran sulfate. After overnight hybridization at 558C, the sections were subsequently washed with 23 SSC at room temperature, 0.23 SSC at 608C, and 23 SSC at room temperature. The hybrids were labeled with streptavidin-alkaline phosphatase (Amersham, Buckinghamshire, United Kingdom), and the complexes were detected with nitroblue tetrazolium as chromogen and 5-chromo-4-chloro-3-indolylphosphate (BCIP) as substrate. A biopsy specimen from the oral mucosa, previously shown to contain HSV-1 DNA, served as a positive control. As negative controls, all sections were treated similarly except that the HSV-1 probe was omitted from the hybridization mixture. ISH for LAT RNA. The digoxigenin (DIG)-labeled singlestranded RNA probe for LAT RNA was a 560-nucleotide SP6 transcript of a plasmid containing the 0.5-kb HpaI-SalI subfragment of the BamHI fragment B of HSV-1 DNA (gift from
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Bernard Roizman, University of Chicago) cloned into a pGEM-3Z vector (Promega, Madison, Wis.). The plasmid DNA was linearized with EcoRI (Boehringer) and subjected to transcription with SP6 polymerase and DIG-11-UTP (Boehringer), as described previously (11). The probe for a0 mRNA was transcribed with T7 polymerase from the same plasmid linearized with SalI. This RNA probe has a 230-nucleotide sequence complementary to a0 mRNA. The correct sizes of the probes were verified by electrophoresis in a formaldehyde gel. The ISH was done as described previously for DIG RNA probes (11). It involved a prehybridization step with 300 mg of denatured salmon sperm DNA per ml and the use of mouse brain nucleic acids (500 mg/ml) as a blocking agent in the hybridization buffer. The hybridization was performed for 18 h at 458C under sealed caps in a humidified incubator. The slides were washed, as described in reference 20, without the use of RNases during the washes. For detection of DIG-labeled hybrids, the sections were incubated with the alkaline phosphatase-conjugated anti-DIG antibody (Boehringer). The complexes were detected with nitroblue tetrazolium–X-phosphate color reagents (11). The slides were observed by using a masked protocol. As positive controls, mouse trigeminal ganglia (TG) infected with HSV-1 were used. The negative controls were sections from uninfected cultures and mice. The specificity of the signals was tested with additional sections with both RNase and DNase treatments. Slides were pretreated with RNase (100 mg/ml; Sigma) or DNase (75 U/ml, RNase-free DNase; Boehringer) for 30 min at 378C before hybridization. The slides were fixed again with modified Carnoy’s fixative (11) for 2 h directly after RNase treatment. Northern blot (RNA) analysis of LAT RNA. Preparations of total RNA of the raft cultures were made with the TRIzol reagent (Gibco BRL). Total RNA from latently infected [5 weeks postinfection, 106 PFU of HSV-1(F) per eye] TG of BALB/c mice was prepared by the guanidine thiocyanate-CsCl centrifugation method. Twenty micrograms of total RNA or 15 mg of DNase-treated total RNA from raft cultures was run in 1% agarose-formaldehyde gel, stained with ethidium bromide, and blotted on GeneScreen Plus (DuPont NEN) nylon membrane. The filter was hybridized with a single-stranded RNA probe, which was prepared by transcription from the same template as the ISH LAT probe but with [32P]CTP (Dupont NEN) as the labeled nucleotide. The hybridization took place at 658C in a solution containing 50% formamide, 33 SSC, 53 Denhardt’s solution, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 250 mg each of Escherichia coli transfer RNA (Sigma) and single-stranded herring sperm DNA (Sigma) per ml. Extraction of DNA. For the PCR, DNA was extracted from the frozen cultures by the method of Miller et al. (15). Shortly, samples were lysed in 1 ml of 10 mM Tris (pH 8.3), 400 mM NaCl, 1% SDS, 2 mM EDTA, and 0.3 mg of proteinase K per ml overnight at 378C. Proteins were precipitated by adding 300 ml of saturated NaCl. After centrifugation, supernatant was removed and DNA was precipitated with ethanol. PCR detection of HSV-1 DNA. The PCR test for HSV DNA was modified from a procedure described for cerebrospinal fluid specimens (2). The primers, detecting HSV-1 strains and several HSV-2 strains (59-ATC ACG GTA GCC CGG CCG TGT GACA and 59-CAT ACC GGA ACG CAC CAC ACAA) and the internal hybridization probe (59-TAC GAG GAG GAG GGG TAT AAC AAA GTC TGT), were derived from the glycoprotein D gene of HSV-1 (2). DNA extracts from frozen cultures were tested in two dilutions each. The positive controls were HSV-1 virions, strain F (ATCC), containing 1 PFU in each reaction. The dilutions were made from a high-
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FIG. 1. (A) HaCat cells grown in the raft culture system. Epithelial cells are organized into a thin sheet six to eight cell layers thick. A layer of flattened superficial cells is present, evidencing the terminal differentiation. (B) Tissue culture of HSV-1-infected HaCat cells. The virus was inoculated onto the epithelium 30 min after lifting the culture into the air liquid interface. The morphology was similar to that of the uninfected control cells. (C and D) HaCat cells grown in the raft culture system and infected with HSV-1 at 1 day before the lifting. Typical cytopathic effects of productive HSV infection with large irregular or multinucleated epithelial cells, ballooning, and reticular degeneration of epithelium are seen. Typical intraepithelial vesicles are also present. HE staining. Magnification 3250.
titer stock (109 PFU/ml). Negative controls contained distilled water. The working spaces for specimen handling, construction of the reaction mixture, PCR cycling, and gel electrophoresis were all physically separate from each other. The specimens were pipetted with Finnpipette positive displacement pipettors. The PCR contained 200 mM each deoxynucleotide, 20 pmol of each primer, 1 U of polymerase (DynaZyme, Finnzymes, Espoo, Finland), and corresponding buffer provided with the enzyme. The PCR steps were 958C for 30 s, 558C for 30 s, and 728C for 60 s, for 41 cycles. The first cycle included an incubation at 958C for 5 min. The 728C incubation in the last cycle was extended by 4 min. The presence of a 221-bp reaction product was observed by ethidium bromide staining after electrophoresis in 2% agarose gels. The specificity of the product was confirmed by hybridization to a DIG-labeled oligonucleotide probe, which was detected with CSPD luminescent reagent (Boehringer) on X-ray film (8). Detection of infectious HSV-1 by immunoperoxidase staining. The supernatants from and homogenates of the raft cultures were cultivated on Vero cells on 12-well culture dishes and studied for the presence of infectious HSV-1 by a previously described rapid detection system (26), which is based on immunoperoxidase staining of the viral proteins after an overnight culture, with a monoclonal antibody to HSV-1. Morphology of the raft cultures. The HaCat cells grown in the organotypic cell culture had the morphologic appearance of a partially differentiated epithelium. Epithelial cells were found to be organized into a thin sheet six to eight cell layers thick. There was some attempt to form a basal cell layer, albeit not one as regular as is normally found in vivo. A thin layer of flattened superficial cells was seen, evidencing the terminal differentiation (Fig. 1A). HSV established a productive infection of the epithelial cells only when the cells were infected before lifting the epithelium at the air-liquid interface. This lifting allows differentiation of the epithelium. Typical cytopathic effects of a productive HSV infection were observed in
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FIG. 2. ISH to detect HSV-1 DNA. (A and B) Strong signals are seen in all epithelial cells showing cytopathic effects of productive infection. Panel A magnification, 3250; panel B magnification, 3500. (C) No ISH signals are present in nonproductive HSV-1 infection. (D) Uninfected control epithelium. Magnification, 3250.
the HaCat cells grown in the organotypic cell culture (Fig. 1C and D). Large irregular or multinucleate epithelial cells with ballooning and reticular degeneration of the epithelium were observed by light microscopy. Also, typical intraepithelial vesicles were detectable. When HSV was applied on the surface of the lifted epithelium, no morphological changes indicating a productive HSV infection were seen in these cultures, but the morphology was similar to that of the uninfected control cells (Fig. 1B). HSV DNA and RNA in the raft cultures. The histopathologic findings were confirmed by ISH for the detection of HSV-1 DNA with biotinylated probes. Cultures with the cytopathic changes typical of HSV infection showed strong positivity for HSV DNA by ISH (Fig. 2A and B). HSV DNA was seen in all epithelial cells, but the most intense hybridization signals were encountered in the multinucleate giant cells. Interestingly, no HSV DNA positivity was found in the fibroblasts. The cultures for which HSV was added on the surface of the lifted epithelium remained negative for HSV DNA by ISH (Fig. 2C). However, PCR analysis of these cultures was repeatedly positive (Fig. 3A and B). In the PCR runs, the low positive PCR
FIG. 3. PCR detection of HSV DNA in the raft cultures. (A) Ethidium bromide staining of the specimens after electrophoresis in 2% agarose gel. (B) Southern hybridization with an internal oligonucleotide probe, 39-end-labeled with DIG-dUTP. Detection on X-ray film was carried out by use of luminescent CSPD reagent. Lanes: 1, distilled water; 2, a cerebrospinal fluid specimen negative for HSV-1; 3, uninfected HaCat raft culture; 4, productively infected HaCat raft culture (20 ng of DNA extract); 5, nonproductively infected HaCat raft culture (20 ng of DNA extract); 6, distilled water; and 7 and 8, 1 PFU of HSV-1 (F). M, molecular size markers. The arrowhead indicates the location of the positive PCR product in the gel.
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FIG. 4. Uninfected organotypic cultures were negative for both LAT and a0 RNA expression (A and B, respectively). Both LAT (C) and a0 RNA (D) probes reacted strongly with cultures infected before lifting at the air-liquid interface, as indicated by ISH. Magnification, 3250.
FIG. 5. Cultures infected after lifting at the air-liquid interface showed no cytopathic effect and reacted with the probe for LAT RNA (A) but were clearly negative for a0 mRNA (B). The LAT signal was cytoplasmic rather than nuclear, and it was removed by pretreatment with RNase (C) but not with DNase (D). Magnification, 3250.
controls [1 PFU of HSV-1(F)] were constantly positive, whereas the negative controls (cerebrospinal fluid specimens negative for HSV and specimens containing only distilled water) were all negative. The PCR specimens were total DNA extracts of the cell cultures containing material from both the connective tissue and the epithelium. The HSV-1 PCR-positive but ISH-negative cultures (viral inoculation 30 min after lifting) were further tested for presence of infectious virus in the samples. Both the culture supernatants and the direct homogenates of the cultures were unable to produce any HSV infection in Vero cells. The standard method of verifying HSV latency in mouse TG by using the homogenates of incubated tissue was not relevant in this study. The latency was further analyzed by studying the expression of viral a0 and LAT RNA in the cultures with single-stranded, DIG-labeled RNA probes. The probes were validated with paraffin sections of BALB/c mouse TG which were either uninfected or represented acute (4 days postinfection) or latent (4 weeks postinfection) phases of HSV-1(F) infection (data not shown). The hybridization protocol favored the detection of RNA instead of viral DNA, since the tissue samples were not heat denatured before hybridization. In control settings, specimens were subjected to treatment with RNase or DNase before hybridization. The uninfected organotypic cultures were regarded as being negative for both LAT and a0 RNA expression (Fig. 4A and B). The cultures with the cytopathic effects of HSV showed strong ISH signals with both probes (Fig. 4C and D), partially reflecting the reaction of the probes with the high quantities of viral DNA, since all the reactions were not removable by RNase pretreatment (data not shown). The cultures infected after lifting (without any cytopathic signs of HSV infection) reacted with the probe for LAT RNA, but were clearly negative for a0 mRNA (Fig. 5A and B). The LAT signal was intracytoplasmic rather than nuclear, and it disappeared after pretreatment with RNase but not with DNase (Fig. 5C and D). The LAT signal could not be localized to any particular layer of the cultured epithelium. The fibroblasts in the collagen matrix could not be assessed because of high background reaction, but in the DNA ISH, the fibroblasts remained definitively negative (Fig. 2). Northern blot analysis showed that the 2.0-kb LAT RNA species is expressed in both lytic and nonproductive forms of
infection (Fig. 6). Both the 1.5-kb and 2.0-kb LAT RNAs were detected in the mouse TG specimen. In the past, a variety of in vitro models have been developed for the study of HSV latency in different cell types (3, 13, 16, 17, 23). However, the restriction of HSV replication by different treatments of cultured cells is not similar to that occurring during establishment of HSV latency in the sensory neurons of animals and humans (19). To further elucidate the complex mechanisms of HSV latency, new models capable of better mimicking the in vivo events are urgently needed. In the present report, we describe for the first time an in vitro model to produce a lytic or nonproductive HSV infection in squamous epithelial (HaCat) cells grown in an organotypic raft culture. In the cultures infected before the total confluency and lifting of the cells to the air-liquid interface, i.e., before differentiation of the cell layers, a lytic HSV infection could be
FIG. 6. Northern blot analysis of the raft cultures for LAT RNA. Twenty micrograms (lanes 1 to 3 and 7) or 15 mg (lanes 4 to 6) of total RNA was extracted from uninfected raft cultures (lanes 1 and 4), nonproductively infected cultures (lanes 2 and 5), productively infected cultures (lanes 3 and 6), or TG of latently infected BALB/c mice (lane 7) and run in a 1% agarose-formaldehyde gel, blotted on nylon filter, and hybridized with a 32P-labeled single-stranded RNA probe for LAT. The RNA specimens in lanes 4 to 6 were treated with DNase prior to electrophoresis. The arrowhead indicates the location of the 2.0-kb LAT RNA species. In the mouse TG specimen the 1.5-kb LAT can also be detected.
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established with typical cytopathic changes and the formation of characteristic intraepithelial vesicles in vitro. Application of HSV on the lifted epithelium led to a nonproductive infection characterized by the lack of infectious virus in the culture supernatant and in the direct homogenate of the culture. PCR for HSV DNA revealed the presence of HSV DNA in this culture, and ISH for LAT RNA showed a cytoplasmic pattern of hybridization. The infection of fibroblasts in the collagen matrix was unlikely, however, since the ISH for HSV DNA was negative in the fibroblasts. Similarly, it was apparent that the positive PCR result could not be due to seed viruses only, attached on the culture surfaces, since the LAT hybridization was evident in all epithelial layers of the culture without intensified signals restricted to the external surface. The 2.0-kb LAT RNA species was detectable by Northern blot analysis in nonproductive and lytic forms of infection (Fig. 6). This inevitably shows that in the nonproductive infection HSV gene expression takes place, although in a restricted manner. Further study of HSV gene expression in this model is in progress. The PCR tests were validated with respect to contamination by including numerous HSV DNA-negative control specimens in the runs, which invariably remained PCR negative. Interestingly, LAT RNA was detected in the cytoplasm of the epithelial cells, instead of the nucleus. In the mouse models, LAT RNA is predominantly localized in the neuronal nucleus (4, 11, 25). Our present approach may provide an applicable model for in vitro studies of latent HSV infection once knowledge of the state of the virus in the raft culture is further established. Thus far, some of the models for in vitro latency have included treatments of the host cells with antiviral agents and interferon (3, 17). The use of a double mutant HSV with deficient a trans-inducing factor and a0 (23) has yielded an interesting state of infection in fibroblasts. However, in our culture model, the HSV-1 is the prototype F strain and the form of infection is modified exclusively by the timing of virus application relative to the epithelial differentiation in the culture. Further analysis of the state of the viral DNA and gene expression, which will finally establish the value of our model for in vitro HSV latency studies, is in progress. We are grateful to Bernard Roizman, University of Chicago, for the HSV clone and for the protocols for producing the stock virus. This study was supported by grants from the Medical Research Council of the Academy of Finland. REFERENCES 1. Asselineau, D., and M. Prunieras. 1984. Reconstruction of simplified skin: control of fabrication. Br. J. Dermatol. Suppl. 111:219–222. 2. Aurelius, E., B. Johansson, B. Sko ¨ldenberg, Å. Staland, and M. Forsgren. 1991. Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid. Lancet 337:189–192. 3. Biswal, N., A. Patel, and S. Max. 1988. Regulation of viral and cellular genes in a human neuroblastoma cell line latently infected with herpes simplex virus type 2. Mol. Brain Res. 3:95–106. 4. Dobson, A. T., F. Sederati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner, and L. T. Feldman. 1989. Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant
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