VIROLOGY
188, 311-318
(1992)
Activation
of Second-Messenger Pathways Reactivates Herpes Simplex Virus in Neuronal Cultures
Latent
R. L. SMITH,* L. I. PIZER,t E. M. JOHNSON, JR.,+ AND C. L. WILCOX-t*’ University of Colorado Health Sciences Center, Departments of *Neurology and tMicrobiology and Immunology, Denver, Colorado 80262; and + Washington University School of Medicine, Department of Pharmacology, St. Louis, Missouri 63 130 Received December
11, 199 1; accepted
February
5, 1992
Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of CAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a CAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways. o 1992 Academic press. I~C.
INTRODUCTION
animal models (Block et al., 1990; Clements and Stow, 1989; Hill eta/., 1990; Ho and Mocarski, 1989; Javieret al., 1988; Leib et al., 1989; Steiner et al., 1989). The function(s) of LATs, if any, is unknown. LATs mayfacilitate reactivaiton, as suggested by results with certain LAT deletion mutants showing decreased frequency of reactivation or delayed reactivation kinetics (Clements and Stow, 1989; Leib et a/., 1989; Steiner eta/., 1989). Previously we demonstrated that the presence of nerve growth factor (NGF) maintains latency of either HSV-1 or HSV-2 in sympathetic and sensory neuronal cultures prepared from rodents or primates (Wilcox et al., 1990). Latent HSV-1 infections can be established without treating the neuronal cultures with an antiviral agent. However, under these conditions, to achieve latent infection rather than productive infection, multiplicities of infections must be very low (0.03-l .O plaque-forming units of HSV-1 per cell), and, consequently, only a small percentage of the neurons harbor latent HSV-1 and many cultures develop a Iytic infection (Wilcox and Johnson, 1987). By limited treatment of the neuronal cultures with acyclovir for 7 days aftei inoculation with HSV, relatively high multiplicities of infection (5-10 plaque-forming units of HSV-1 per cell) can be used to establish latency in a high percentage of the neurons in the cultures (Wilcox and Johnson, 1987). As long as NGF is present, neither viral antigens present during the productive infection nor infectious virus is detected (Wilcox and Johnson, 1988). Cultures harboring latent virus are routinely maintained for 2-8
In the human disease,herpes simplex virus type-l (HSV-1) infects the skin or mucosal membrane and subsequently gains access to the superficial nerve terminals (Hill, 1985; Price, 1986). HSV-1 travels by rapid retrograde axonal transport from the site of inoculation to the corresponding ganglia, where the virus establishes a latent infection. Considerable evidence indicates that the predominant sites of latency are NGFdependent, peripheral, neural crest-derived neurons (Hill, 1985; Price, 1986; Thoenen and Barde, 1980). Following reactivation, the virus travels by antegrade axonal transport back to the corresponding epithelial surface and produces the characteristic recurrent lesions. While a variety of stimuli lead to reactivation, the molecular mechanisms controlling latency and reactivation of HSV-1 are not understood. During the latent HSV-1 infection, the virus appears to be maintained in an episomal state (Rock and Fraser, 1985), and viral gene expression is repressed except from a limited region of the genome, termed the latency-associated transcripts (LATs) (reviewed in Stevens, 1989). It appears that expression of LATs are not essential for productive infection, latency, or reactivation, since mutants with deletions in the region of the gene encoding the LATs are capable of establishing, maintaining, and reactivating from latent infections in ’ To whom reprint requests should be addressed. 311
0042-6822192
$3.00
Copyright 0 1992 by Academic Press, Inc. All rlghts of reproduction in any form reserved.
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weeks without evidence of infectious virus. Upon removal of NGF, viral antigens are detected in essentially all of the neurons followed by the appearance of infectious virus after an additional 24-48 hr (Wilcox and Johnson, 1988). Recent studies indicate that during the latent infection of neurons in vitro, viral transcription is limited to the LATs (Doerig et al., 1991 a) and a latency-associated viral antigen apparently encoded by the region corresponding to the LATs is present (Doerig eta/., 1991 b). Thus, thein vitro model of HSV-1 latency in neurons reproduces many of the characteristics of the animal models and the human disease. The in vivo models have been invaluable for describing many aspects of the HSV-1 infection; however, the numbers of neurons in a ganglion harboring latent HSV-1 are few, estimated to be between 0.4 and 10% of the neurons (Hill, 1985; Stevens, 1989). The usual stimulus for reactivation of latent HSV-1 in the mouse model is explantation of the ganglia, thus making studies of mechanisms controlling latency difficult. The in vitro model of HSV latency in neurons provides a system in which mechanistic questions of HSV-1 latency can be addressed with greater ease. In humans, different physiologic stimuli lead to reactivation of latent HSV-1, including fever, stress, ultraviolet irradiation, menstruation, and damage or manipulation of the ganglia or nerve roots (Hill, 1985; Price, 1986). However, the mechanisms controlling viral latency and reactivation remain poorly understood. Determining the role of cellular pathways which mediate HSV-1 reactivation is important for understanding HSV-1 latency and reactivation. The demonstration of NGF regulation of HSV-1 latency in neurons in culture indicates that latent virus is capable of responding to a cellular signaling pathway involving a neurotrophic factor (Wilcox and Johnson, 1987). Here we report studies using the in vitro neuronal model of HSV latency to examine the effects of CAMP-mediated pathways, protein kinase C activation, and NGF on the regulation of HSV-1 latency.
MATERIALS AND METHODS Virus and neuronal culture preparation HSV-1 (KOS) was grown and quantified by plaqueformation assays on confluent Vero cells obtained from the American Type Culture Collection (Rockville, MD). Neuronal cultures were prepared from either sympathetic (superior cervical ganglia) or sensory ganglia (dorsal root ganglia) of embryonic rats as previously described (Wilcox and Johnson, 1987; Wilcox et a/., 1990). The neuronal cultures used in this study contained approximately 1O3 neurons in each well of a 24well cluster dish. Latently infected neuronal cultures
ET AL.
were prepared as previously described (Wilcox et a/., 1990). Briefly, 10 to 12 days after plating, medium containing 50 PM acyclovir (Burroughs Wellcome Co., Research Triangle Park, NC) was added to the cultures 12 hr prior to and for 7 days following inoculation with 5-l 0 PFU of HSV-1 per cell. Seven days following inoculation, the medium containing acyclovir was replaced with standard culture medium, which lacks acyclovir. The standard neuronal culture medium consists of 10% newborn bovine serum (GIBCO; Grand Island, NY) and 50 rig/ml of 2.5 S mouse NGF in Eagle’s minimum essential medium (GIBCO). Fourteen days or longer after viral inoculation, the cultures were used in the studies described.
Reactivation of latent HSV-1 Neuronal cultures harboring latent HSV-1 were treated as described in the text and legends. NGF deprivation was achieved by adding lo/o guinea pig antimouse NGF serum (titer >20,000) as has been previously described (Wilcox and Johnson, 1987). Drugs were prepared at the concentrations indicated by dilution in standard neuronal culture medium which contained NGF (50 rig/ml). Neuronal cultures were tested for the presence of infectious virus in plaque-formation assays using culture supernatants or total culture lysates on Vero indicator cells.
RESULTS Stimulation of CAMP-mediated pathways causes reactivation of latent HSV-1 To determine if CAMP-mediated pathways are involved in controlling HSV-1 latency, neuronal cultures harboring latent HSV-1 were incubated with the membrane-permeable CAMP analog, chlorophenylthioCAMP (CPT-CAMP) at a concentration of 0.5 mM, which has been shown to activate CAMP-dependent processes in neurons (Rydel and Greene, 1988). Reactivation occurred in 100% of the cultures treated with this highly selective CAMP analog (Table 1). Incubation with CPT-CAMP for as briefly as 1 hr resulted in reactivation in 100% of the cultures. Reactivation was also observed when neurons were treated with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), orthe adenylate cyclase activator, forskolin (Table 1). The concentrations of forskolin or IBMX required to produce reactivation were similar to the concentrations required to raise CAMP levels in cells in culture (Greene et al., 1986). Reactivation was not observed in cultures harboring latent HSV-1 which were untreated. Mock-infected neuronal cultures were maintained in the presence of CPT-CAMP, IBMX, or forskolin for
SECOND-MESSENGER TABLE 1 REACTIVATIONOFLATENT HSV-1 EIYCAMPAGONISTS %Reactivationa (No. positive/No. tested) Treatment
SCGN
DRGN
Untreated controls CPT-cAMPb (500 fin/l) Forskolin (100 4’0 (50 44 (5 WW lBMXd (1 mM)
0 (O/l 2) 100 (818)
0 (O/8) 100 (8/8)
100 100 0 100
100 (8/8) ND" ND 100 (818)
(8/8) (818) (O/8) (818)
a Neuronal cultures, either sympathetic (SCGN) or sensory (DRGN), were infected with HSV-1 as described under Materials and Methods. Two weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested In plaque-formation assays for infectious virus. b Chlorophenylthio-cyclic AMP. c Not determined. d lsobutylmethylxyanthine.
7-l 0 days without loss of viability of the neurons (data not shown). The data indicate that the agents are not toxic to neurons even with prolonged exposure, and these results are supported by published observations of prolonged treatments of neurons with CPT-CAMP (Rydel and Greene, 1988). These results strongly suggest that CAMP-mediated processes produce reactivation of latent HSV-1.
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AND HSV-1
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Prolonged treatment with high concentrations of phorbol esters dramatically down-regulates the cellular level of PKC (Young eta/., 1987; Ase eta/., 1988). In rat neurons in culture, treatment with PMA reduces PKC activity below detectable levels within 1O-20 hr but produces minimal changes in levels of CAMP-dependent and Ca2+-stimulated protein kinase activity (Matthies et al., 1987). To determine if HSV-1 latency and reactivation occur under conditions which downregulate PKC, neurons were maintained in a high concentration of PMA (10m6 m for 1 week prior to establishment of latent HSV-1 infection and then were continuously treated with 1Om6M PMA. Viral latency was established and maintained in the presence of PMA in sensory neuronal cultures. Reactivation occurred in 100% of the cultures following treatment with either 1% anti-NGF (8/8 cultures) or 500 pn/l CPT-CAMP (8/8 cultures). These results suggest that PKC is not necessary for the establishment, maintenance, or reactivation of latent HSV-1 and suggest that reactivation produced by CAMP and anti-NGF is mediated by pathways independent of PKC. Comparison of reactivation kinetics of latent HSV-1 after treatment with NGF deprivation, CPT-CAMP, or PMA We observed that neuronal cultures harboring latent virus developed cytopathic effects at different rates following treatments with activators of CAMP-mediated pathways or PKC when compared with NGF deprivation. To characterize the effects of different stimuli on
Activation of protein kinase C results in reactivation of latent HSV-1 Protein kinase C (PKC) has important regulatory functions in many cell types. Therefore, to determine if activation of protein kinase C-mediated pathways rem suits in reactivation of latent HSV-1, neuronal cultures harboring latent HSV-1 were treated with the potent activator of PKC, phorbol myristate acetate (PMA). Reactivation occurred at concentrations of 10-l’ to 10-l’ M PMA with slightly greater sensitivity to reactivation by PMA in sensory neuron cultures (Table 2), consistent with concentrations shown to activate PKC (Nishizuka, 1986). The vehicle control (7 X 1O-’ M dimethyl sulfoxide) and the inactive 4a-phorbol analog of PMA had essentially no effect on cultures harboring latent virus. The 10% reactivation with the 4a-PMA analog in the sensory neuronal cultures was most likely the result of spontaneous reactivation, which we have rarely observed with sensory but not sympathetic neuron cultures (Wilcox et a/., 1990). These results indicate that a highly specific activator of protein kinase C can reactivate latent HSV-1.
TABLE 2 TREATMENTWITH
PHORBOLESTER
REACTIVATES LATENT HSV-1
%Reactivationa (No. positive/No. tested) Treatment Vehicle controlb 4aPDDC (1 O-6 M) PMAd(lO-’ M, 1 hr) PMA (10-l’ M) PMA (10-l’ M)
SCGN 0 0 100 100 0
(O/8) (O/8) (12112) (4/4) (O/4)
DRGN 0 10 100 100 100
(O/8) (l/10) (8/8) (5/5) (5/5)
’ Neuronal cultures, either sympathetic (SCGN) or sensory (DRGN), were infected with HSV-1. Two weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested in plaque-formation assays for infectious virus. b Latently infected neuronal cultures were treated with 7 X 1 O-’ M drmethyl sulfoxide. which was the final concentration in the medium used to prepare the phorbol esters. ’ 4-a-Phorbol-12decanoate-13.decanoate. d [email protected].
SMITH
314
-I 0
1
2 3 Days after treatment
4
I 5
FIG. 1. Time course of reactivation of latent HSV-1 after treatment with anti-NGF. CPT-CAMP, or PMA for 1 hr. Two weeks postinoculation, sensory neuronal cultures containing approximately 1 O3 neurons harboring latent HSV-1 were treated as indicated. Viral titers of culture lysates were determined in plaque-formation assays. Shown are the mean viral titers from four cultures f the SEM harvested at the times indicated after cultures were untreated (O), treated with anti-NGF (w), treated with 500 pM CPT-CAMP (A), or treated with 10-g M PMA (0).
reactivation, cultures harboring latent HSV-1 were treated with anti-NGF, CPT-CAMP, or PMA for 1 hr. The drugs were removed by washing the cultures with drug-free medium. At various times after treatment the cultures were analyzed for the presence of infectious virus. Untreated cultures harboring latent HSV-1 did not show evidence of infectious virus. Mock-infected cultures treated with anti-NGF, CPT-CAMP, or PMA did not produce infectious virus (data not shown). Cultures harboring latent HSV-1 produced detectable infectious virus at different times after treatment. The kinetics of reactivation were similarly accelerated by activation of CAMP-mediated pathways or PKC activation compared to those cultures treated with anti-NGF (Fig. 1). Following treatment with CPT-CAMP or PMA, cultures harboring latent HSV-1 produced infectious virus more than 24 hr prior to similar cultures treated with antiNGF (Fig. 1). Treatment of neuronal cultures harboring latent HSV-1 with either CPT-CAMP or PMA appears to accelerate the reactivation process compared with reactivation following anti-NGF treatment.
ET AL.
Neuronal cultures were treated with 2-AP to determine if this compound would affect HSV-1 latency or reactivation. 2-AP alone at a concentration of 1 ml\/l had no effect on neuronal cultures harboring latent HSV-1. In cultures treated with anti-NGF alone reactivation occurred in 100% of the cultures (Table 3). However, when neurons harboring latent HSV-1 were treated with anti-NGF in the presence of 2-AP, reactivation was blocked (Table 3). Similarly, 2-AP treatment blocked reactivation produced by CPT-CAMP. One week after removal of 2-AP, anti-NGF treatment reactivated latent virus from 100% of the cultures, indicating that both the neurons and the virus were able to produce reactivation and that the effects of 2-AP were reversible (Table 3). In contrast to the results with antiNGF and CPT-CAMP, however, 2-AP treatment did not prevent HSV-1 reactivation following incubation with PMA. 2-Aminopurine exerted no apparent toxic effects on neurons at the concentrations used to prevent reactivation. Consistent with the report of Greene et al. (1990) neuronal cultures were maintained for several weeks in the presence of 1 mM 2-AP without loss of viability or morphologic changes (data not shown). Furthermore, 2-AP did not directly interfere with viral replication in either Vero cells or sensory neuronal cultures. During a productive infection in Vero cells or neuronal cultures, 48 hr after inoculation with HSV-1 (multiplicity of infection of lo), viral titers were essentially identical with (6.2 X 1O* PFU/ml in Vero cells; 1.05 X 1O4PFU/ml in neuronal cultures) or without (6.5 X lo* PFU/ml in TABLE 3 THE EFFECTS OF 2-AMINOPURINE ON THE REACTIVATION OF LATENT HSV-i
Treatment
%Reactivation” (No. positive/No. tested) 0 (O/22) 100 (32/32)
A novel protein kinase inhibitor prevents reactivation of latent HSV caused by NGF deprivation or CPT-CAMP
Nonimmune serum (l%, 1 hr) Anti-NGF serum (lo/o, 1 hr) Anti-NGF serum (l%, 1 hr) + 2-APb (1 mM. 2 hr) CPT-cAMPC (500 pM, 1 hr) CPT-CAMP (500 PM, 1 hr) + 2-AP (1 mM, 2 hr) PMA“ (1 O-’ M. 1 hr) PMA (1 O-g M, 1 hr) + 2-AP (1 mM. 2 hr)
2-Aminopurine (2-AP) is known to inhibit NGF-stimulated protein kinases (Volonte et a/., 1989), as well as elF2a protein kinases (De Benedetti and Baglioni, 1983) but has no effect on protein kinase A or C (Volonte et al., 1989). 2-AP also blocks some of the actions of NGF (Volonte et al., 1989; Greene et al., 1990).
B Sensory neuronal cultures were infection with HSV-1. TWO weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested in plaque-formation assays for infectious virus. b 2-Aminopurine. ’ Chlorophenylthio-cyclic AMP. d 4-fl-Phorbol-12.myristate-13.acetate.
0 (O/l 0) 100 (8/8) 0 (O/l 0) 100 (8/8) 100 (lO/lO)
SECOND-MESSENGER
Vero cells; 1 .l 1 X 1O4 PFU/ml in neuronal treatment with 1 mM 2-AP.
cultures)
DISCUSSION Role of NGF in HSV latency The predominate sites of HSV latency are neural crest-derived sensory and sympathetic neurons which depend on NGF for survival during development and continue to require NGF for maintenance of normal function (Thoenen and Barde, 1980). NGF is produced in small quantities by the target tissue, providing the innervating neurons with trophic support (Brandtlow et al., 1987). While some of the stimuli which reactivate latent HSV-1 in viva are consistent with producing deprivation of trophic support, including central rhizotomy, axotomy, and damage to the target tissue (Hill, 1985; Price, 1986) many stimuli which produce reactivation in humans, such as stress and fever, may have distinct pathways. We have previously demonstrated that even brief NGF deprivation produces reactivation of latent HSV in vitro (Wilcox and Johnson, 1987, 1988; Wilcox eta/., 1990). The role of NGF in the regulation of HSV latency in viva remains to be determined. However, in the mouse, NGF treatment greatly ameliorates the productive HSV-1 infection in the ganglia (Johnson et al., 1988). Determining the molecular basis of NGF dependence of HSV-1 latency remains a major challenge. Recently, understanding of NGF signaling pathways has been advanced by the demonstration of an NGF receptor that is a ligand-stimulated tyrosine kinase and is homologous to the proto-oncogene trk (Kaplan eta/., 1991a,b; Klein el al., 1991). The intracellular signal transduction that follows NGF binding remains unknown; however, activation of specific serine protein kinases (Aletta et al., 1988; Volonte et a/., 1989) and altered gene expression occur within minutes of NGF addition to responsive cells (Greenberg et a/., 1985). Study of these NGF-stimulated serine protein kinases is aided by their selective inhibition by purine analogs, including 2-AP, which also block some of the biochemical and morphologic changes associated with NGFmediated responses (Greene et al., 1990). Therefore, there are a wide range of signaling mechanisms that could potentially mediate HSV-1 reactivation in response to NGF deprivation. Activation of CAMP-dependent reactivates latent HSV-1
processes
or PKC
In this report we used the in vitro model of HSV latency in neurons to demonstrate reactivation in response to cellular second-messenger pathways. The
PATHWAYS
AND HSV-1
315
observation that the highly specific CAMP analog, CPT-CAMP, reactivates latent HSV-1 at concentrations previously shown to activate CAMP-dependent pathways in neurons in culture (Rydel and Greene, 1988) strongly suggests that CAMP-dependent mechanisms can control viral latency. This result is strengthened by the observation that both IBMX and forskolin, which activate CAMP-dependent pathways via inhibition of phosphodiesterase and direct activation of adenylate cyciase, respectively, also reactivated latent HSV-1. With forskolin, some caution in the interpretation is required since forskolin is reported to have effects on potassium channels which are independent of CAMPmediated effects (Hoshi et al., 1988). These results indicate that latent virus can reactivate in response to CAMP-mediated signals. The effects of NGF deprivation and CAMP analogs on HSV reactivation share common features: both treatments can be blocked by 2-AP and both NGF deprivation and CPT-CAMP treatments produce reactivation in neurons previously treated with high concentrations of phorbol esters, suggesting that PKC is not required for either effect. The effects of CAMP agonist on NGF-mediated responses remain contradictory. Agonist of CAMP can partially replace NGF for neuronal survival and morphologic differentiation (Rydell and Greene, 1988) and produce increased phosphotylation of some of the same intracellular targets (McTigue et al., 1985; Cremins et al., 1986). However, CAMP agonist blocks some of the actions of NGF (Greene et a/., 1986). NGF treatment may minimally increase neuronal CAMP in neuronal cultures derived from dorsal root ganglia (Narumi and Fujita, 1978; Skaper et al., 1979) and superior cervical ganglia (Nikodijevic et a/., 1975); the mechanism remains incompletely understood, but does not appear to involve direct stimulation of adenylate cyclase (Race and Wagner, 1985). However, other reports suggest NGF deprivation has little effect on CAMP levels in neurons in culture (Johnson, unpublished observation; Narumi and Fujita, 1978; Skaper et al., 1979). Consequently, HSV-1 reactivation caused by NGF deprivation is unlikely to be the result of elevation of the neuronal CAMP level. Furthermore, the ability of CPT-CAMP to produce reactivation even in the presence of NGF suggests that CAMP signaling can occur independent of changes in NGF. Phorbol esters produce pleotrophic effects on cells, which are mediated by high affinity receptors, and there is considerable evidence that the major high affinity receptor is PKC, which is activated by these compounds (Nishizuka, 1986, review). PKC is involved in cellular second-messenger pathways involving release of diacylglycerol and inositol phosphates (Nishizuka, 1988; Cantley et al., 1991). Therefore, the ability of
316
SMITH
phorbol esters to reactivate latent HSV-1 indicates that acute activation of signaling pathways involving activation of PKC can reactivate latent HSV-1. Previous studies have demonstrated that rat neurons in culture survive after down-regulation of PKC by prolonged treatment with high concentrations of phorbol esters (Matthies et al., 1987). In this study survival of rat neurons was not effected by long term treatment with high concentrations of phorbol esters, and the ability of the neuronal cultures to support latent infections or reactivate in response to CAMP analogs or NGF deprivation was not altered by phorbol ester treatment, suggesting that PKC is not necessary for the establishment of HSV-1 latency or reactivation. This suggests that the actions of PKC on latency are independent of CAMP or NGF deprivation. The data suggest that both PKC- and CAMP-dependent pathways can function to control reactivation of latent HSV-1 in the neuronal cultures and may act independently to produce reactivation. The significant delay in the appearance of infectious virus produced by NGF deprivation suggests that the signal resulting from NGF deprivation may be distinct from that resulting from CPT-CAMP or PMA treatment. Alternatively, the ganglia from which the cultures were derived contain heterogeneous populations of neurons; however, it is unknown if differences in neurons persist in culture. It is possible that different populations of neurons could respond to different reactivation stimuli. The effects of 2-aminopurine on reactivation of latent HSV-1 NGF-stimulated serine-protein kinases have been described; these include the protein kinase N and MAP-l kinase (Aletta et al., 1988; Volonte et al., 1989). 2-AP blocks activation of these NGF-dependent kinases both in vivo and in vitro (Aletta et a/., 1988; Volonte et al., 1989). It would be expected that if the action of 2-AP is to inhibit NGF-dependent systems, then 2-AP would produce reactivation of latent virus by mimicking NGF deprivation. However, we observed the opposite effect; the treatment of neuronal cultures with 2-AP prevented reactivation caused by either anti-NGF or CPT-CAMP. The reactivation produced by PMA was not prevented by 2-AP treatment. The failure of 2-AP to prevent reactivation by PMA may result from the difficulty in completely removing the lipophilic phorbol ester. An alternative explanation is that PMA reactivates HSV-1 by a distinct pathway which is not affected by 2-AP, and the mechanism of PMA action therefore differs from reactivation by CAMP agonist or NGF deprivation. The concentration of 2-AP effective in preventing HSV-1 reactivation was similar to the concentrations
ET AL.
reported to block NGF-mediated responses (Greene et al., 1990) and inhibit the NGF-stimulated protein kinase N and MAP 1.2 kinases (Aletta et al., 1988; Volonte et a/., 1989). 2-AP also is known to inhibit the elF2a kinases at a similar concentration (De Benedetti and Baglioni, 1983), and, presumably, based on this action leads to selective mRNA translation (Kaufman and Murtha, 1987). 2-AP prevents c-fos message induction by NGF in PC-l 2 cells (Volonte eta/., 1989) and inhibits induction of the proto-oncogenes c-fos and c-myc by interferon in an osteosarcoma cell line (Zinn et a/., 1987). It remains unclear if any of these reported actions are related to the observed effects of 2-AP on the inhibition of HSV-1 reactivation. In contrast, the actions of 2-AP are not likely to be the result of nonspecific toxicity. This conclusion is supported by our observations and by recent reports that rat sensory neurons can be maintained in the presence of 10 mM 2-AP for days without loss of viability (Greene et al., 1990). In addition HSV-1 reactivated readily after removal of 2-AP, indicating neuronal viability. Furthermore, 2-AP does not alter the pattern of phosphorylation observed in HeLa cells or in NGF-responsive PC-l 2 cells and does not alter protein synthesis (Zinn et a/., 1987; Volonte et a/., 1989). In vitro, 2-AP does not inhibit activity of CAMP-dependent protein kinase or PKC (Volonte et a/., 1989). Our results indicate that at least two cellular secondmessenger pathways reactivate latent HSV-1. This is consistent with the wide range of physiological stimuli capable of reactivating HSV-1 in vivo. The ability of a latent virus to respond to stress in the host at an early stage may be important to viral propagation and survival. Our results also suggest that agents such as 2AP may be of value in identifying specific cellular mechanisms responsible for the control of HSV-1 latency. ACKNOWLEDGMENTS We thank G. Huitt for expert technical assistance and Drs. J. Schaack, D. Gilden. and K. Escudero for critical review of the manuscript. This work was supported in part by National Institutes of Health Grants NS29046 and AGO7347 and National Multiple Sclerosis Society Grant RG2344-A.
REFERENCES ALET~A. J. M., LEWIS, S. A., COWAN, N. J., and GREENE, L. A. (1988). Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2. /. Cell Biol. 106, 1573-l 581. ASE, K., BERRY,N., KISHIMOTO,A., and NISHIZUKA,Y. (1988). Differential down-regulation of protein kinase C subspecies in KM3 cells. FEES’ Left. 236, 396-400. BANDTLOW, C. E., HEUMANN, R., SCHWAB, M. E., and THOENEN, H. (1987). Cellular localization of nerve growth factor synthesis by in situ hybridization. EMBO /. 6, 891-899.
SECOND-MESSENGER BARINGER,R. J., and SWOVELAND,P. (1973). Recovery of herpes simplex virus from human trigeminal ganglions. N. fngl. /. Med. 288, 648-650. BASTIAN, F. O., RAESON, A. S., YEE, C. L., and TRALKA, T. S. (1972). Herpes hominis: Isolation from human trigeminal ganglions. Science 178, 306-307. BLOCK, T. M., SPIVACK, J. G., STEINER, I., DESHMANE, S., MCINTOSH, M. T.. LIRE~E, M. R. P.. and FRASER,N. W. (1990). A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics. /. Viral. 64, 3417-3426. CANTLEY, L. C., AUGER, K. R., CARPENTER,C., DUCKWORTH, B., GMZIANI, A., KAPELLER,R., and SOLTOFF, S. (1991). Oncogenes and signal transduction. Cell 64, 281-302. COOK, M. L., BASTONE,V. B., and STEVENS,J. G. (1974). Evidence that neurons harbor latent virus. Infect. Immun. 9, 946-951. CLEMENTS, G. B., and STOW, N. D. (1989). A herpes simplex virus type 1 mutant containing a deletion within immediate early gene 1 is latency-competent in mice. /. Gen. \/irol. 70, 2501-2506. CREMINS, J., WAGNER, J. A., and HALEGOUA, S. (1986). Nerve growth factor action IS mediated by cyclic AMP and Ca++/phospholiplddependent protein kinases. J. Cell Biol. 103, 887-893. DE BENEDETTI,A., and BAGLIONI, C. (1983). Phosphorylation of initiation factor elF-2a, binding of mRNA to 48 S complexes, and its reutilization In Initiation of protein synthesis. /. Biol. Chem. 258, 14,552-14,562. DOERIG, C., PIZER. L. I., and WILCOX, C. L. (1991a). An antigen encoded by the latency-associated transcript in neuronal cell cultures latently infected with herpes simplex virus type 1. /. Viral. 65, 2724-2727. DOERIG, C., PIZER, L. I., and WILCOX, C. L. (1991 b). Detection of the latency-associated transcript in neuronal cultures during the latent infection with herpes simplexvirus type 1. Virology 183,423%426. GREENBERG,M. E., GREENE,L. A., ~~~ZIFF, E. B. (1985). Nervegrowth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcnption in PC1 2 cells. J. Biol. Chem. 260, 14,101-14,110. GREENE, L. A., DREXLER,S. A., CONNOLLY, J. L., RUKENSTEIN,A., and GREENE, S. H. (1986). Selective inhibition of responses to nerve growth factor and of microtubule-associated protein phosphorylation by activators of adenylate cyclase. J. Cell Biol. 103, 19671978. GREENE,L. A., VOLONTE, C., and CHALAZONITIS,A. (1990). Purine analogues inhibit nerve growth factor-promoted neunte outgrowth by sympathetic and sensory neurons. J. Neurosci 10, 1479-1485. HILL, J. M.. SEDARATI, F., JAVIER, R. T., WAGNER, E. K., and STEVENS. J. G. (1990). Herpes simplex latent phase transcription facilitates in viva reactivation. Virology 174, 1 17-l 25. HILL, T. J. (1985). Herpes simplex virus latency. In “The Herpesviruses” (B. Rolzman, Ed.). Plenum, New York. Ho, D. Y., and MOCARSKI, E. S. (1989). Herpes simplex virus latent RNA IS not required for latent Infection in the mouse. Proc. Nat/. Acad. SC;. USA 86,7596-7600. HOSHI, T., GARBER.S. S.. and ALDRICH, R. W. (1988). Effects of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation. Science 240, 1652-l 654. JAVIER, R. T., STEVENS, J. G., DISSETTE, V. B., and WAGNER, E. K. (1988). A herpes simplex virus transcript abundant in latently Infected neurons is dispensable for the establishment of the latent state. Virology 166, 254-257. JOHNSON, E. M., JR., MANNING, P. T., and WILCOX, C. L. (1988). The biology of nerve growth factor in viva. ln “Neurobiology of Amino Acids Peptides and Trophic Factors” (J. A. Ferrendelli, R. Collins, and E. M. Johnson, Jr., Eds.). Kluwer Academic, Norwell, MA.
PATHWAYS
AND HSV-1
317
KAPLAN, D. R., HEMPSTEAD, B. L., MARTIN-ZANCA, D., CHAO, M. V., and PARADA, L. F. (1991a). The trk proto-oncogene product: A signal transducing receptor for nerve growth factor. Science 252, 554-557. KAPLAN, D. R., MARTIN-ZANCA, D., and PARADA, L. F. (199 1b). Tyrosine phosphorylation and tyrosine kinase activity of the frk protooncogene product induced by NGF. Nature 350, 158-I 60. KAUFMAN, R. J., and MURTHA, P. (1987). Translational Control Mediated by Eucaryotic initiation factor-z is restricted to specific mRNAs in transfected cells. Molec. Cell. Biol. 7, 1568-l 571. KLEIN, R., JING, S., NANDURI, V., O’ROURKE, E., and BARBACID, M. (1991). The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65, 189-l 97. LEIB, D. A., BOGARD, C. L., KLOSZVNENCHAK,M., HICKS, K. A., COEN, D. M., KNIPE, D. M., and SCHAFFER,P. A. (1989). A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with a reduced frequency. J. Viral. 63, 2893-2900. LEIB, D. A., NADEAU, K. C.. RUNDLE, S. A., and SCHAFFER,P. A. (1991). The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional CAMP-response element: Role of the latency-associated transcripts and CAMP in reactivation of viral latency. Proc. Nat/. Acad. Sci. USA 88, 48-52. MA~HIES, H. 1. G., PALFREY,H. C., HIRNING, L. D., and MILLER, R. J. (1987). Down regulation of protein kinase C in neuronal cells: Effects on neurotransmitter release. J. Neurosci. 7, 1 198-l 206. MCTIGUE, M., CREMININS, J., and HALEGOUA, S. (1985). Nerve growth factor and other agents mediate phosphorylation and activation of tyroslne hydroxylase. J. Bioi. Chem. 260, 9047-9056. NARUMI, S., and FUJITA, I. (1978). Stimulatory effects of substance P and nerve growth factor on neurite outgrowth in embryonic chick dorsal root ganglia. Neuropharmacology 17, 73-76. NIKODIJEVIC,B., NIKODIJEVIC,O., Yu, M. W., POLLARD, H., and GUROFF. G. (1975). The effects of substance P and nerve growth factor on cyclic AMP levels in superior cemical ganglia of the rat. Proc. Nat/. Acad. Sci. USA 72, 4769-4771. NISHIZUKA, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-312. NISHIZUKA, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661665. PRICE, R. W. (1986). Neurobiology of human herpesvirus infections. CRC Crit. Rev. C/in. Neurobioi. 2, 61-123. PRICE, R. W.. KATZ, B. J., and NOTKINS.A. L. (1975). Latent infection of the peripheral ANS with herpes simplex virus. Nature (London) 257, 686-688. RACE, H. M., and WAGNER, J. A. (1985). Nerve growth factor affects CAMP metabolism but not by directly stimulation adenylate cyclase activity. J. Neurochem. 44, 1588-l 592. ROCK, D. L., and FRASER,N. W. (1985). Latent herpes simplex type 1 DNA contains two copies of the virion DNA joint region. J. Viral. 55, 849-852. ROWLAND, E. A., MULLER, T. H., GOLDSTEIN, M.. and GREENE, L. A. (1987). Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC 12 cells. J. Biol. Chem. 262, 7504-7513. RYDEL, R. E., and GREENE, L. A. (1988). CAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. Nat/. Acad. Sci. USA 85, 1257-1261. SKAPER, S. D., BOTTENSTEIN,J. E., and VARON, S. (1979). Effects of nerve growth factor on cyclic AMP levels in embryonic chtck dor-
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sal root ganglia following factor deprivation. 1. Neurochem. 32, 1845-1851. STEINER,I., SPIVACK,J. G., LIRETTE,R. P., BROWN,S. M., MCLEAN, A. I?., SUBAK-SHARPE, 1. H., and FASER,N. W. (1989). Herpes simplex virus type 1 transcripts are evidently not essential for latent infection. EM60 1. 8, 505-51 1. STEVENS,1. G. (1989). Human herpesviruses: A consideration of the latent state. Microbial. Rev. 53, 3 18-332. THOENEN,H., and BARDE,Y.-A. (1980). Physiology of nerve growth factor. Physiol. Rev. 60, 1284-l 335. VOLONTE,C. A., RUKENSTEIN, LOEB,D. M., and GREENE,L. A. (1989). Differential inhibition of nerve growth factor responses by purine analogues: Correlation with inhibition of a nerve growth factor-activated protein kinase. J. Cell Biol. 109, 2395-2403. WILCOX,C. L., and JOHNSON,E. M., JR.(1987). Nerve growth factor
deprivation results in the reactivation of latent herpes simplexvirus in vitro. J. Viral. 61, 231 1-2315. WILCOX,C. L., and JOHNSON,E. M., JR. (1988). Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro. 1. Viral. 62, 393-399. WILCOX,C. L., SMITH. R. L., FREED,C. R., and JOHNSON,E. M., JR. (1990). Nerve growth factor dependence of herpes simplex latency in peripheral sympathetic and sensory neurons in vitro. 1. Neurosci. 10, 1268-l 275. YOUNG,S., PARKER,P. J., ULLRICH,A., and STABEL,S. (1987). Downregulation of protein kinase C is due to an increased rate of degradation. Biochem. 1. 244, 775-779. ZINN, K., KELLER,A., WHITTEMORE, L., and MANIATIS,T. (1987). 2Aminopurine selectively inhibits the induction of @Interferon, c-ios and c-myc gene expression. Science 240, 2 1O-21 3.
188, 311-318
(1992)
Activation
of Second-Messenger Pathways Reactivates Herpes Simplex Virus in Neuronal Cultures
Latent
R. L. SMITH,* L. I. PIZER,t E. M. JOHNSON, JR.,+ AND C. L. WILCOX-t*’ University of Colorado Health Sciences Center, Departments of *Neurology and tMicrobiology and Immunology, Denver, Colorado 80262; and + Washington University School of Medicine, Department of Pharmacology, St. Louis, Missouri 63 130 Received December
11, 199 1; accepted
February
5, 1992
Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of CAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a CAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways. o 1992 Academic press. I~C.
INTRODUCTION
animal models (Block et al., 1990; Clements and Stow, 1989; Hill eta/., 1990; Ho and Mocarski, 1989; Javieret al., 1988; Leib et al., 1989; Steiner et al., 1989). The function(s) of LATs, if any, is unknown. LATs mayfacilitate reactivaiton, as suggested by results with certain LAT deletion mutants showing decreased frequency of reactivation or delayed reactivation kinetics (Clements and Stow, 1989; Leib et a/., 1989; Steiner eta/., 1989). Previously we demonstrated that the presence of nerve growth factor (NGF) maintains latency of either HSV-1 or HSV-2 in sympathetic and sensory neuronal cultures prepared from rodents or primates (Wilcox et al., 1990). Latent HSV-1 infections can be established without treating the neuronal cultures with an antiviral agent. However, under these conditions, to achieve latent infection rather than productive infection, multiplicities of infections must be very low (0.03-l .O plaque-forming units of HSV-1 per cell), and, consequently, only a small percentage of the neurons harbor latent HSV-1 and many cultures develop a Iytic infection (Wilcox and Johnson, 1987). By limited treatment of the neuronal cultures with acyclovir for 7 days aftei inoculation with HSV, relatively high multiplicities of infection (5-10 plaque-forming units of HSV-1 per cell) can be used to establish latency in a high percentage of the neurons in the cultures (Wilcox and Johnson, 1987). As long as NGF is present, neither viral antigens present during the productive infection nor infectious virus is detected (Wilcox and Johnson, 1988). Cultures harboring latent virus are routinely maintained for 2-8
In the human disease,herpes simplex virus type-l (HSV-1) infects the skin or mucosal membrane and subsequently gains access to the superficial nerve terminals (Hill, 1985; Price, 1986). HSV-1 travels by rapid retrograde axonal transport from the site of inoculation to the corresponding ganglia, where the virus establishes a latent infection. Considerable evidence indicates that the predominant sites of latency are NGFdependent, peripheral, neural crest-derived neurons (Hill, 1985; Price, 1986; Thoenen and Barde, 1980). Following reactivation, the virus travels by antegrade axonal transport back to the corresponding epithelial surface and produces the characteristic recurrent lesions. While a variety of stimuli lead to reactivation, the molecular mechanisms controlling latency and reactivation of HSV-1 are not understood. During the latent HSV-1 infection, the virus appears to be maintained in an episomal state (Rock and Fraser, 1985), and viral gene expression is repressed except from a limited region of the genome, termed the latency-associated transcripts (LATs) (reviewed in Stevens, 1989). It appears that expression of LATs are not essential for productive infection, latency, or reactivation, since mutants with deletions in the region of the gene encoding the LATs are capable of establishing, maintaining, and reactivating from latent infections in ’ To whom reprint requests should be addressed. 311
0042-6822192
$3.00
Copyright 0 1992 by Academic Press, Inc. All rlghts of reproduction in any form reserved.
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weeks without evidence of infectious virus. Upon removal of NGF, viral antigens are detected in essentially all of the neurons followed by the appearance of infectious virus after an additional 24-48 hr (Wilcox and Johnson, 1988). Recent studies indicate that during the latent infection of neurons in vitro, viral transcription is limited to the LATs (Doerig et al., 1991 a) and a latency-associated viral antigen apparently encoded by the region corresponding to the LATs is present (Doerig eta/., 1991 b). Thus, thein vitro model of HSV-1 latency in neurons reproduces many of the characteristics of the animal models and the human disease. The in vivo models have been invaluable for describing many aspects of the HSV-1 infection; however, the numbers of neurons in a ganglion harboring latent HSV-1 are few, estimated to be between 0.4 and 10% of the neurons (Hill, 1985; Stevens, 1989). The usual stimulus for reactivation of latent HSV-1 in the mouse model is explantation of the ganglia, thus making studies of mechanisms controlling latency difficult. The in vitro model of HSV latency in neurons provides a system in which mechanistic questions of HSV-1 latency can be addressed with greater ease. In humans, different physiologic stimuli lead to reactivation of latent HSV-1, including fever, stress, ultraviolet irradiation, menstruation, and damage or manipulation of the ganglia or nerve roots (Hill, 1985; Price, 1986). However, the mechanisms controlling viral latency and reactivation remain poorly understood. Determining the role of cellular pathways which mediate HSV-1 reactivation is important for understanding HSV-1 latency and reactivation. The demonstration of NGF regulation of HSV-1 latency in neurons in culture indicates that latent virus is capable of responding to a cellular signaling pathway involving a neurotrophic factor (Wilcox and Johnson, 1987). Here we report studies using the in vitro neuronal model of HSV latency to examine the effects of CAMP-mediated pathways, protein kinase C activation, and NGF on the regulation of HSV-1 latency.
MATERIALS AND METHODS Virus and neuronal culture preparation HSV-1 (KOS) was grown and quantified by plaqueformation assays on confluent Vero cells obtained from the American Type Culture Collection (Rockville, MD). Neuronal cultures were prepared from either sympathetic (superior cervical ganglia) or sensory ganglia (dorsal root ganglia) of embryonic rats as previously described (Wilcox and Johnson, 1987; Wilcox et a/., 1990). The neuronal cultures used in this study contained approximately 1O3 neurons in each well of a 24well cluster dish. Latently infected neuronal cultures
ET AL.
were prepared as previously described (Wilcox et a/., 1990). Briefly, 10 to 12 days after plating, medium containing 50 PM acyclovir (Burroughs Wellcome Co., Research Triangle Park, NC) was added to the cultures 12 hr prior to and for 7 days following inoculation with 5-l 0 PFU of HSV-1 per cell. Seven days following inoculation, the medium containing acyclovir was replaced with standard culture medium, which lacks acyclovir. The standard neuronal culture medium consists of 10% newborn bovine serum (GIBCO; Grand Island, NY) and 50 rig/ml of 2.5 S mouse NGF in Eagle’s minimum essential medium (GIBCO). Fourteen days or longer after viral inoculation, the cultures were used in the studies described.
Reactivation of latent HSV-1 Neuronal cultures harboring latent HSV-1 were treated as described in the text and legends. NGF deprivation was achieved by adding lo/o guinea pig antimouse NGF serum (titer >20,000) as has been previously described (Wilcox and Johnson, 1987). Drugs were prepared at the concentrations indicated by dilution in standard neuronal culture medium which contained NGF (50 rig/ml). Neuronal cultures were tested for the presence of infectious virus in plaque-formation assays using culture supernatants or total culture lysates on Vero indicator cells.
RESULTS Stimulation of CAMP-mediated pathways causes reactivation of latent HSV-1 To determine if CAMP-mediated pathways are involved in controlling HSV-1 latency, neuronal cultures harboring latent HSV-1 were incubated with the membrane-permeable CAMP analog, chlorophenylthioCAMP (CPT-CAMP) at a concentration of 0.5 mM, which has been shown to activate CAMP-dependent processes in neurons (Rydel and Greene, 1988). Reactivation occurred in 100% of the cultures treated with this highly selective CAMP analog (Table 1). Incubation with CPT-CAMP for as briefly as 1 hr resulted in reactivation in 100% of the cultures. Reactivation was also observed when neurons were treated with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), orthe adenylate cyclase activator, forskolin (Table 1). The concentrations of forskolin or IBMX required to produce reactivation were similar to the concentrations required to raise CAMP levels in cells in culture (Greene et al., 1986). Reactivation was not observed in cultures harboring latent HSV-1 which were untreated. Mock-infected neuronal cultures were maintained in the presence of CPT-CAMP, IBMX, or forskolin for
SECOND-MESSENGER TABLE 1 REACTIVATIONOFLATENT HSV-1 EIYCAMPAGONISTS %Reactivationa (No. positive/No. tested) Treatment
SCGN
DRGN
Untreated controls CPT-cAMPb (500 fin/l) Forskolin (100 4’0 (50 44 (5 WW lBMXd (1 mM)
0 (O/l 2) 100 (818)
0 (O/8) 100 (8/8)
100 100 0 100
100 (8/8) ND" ND 100 (818)
(8/8) (818) (O/8) (818)
a Neuronal cultures, either sympathetic (SCGN) or sensory (DRGN), were infected with HSV-1 as described under Materials and Methods. Two weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested In plaque-formation assays for infectious virus. b Chlorophenylthio-cyclic AMP. c Not determined. d lsobutylmethylxyanthine.
7-l 0 days without loss of viability of the neurons (data not shown). The data indicate that the agents are not toxic to neurons even with prolonged exposure, and these results are supported by published observations of prolonged treatments of neurons with CPT-CAMP (Rydel and Greene, 1988). These results strongly suggest that CAMP-mediated processes produce reactivation of latent HSV-1.
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AND HSV-1
313
Prolonged treatment with high concentrations of phorbol esters dramatically down-regulates the cellular level of PKC (Young eta/., 1987; Ase eta/., 1988). In rat neurons in culture, treatment with PMA reduces PKC activity below detectable levels within 1O-20 hr but produces minimal changes in levels of CAMP-dependent and Ca2+-stimulated protein kinase activity (Matthies et al., 1987). To determine if HSV-1 latency and reactivation occur under conditions which downregulate PKC, neurons were maintained in a high concentration of PMA (10m6 m for 1 week prior to establishment of latent HSV-1 infection and then were continuously treated with 1Om6M PMA. Viral latency was established and maintained in the presence of PMA in sensory neuronal cultures. Reactivation occurred in 100% of the cultures following treatment with either 1% anti-NGF (8/8 cultures) or 500 pn/l CPT-CAMP (8/8 cultures). These results suggest that PKC is not necessary for the establishment, maintenance, or reactivation of latent HSV-1 and suggest that reactivation produced by CAMP and anti-NGF is mediated by pathways independent of PKC. Comparison of reactivation kinetics of latent HSV-1 after treatment with NGF deprivation, CPT-CAMP, or PMA We observed that neuronal cultures harboring latent virus developed cytopathic effects at different rates following treatments with activators of CAMP-mediated pathways or PKC when compared with NGF deprivation. To characterize the effects of different stimuli on
Activation of protein kinase C results in reactivation of latent HSV-1 Protein kinase C (PKC) has important regulatory functions in many cell types. Therefore, to determine if activation of protein kinase C-mediated pathways rem suits in reactivation of latent HSV-1, neuronal cultures harboring latent HSV-1 were treated with the potent activator of PKC, phorbol myristate acetate (PMA). Reactivation occurred at concentrations of 10-l’ to 10-l’ M PMA with slightly greater sensitivity to reactivation by PMA in sensory neuron cultures (Table 2), consistent with concentrations shown to activate PKC (Nishizuka, 1986). The vehicle control (7 X 1O-’ M dimethyl sulfoxide) and the inactive 4a-phorbol analog of PMA had essentially no effect on cultures harboring latent virus. The 10% reactivation with the 4a-PMA analog in the sensory neuronal cultures was most likely the result of spontaneous reactivation, which we have rarely observed with sensory but not sympathetic neuron cultures (Wilcox et a/., 1990). These results indicate that a highly specific activator of protein kinase C can reactivate latent HSV-1.
TABLE 2 TREATMENTWITH
PHORBOLESTER
REACTIVATES LATENT HSV-1
%Reactivationa (No. positive/No. tested) Treatment Vehicle controlb 4aPDDC (1 O-6 M) PMAd(lO-’ M, 1 hr) PMA (10-l’ M) PMA (10-l’ M)
SCGN 0 0 100 100 0
(O/8) (O/8) (12112) (4/4) (O/4)
DRGN 0 10 100 100 100
(O/8) (l/10) (8/8) (5/5) (5/5)
’ Neuronal cultures, either sympathetic (SCGN) or sensory (DRGN), were infected with HSV-1. Two weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested in plaque-formation assays for infectious virus. b Latently infected neuronal cultures were treated with 7 X 1 O-’ M drmethyl sulfoxide. which was the final concentration in the medium used to prepare the phorbol esters. ’ 4-a-Phorbol-12decanoate-13.decanoate. d [email protected].
SMITH
314
-I 0
1
2 3 Days after treatment
4
I 5
FIG. 1. Time course of reactivation of latent HSV-1 after treatment with anti-NGF. CPT-CAMP, or PMA for 1 hr. Two weeks postinoculation, sensory neuronal cultures containing approximately 1 O3 neurons harboring latent HSV-1 were treated as indicated. Viral titers of culture lysates were determined in plaque-formation assays. Shown are the mean viral titers from four cultures f the SEM harvested at the times indicated after cultures were untreated (O), treated with anti-NGF (w), treated with 500 pM CPT-CAMP (A), or treated with 10-g M PMA (0).
reactivation, cultures harboring latent HSV-1 were treated with anti-NGF, CPT-CAMP, or PMA for 1 hr. The drugs were removed by washing the cultures with drug-free medium. At various times after treatment the cultures were analyzed for the presence of infectious virus. Untreated cultures harboring latent HSV-1 did not show evidence of infectious virus. Mock-infected cultures treated with anti-NGF, CPT-CAMP, or PMA did not produce infectious virus (data not shown). Cultures harboring latent HSV-1 produced detectable infectious virus at different times after treatment. The kinetics of reactivation were similarly accelerated by activation of CAMP-mediated pathways or PKC activation compared to those cultures treated with anti-NGF (Fig. 1). Following treatment with CPT-CAMP or PMA, cultures harboring latent HSV-1 produced infectious virus more than 24 hr prior to similar cultures treated with antiNGF (Fig. 1). Treatment of neuronal cultures harboring latent HSV-1 with either CPT-CAMP or PMA appears to accelerate the reactivation process compared with reactivation following anti-NGF treatment.
ET AL.
Neuronal cultures were treated with 2-AP to determine if this compound would affect HSV-1 latency or reactivation. 2-AP alone at a concentration of 1 ml\/l had no effect on neuronal cultures harboring latent HSV-1. In cultures treated with anti-NGF alone reactivation occurred in 100% of the cultures (Table 3). However, when neurons harboring latent HSV-1 were treated with anti-NGF in the presence of 2-AP, reactivation was blocked (Table 3). Similarly, 2-AP treatment blocked reactivation produced by CPT-CAMP. One week after removal of 2-AP, anti-NGF treatment reactivated latent virus from 100% of the cultures, indicating that both the neurons and the virus were able to produce reactivation and that the effects of 2-AP were reversible (Table 3). In contrast to the results with antiNGF and CPT-CAMP, however, 2-AP treatment did not prevent HSV-1 reactivation following incubation with PMA. 2-Aminopurine exerted no apparent toxic effects on neurons at the concentrations used to prevent reactivation. Consistent with the report of Greene et al. (1990) neuronal cultures were maintained for several weeks in the presence of 1 mM 2-AP without loss of viability or morphologic changes (data not shown). Furthermore, 2-AP did not directly interfere with viral replication in either Vero cells or sensory neuronal cultures. During a productive infection in Vero cells or neuronal cultures, 48 hr after inoculation with HSV-1 (multiplicity of infection of lo), viral titers were essentially identical with (6.2 X 1O* PFU/ml in Vero cells; 1.05 X 1O4PFU/ml in neuronal cultures) or without (6.5 X lo* PFU/ml in TABLE 3 THE EFFECTS OF 2-AMINOPURINE ON THE REACTIVATION OF LATENT HSV-i
Treatment
%Reactivation” (No. positive/No. tested) 0 (O/22) 100 (32/32)
A novel protein kinase inhibitor prevents reactivation of latent HSV caused by NGF deprivation or CPT-CAMP
Nonimmune serum (l%, 1 hr) Anti-NGF serum (lo/o, 1 hr) Anti-NGF serum (l%, 1 hr) + 2-APb (1 mM. 2 hr) CPT-cAMPC (500 pM, 1 hr) CPT-CAMP (500 PM, 1 hr) + 2-AP (1 mM, 2 hr) PMA“ (1 O-’ M. 1 hr) PMA (1 O-g M, 1 hr) + 2-AP (1 mM. 2 hr)
2-Aminopurine (2-AP) is known to inhibit NGF-stimulated protein kinases (Volonte et a/., 1989), as well as elF2a protein kinases (De Benedetti and Baglioni, 1983) but has no effect on protein kinase A or C (Volonte et al., 1989). 2-AP also blocks some of the actions of NGF (Volonte et al., 1989; Greene et al., 1990).
B Sensory neuronal cultures were infection with HSV-1. TWO weeks postinoculation, cultures were treated as indicated. Five days after treatment, culture supernatants were tested in plaque-formation assays for infectious virus. b 2-Aminopurine. ’ Chlorophenylthio-cyclic AMP. d 4-fl-Phorbol-12.myristate-13.acetate.
0 (O/l 0) 100 (8/8) 0 (O/l 0) 100 (8/8) 100 (lO/lO)
SECOND-MESSENGER
Vero cells; 1 .l 1 X 1O4 PFU/ml in neuronal treatment with 1 mM 2-AP.
cultures)
DISCUSSION Role of NGF in HSV latency The predominate sites of HSV latency are neural crest-derived sensory and sympathetic neurons which depend on NGF for survival during development and continue to require NGF for maintenance of normal function (Thoenen and Barde, 1980). NGF is produced in small quantities by the target tissue, providing the innervating neurons with trophic support (Brandtlow et al., 1987). While some of the stimuli which reactivate latent HSV-1 in viva are consistent with producing deprivation of trophic support, including central rhizotomy, axotomy, and damage to the target tissue (Hill, 1985; Price, 1986) many stimuli which produce reactivation in humans, such as stress and fever, may have distinct pathways. We have previously demonstrated that even brief NGF deprivation produces reactivation of latent HSV in vitro (Wilcox and Johnson, 1987, 1988; Wilcox eta/., 1990). The role of NGF in the regulation of HSV latency in viva remains to be determined. However, in the mouse, NGF treatment greatly ameliorates the productive HSV-1 infection in the ganglia (Johnson et al., 1988). Determining the molecular basis of NGF dependence of HSV-1 latency remains a major challenge. Recently, understanding of NGF signaling pathways has been advanced by the demonstration of an NGF receptor that is a ligand-stimulated tyrosine kinase and is homologous to the proto-oncogene trk (Kaplan eta/., 1991a,b; Klein el al., 1991). The intracellular signal transduction that follows NGF binding remains unknown; however, activation of specific serine protein kinases (Aletta et al., 1988; Volonte et a/., 1989) and altered gene expression occur within minutes of NGF addition to responsive cells (Greenberg et a/., 1985). Study of these NGF-stimulated serine protein kinases is aided by their selective inhibition by purine analogs, including 2-AP, which also block some of the biochemical and morphologic changes associated with NGFmediated responses (Greene et al., 1990). Therefore, there are a wide range of signaling mechanisms that could potentially mediate HSV-1 reactivation in response to NGF deprivation. Activation of CAMP-dependent reactivates latent HSV-1
processes
or PKC
In this report we used the in vitro model of HSV latency in neurons to demonstrate reactivation in response to cellular second-messenger pathways. The
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observation that the highly specific CAMP analog, CPT-CAMP, reactivates latent HSV-1 at concentrations previously shown to activate CAMP-dependent pathways in neurons in culture (Rydel and Greene, 1988) strongly suggests that CAMP-dependent mechanisms can control viral latency. This result is strengthened by the observation that both IBMX and forskolin, which activate CAMP-dependent pathways via inhibition of phosphodiesterase and direct activation of adenylate cyciase, respectively, also reactivated latent HSV-1. With forskolin, some caution in the interpretation is required since forskolin is reported to have effects on potassium channels which are independent of CAMPmediated effects (Hoshi et al., 1988). These results indicate that latent virus can reactivate in response to CAMP-mediated signals. The effects of NGF deprivation and CAMP analogs on HSV reactivation share common features: both treatments can be blocked by 2-AP and both NGF deprivation and CPT-CAMP treatments produce reactivation in neurons previously treated with high concentrations of phorbol esters, suggesting that PKC is not required for either effect. The effects of CAMP agonist on NGF-mediated responses remain contradictory. Agonist of CAMP can partially replace NGF for neuronal survival and morphologic differentiation (Rydell and Greene, 1988) and produce increased phosphotylation of some of the same intracellular targets (McTigue et al., 1985; Cremins et al., 1986). However, CAMP agonist blocks some of the actions of NGF (Greene et a/., 1986). NGF treatment may minimally increase neuronal CAMP in neuronal cultures derived from dorsal root ganglia (Narumi and Fujita, 1978; Skaper et al., 1979) and superior cervical ganglia (Nikodijevic et a/., 1975); the mechanism remains incompletely understood, but does not appear to involve direct stimulation of adenylate cyclase (Race and Wagner, 1985). However, other reports suggest NGF deprivation has little effect on CAMP levels in neurons in culture (Johnson, unpublished observation; Narumi and Fujita, 1978; Skaper et al., 1979). Consequently, HSV-1 reactivation caused by NGF deprivation is unlikely to be the result of elevation of the neuronal CAMP level. Furthermore, the ability of CPT-CAMP to produce reactivation even in the presence of NGF suggests that CAMP signaling can occur independent of changes in NGF. Phorbol esters produce pleotrophic effects on cells, which are mediated by high affinity receptors, and there is considerable evidence that the major high affinity receptor is PKC, which is activated by these compounds (Nishizuka, 1986, review). PKC is involved in cellular second-messenger pathways involving release of diacylglycerol and inositol phosphates (Nishizuka, 1988; Cantley et al., 1991). Therefore, the ability of
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phorbol esters to reactivate latent HSV-1 indicates that acute activation of signaling pathways involving activation of PKC can reactivate latent HSV-1. Previous studies have demonstrated that rat neurons in culture survive after down-regulation of PKC by prolonged treatment with high concentrations of phorbol esters (Matthies et al., 1987). In this study survival of rat neurons was not effected by long term treatment with high concentrations of phorbol esters, and the ability of the neuronal cultures to support latent infections or reactivate in response to CAMP analogs or NGF deprivation was not altered by phorbol ester treatment, suggesting that PKC is not necessary for the establishment of HSV-1 latency or reactivation. This suggests that the actions of PKC on latency are independent of CAMP or NGF deprivation. The data suggest that both PKC- and CAMP-dependent pathways can function to control reactivation of latent HSV-1 in the neuronal cultures and may act independently to produce reactivation. The significant delay in the appearance of infectious virus produced by NGF deprivation suggests that the signal resulting from NGF deprivation may be distinct from that resulting from CPT-CAMP or PMA treatment. Alternatively, the ganglia from which the cultures were derived contain heterogeneous populations of neurons; however, it is unknown if differences in neurons persist in culture. It is possible that different populations of neurons could respond to different reactivation stimuli. The effects of 2-aminopurine on reactivation of latent HSV-1 NGF-stimulated serine-protein kinases have been described; these include the protein kinase N and MAP-l kinase (Aletta et al., 1988; Volonte et al., 1989). 2-AP blocks activation of these NGF-dependent kinases both in vivo and in vitro (Aletta et a/., 1988; Volonte et al., 1989). It would be expected that if the action of 2-AP is to inhibit NGF-dependent systems, then 2-AP would produce reactivation of latent virus by mimicking NGF deprivation. However, we observed the opposite effect; the treatment of neuronal cultures with 2-AP prevented reactivation caused by either anti-NGF or CPT-CAMP. The reactivation produced by PMA was not prevented by 2-AP treatment. The failure of 2-AP to prevent reactivation by PMA may result from the difficulty in completely removing the lipophilic phorbol ester. An alternative explanation is that PMA reactivates HSV-1 by a distinct pathway which is not affected by 2-AP, and the mechanism of PMA action therefore differs from reactivation by CAMP agonist or NGF deprivation. The concentration of 2-AP effective in preventing HSV-1 reactivation was similar to the concentrations
ET AL.
reported to block NGF-mediated responses (Greene et al., 1990) and inhibit the NGF-stimulated protein kinase N and MAP 1.2 kinases (Aletta et al., 1988; Volonte et a/., 1989). 2-AP also is known to inhibit the elF2a kinases at a similar concentration (De Benedetti and Baglioni, 1983), and, presumably, based on this action leads to selective mRNA translation (Kaufman and Murtha, 1987). 2-AP prevents c-fos message induction by NGF in PC-l 2 cells (Volonte eta/., 1989) and inhibits induction of the proto-oncogenes c-fos and c-myc by interferon in an osteosarcoma cell line (Zinn et a/., 1987). It remains unclear if any of these reported actions are related to the observed effects of 2-AP on the inhibition of HSV-1 reactivation. In contrast, the actions of 2-AP are not likely to be the result of nonspecific toxicity. This conclusion is supported by our observations and by recent reports that rat sensory neurons can be maintained in the presence of 10 mM 2-AP for days without loss of viability (Greene et al., 1990). In addition HSV-1 reactivated readily after removal of 2-AP, indicating neuronal viability. Furthermore, 2-AP does not alter the pattern of phosphorylation observed in HeLa cells or in NGF-responsive PC-l 2 cells and does not alter protein synthesis (Zinn et a/., 1987; Volonte et a/., 1989). In vitro, 2-AP does not inhibit activity of CAMP-dependent protein kinase or PKC (Volonte et a/., 1989). Our results indicate that at least two cellular secondmessenger pathways reactivate latent HSV-1. This is consistent with the wide range of physiological stimuli capable of reactivating HSV-1 in vivo. The ability of a latent virus to respond to stress in the host at an early stage may be important to viral propagation and survival. Our results also suggest that agents such as 2AP may be of value in identifying specific cellular mechanisms responsible for the control of HSV-1 latency. ACKNOWLEDGMENTS We thank G. Huitt for expert technical assistance and Drs. J. Schaack, D. Gilden. and K. Escudero for critical review of the manuscript. This work was supported in part by National Institutes of Health Grants NS29046 and AGO7347 and National Multiple Sclerosis Society Grant RG2344-A.
REFERENCES ALET~A. J. M., LEWIS, S. A., COWAN, N. J., and GREENE, L. A. (1988). Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2. /. Cell Biol. 106, 1573-l 581. ASE, K., BERRY,N., KISHIMOTO,A., and NISHIZUKA,Y. (1988). Differential down-regulation of protein kinase C subspecies in KM3 cells. FEES’ Left. 236, 396-400. BANDTLOW, C. E., HEUMANN, R., SCHWAB, M. E., and THOENEN, H. (1987). Cellular localization of nerve growth factor synthesis by in situ hybridization. EMBO /. 6, 891-899.
SECOND-MESSENGER BARINGER,R. J., and SWOVELAND,P. (1973). Recovery of herpes simplex virus from human trigeminal ganglions. N. fngl. /. Med. 288, 648-650. BASTIAN, F. O., RAESON, A. S., YEE, C. L., and TRALKA, T. S. (1972). Herpes hominis: Isolation from human trigeminal ganglions. Science 178, 306-307. BLOCK, T. M., SPIVACK, J. G., STEINER, I., DESHMANE, S., MCINTOSH, M. T.. LIRE~E, M. R. P.. and FRASER,N. W. (1990). A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics. /. Viral. 64, 3417-3426. CANTLEY, L. C., AUGER, K. R., CARPENTER,C., DUCKWORTH, B., GMZIANI, A., KAPELLER,R., and SOLTOFF, S. (1991). Oncogenes and signal transduction. Cell 64, 281-302. COOK, M. L., BASTONE,V. B., and STEVENS,J. G. (1974). Evidence that neurons harbor latent virus. Infect. Immun. 9, 946-951. CLEMENTS, G. B., and STOW, N. D. (1989). A herpes simplex virus type 1 mutant containing a deletion within immediate early gene 1 is latency-competent in mice. /. Gen. \/irol. 70, 2501-2506. CREMINS, J., WAGNER, J. A., and HALEGOUA, S. (1986). Nerve growth factor action IS mediated by cyclic AMP and Ca++/phospholiplddependent protein kinases. J. Cell Biol. 103, 887-893. DE BENEDETTI,A., and BAGLIONI, C. (1983). Phosphorylation of initiation factor elF-2a, binding of mRNA to 48 S complexes, and its reutilization In Initiation of protein synthesis. /. Biol. Chem. 258, 14,552-14,562. DOERIG, C., PIZER. L. I., and WILCOX, C. L. (1991a). An antigen encoded by the latency-associated transcript in neuronal cell cultures latently infected with herpes simplex virus type 1. /. Viral. 65, 2724-2727. DOERIG, C., PIZER, L. I., and WILCOX, C. L. (1991 b). Detection of the latency-associated transcript in neuronal cultures during the latent infection with herpes simplexvirus type 1. Virology 183,423%426. GREENBERG,M. E., GREENE,L. A., ~~~ZIFF, E. B. (1985). Nervegrowth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcnption in PC1 2 cells. J. Biol. Chem. 260, 14,101-14,110. GREENE, L. A., DREXLER,S. A., CONNOLLY, J. L., RUKENSTEIN,A., and GREENE, S. H. (1986). Selective inhibition of responses to nerve growth factor and of microtubule-associated protein phosphorylation by activators of adenylate cyclase. J. Cell Biol. 103, 19671978. GREENE,L. A., VOLONTE, C., and CHALAZONITIS,A. (1990). Purine analogues inhibit nerve growth factor-promoted neunte outgrowth by sympathetic and sensory neurons. J. Neurosci 10, 1479-1485. HILL, J. M.. SEDARATI, F., JAVIER, R. T., WAGNER, E. K., and STEVENS. J. G. (1990). Herpes simplex latent phase transcription facilitates in viva reactivation. Virology 174, 1 17-l 25. HILL, T. J. (1985). Herpes simplex virus latency. In “The Herpesviruses” (B. Rolzman, Ed.). Plenum, New York. Ho, D. Y., and MOCARSKI, E. S. (1989). Herpes simplex virus latent RNA IS not required for latent Infection in the mouse. Proc. Nat/. Acad. SC;. USA 86,7596-7600. HOSHI, T., GARBER.S. S.. and ALDRICH, R. W. (1988). Effects of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation. Science 240, 1652-l 654. JAVIER, R. T., STEVENS, J. G., DISSETTE, V. B., and WAGNER, E. K. (1988). A herpes simplex virus transcript abundant in latently Infected neurons is dispensable for the establishment of the latent state. Virology 166, 254-257. JOHNSON, E. M., JR., MANNING, P. T., and WILCOX, C. L. (1988). The biology of nerve growth factor in viva. ln “Neurobiology of Amino Acids Peptides and Trophic Factors” (J. A. Ferrendelli, R. Collins, and E. M. Johnson, Jr., Eds.). Kluwer Academic, Norwell, MA.
PATHWAYS
AND HSV-1
317
KAPLAN, D. R., HEMPSTEAD, B. L., MARTIN-ZANCA, D., CHAO, M. V., and PARADA, L. F. (1991a). The trk proto-oncogene product: A signal transducing receptor for nerve growth factor. Science 252, 554-557. KAPLAN, D. R., MARTIN-ZANCA, D., and PARADA, L. F. (199 1b). Tyrosine phosphorylation and tyrosine kinase activity of the frk protooncogene product induced by NGF. Nature 350, 158-I 60. KAUFMAN, R. J., and MURTHA, P. (1987). Translational Control Mediated by Eucaryotic initiation factor-z is restricted to specific mRNAs in transfected cells. Molec. Cell. Biol. 7, 1568-l 571. KLEIN, R., JING, S., NANDURI, V., O’ROURKE, E., and BARBACID, M. (1991). The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65, 189-l 97. LEIB, D. A., BOGARD, C. L., KLOSZVNENCHAK,M., HICKS, K. A., COEN, D. M., KNIPE, D. M., and SCHAFFER,P. A. (1989). A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with a reduced frequency. J. Viral. 63, 2893-2900. LEIB, D. A., NADEAU, K. C.. RUNDLE, S. A., and SCHAFFER,P. A. (1991). The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional CAMP-response element: Role of the latency-associated transcripts and CAMP in reactivation of viral latency. Proc. Nat/. Acad. Sci. USA 88, 48-52. MA~HIES, H. 1. G., PALFREY,H. C., HIRNING, L. D., and MILLER, R. J. (1987). Down regulation of protein kinase C in neuronal cells: Effects on neurotransmitter release. J. Neurosci. 7, 1 198-l 206. MCTIGUE, M., CREMININS, J., and HALEGOUA, S. (1985). Nerve growth factor and other agents mediate phosphorylation and activation of tyroslne hydroxylase. J. Bioi. Chem. 260, 9047-9056. NARUMI, S., and FUJITA, I. (1978). Stimulatory effects of substance P and nerve growth factor on neurite outgrowth in embryonic chick dorsal root ganglia. Neuropharmacology 17, 73-76. NIKODIJEVIC,B., NIKODIJEVIC,O., Yu, M. W., POLLARD, H., and GUROFF. G. (1975). The effects of substance P and nerve growth factor on cyclic AMP levels in superior cemical ganglia of the rat. Proc. Nat/. Acad. Sci. USA 72, 4769-4771. NISHIZUKA, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-312. NISHIZUKA, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661665. PRICE, R. W. (1986). Neurobiology of human herpesvirus infections. CRC Crit. Rev. C/in. Neurobioi. 2, 61-123. PRICE, R. W.. KATZ, B. J., and NOTKINS.A. L. (1975). Latent infection of the peripheral ANS with herpes simplex virus. Nature (London) 257, 686-688. RACE, H. M., and WAGNER, J. A. (1985). Nerve growth factor affects CAMP metabolism but not by directly stimulation adenylate cyclase activity. J. Neurochem. 44, 1588-l 592. ROCK, D. L., and FRASER,N. W. (1985). Latent herpes simplex type 1 DNA contains two copies of the virion DNA joint region. J. Viral. 55, 849-852. ROWLAND, E. A., MULLER, T. H., GOLDSTEIN, M.. and GREENE, L. A. (1987). Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC 12 cells. J. Biol. Chem. 262, 7504-7513. RYDEL, R. E., and GREENE, L. A. (1988). CAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. Nat/. Acad. Sci. USA 85, 1257-1261. SKAPER, S. D., BOTTENSTEIN,J. E., and VARON, S. (1979). Effects of nerve growth factor on cyclic AMP levels in embryonic chtck dor-
318
SMITH ET AL.
sal root ganglia following factor deprivation. 1. Neurochem. 32, 1845-1851. STEINER,I., SPIVACK,J. G., LIRETTE,R. P., BROWN,S. M., MCLEAN, A. I?., SUBAK-SHARPE, 1. H., and FASER,N. W. (1989). Herpes simplex virus type 1 transcripts are evidently not essential for latent infection. EM60 1. 8, 505-51 1. STEVENS,1. G. (1989). Human herpesviruses: A consideration of the latent state. Microbial. Rev. 53, 3 18-332. THOENEN,H., and BARDE,Y.-A. (1980). Physiology of nerve growth factor. Physiol. Rev. 60, 1284-l 335. VOLONTE,C. A., RUKENSTEIN, LOEB,D. M., and GREENE,L. A. (1989). Differential inhibition of nerve growth factor responses by purine analogues: Correlation with inhibition of a nerve growth factor-activated protein kinase. J. Cell Biol. 109, 2395-2403. WILCOX,C. L., and JOHNSON,E. M., JR.(1987). Nerve growth factor
deprivation results in the reactivation of latent herpes simplexvirus in vitro. J. Viral. 61, 231 1-2315. WILCOX,C. L., and JOHNSON,E. M., JR. (1988). Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro. 1. Viral. 62, 393-399. WILCOX,C. L., SMITH. R. L., FREED,C. R., and JOHNSON,E. M., JR. (1990). Nerve growth factor dependence of herpes simplex latency in peripheral sympathetic and sensory neurons in vitro. 1. Neurosci. 10, 1268-l 275. YOUNG,S., PARKER,P. J., ULLRICH,A., and STABEL,S. (1987). Downregulation of protein kinase C is due to an increased rate of degradation. Biochem. 1. 244, 775-779. ZINN, K., KELLER,A., WHITTEMORE, L., and MANIATIS,T. (1987). 2Aminopurine selectively inhibits the induction of @Interferon, c-ios and c-myc gene expression. Science 240, 2 1O-21 3.
