INTRODUCTION

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Herpes simplex virus type I (HSV-1) establishes life-long infections of the human host characterized by productive and latent phases. During productive infection, nearly all of the more than 75 genes encoded by the virus are expressed. These genes have been categorized into three major kinetic classes, immediate-early (IE), early (E), and late (L), depending on the time of their peak expression and the sensitivity of their expression to inhibitors of DNA, RNA, and protein synthesis (33, 34). The cascade of productive viral gene expression is regulated by virus-encoded proteins present in virions and five IE proteins which, in combination with host transcriptional machinery, direct the expression of viral E and L genes (78).

HSV-1 initiates productive infection in epithelial cells of the skin and mucosal membranes at the site of infection (reviewed in references 25;0 and 60). Virus particles produced in epithelial cells during productive infection enter neuronal termini that innervate the skin in the area surrounding infection and then travel via retrograde axonal transport to neuronal cell bodies located in sensory ganglia (14). Viral DNA enters the neuronal nucleus, where limited genome amplification ensues and latency is ultimately established (14).

During latency, productive viral gene expression is almost completely repressed, with transcription being limited to a small region of the genome encoding the latency-associated transcripts (LATs) (17, 24). In the absence of viral transcription, viral DNA replication ceases, and viral genomes are condensed into chromatin-like structures containing nucleosomes (18). Months or years later, viral or cellular molecules induced in response to stress stimulate the resumption of viral gene expression (reactivation), resulting in the synthesis of new virus particles (25). These particles travel back to the initial site of infection via the axonal route and initiate lytic infection of epithelial cells, resulting in recurrent clinical disease (14). Although the general course of events involved in the establishment and reactivation of latency is clear, the molecular processes that underlie establishment and reactivation are poorly understood.

Mutant viruses have been widely used to identify viral genes involved in the establishment and reactivation of latent infection (13, 36, 42, 50, 73). These studies have demonstrated that infected-cell polypeptide 0 (ICP0), a viral IE regulatory protein, is important for efficient reactivation in mouse and rabbit models of HSV-1 latency (6, 13). ICP0 stimulates transcription of viral IE, E, and L genes when productive infection is initiated at low multiplicities (9, 10, 37). ICP0 activates a broad spectrum of viral and cellular promoters without apparent sequence specificity in transient expression assays and stimulates de novo gene expression after transfection of infectious DNA into permissive cells (7, 20, 21, 54, 55). In an in vitro cell culture model of HSV latency, ICP0 is both necessary and sufficient for reactivation (29, 61, 85). In addition, ICP0 may also play an important role in establishment of latent infection (80).

Cellular proteins likely play an important part in ICP0's broad transactivating activity. In support of this hypothesis, ICP0 transiently colocalizes with antigens implicated in cell growth control in nuclear substructures called nuclear domain 10 (ND10). Colocalization of ICP0 with ND10 antigens correlates with their redistribution in the infected cell (48, 49). In addition, ICP0 coimmunoprecipitates with a 135-kDa protein homologous to members of the ubiquitin-specific protease family of proteins (22, 52). Redistribution of ND10 antigens and coimmunoprecipitation with the 135-kDa protein requires intact transcriptional activating domains of ICP0, suggesting that these phenomena are functionally related (48, 51). ICP0 also destabilizes the catalytic subunit of a DNA-dependent protein kinase (DNA-PK) following viral infection (41). Thus, ICP0's broad transactivating activity likely involves interactions with cellular proteins.

In studies of ICP0 null mutant viruses, several cell cycle-regulated and cell-type-specific cellular activities that are able to substitute for the transactivating activity of ICP0 have been identified (8, 84). Vero cells growth arrested in G₀/G₁ by isoleucine deprivation express an activity after release from growth arrest that enhances the plating efficiency of an ICP0 null mutant virus (8). Similarly, Vero cells and cells of neural origin express an activity after release from growth arrest that activates HSV-1 gene expression in transient expression assays (57). Finally, U2OS cells, an osteosarcoma cell line, constitutively express high levels of an activity that stimulates the growth of an ICP0 null mutant virus (84). These observations demonstrate that cellular functions can substitute for the transactivating activity of ICP0.

Initiation of viral gene expression at the onset of reactivation occurs in the absence of ICP0 and other viral transcriptional activators. Since ICP0 is an important activator of viral gene expression, the existence of cellular activities able to substitute for ICP0 suggests a possible mechanism by which viral gene expression is activated during reactivation. In this study, we describe an activity in cells of neural lineage (PC12) induced by treatment with two physiologically relevant growth factors, nerve growth factor (NGF) and fibroblast growth factor (FGF), that enhances gene expression and replication of an ICP0 null mutant, n212. The ICP0-like, n212-complementing activity is expressed transiently and can be blocked by inhibitors of NGF-dependent signal transduction. Moreover, like ICP0, the cellular complementing activity functions at the level of mRNA accumulation. We suggest that the NGF/FGF-induced n212-complementing activity may be responsible for the initiation of viral gene expression during reactivation from neuronal latency when no ICP0 is present.

	MATERIALS AND METHODS

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Cells and viruses. PC12 cells were the generous gift of John Wagner (Cornell University Medical College, New York, N.Y.) and were cultured in Dulbecco's modification of Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 5% horse serum as described previously (26). MM17-26 cells were kindly provided by Geoffrey Cooper (Dana-Farber Cancer Institute, Boston, Mass.) and were cultured as described for PC12 cells. Vero cells, L7 cells, which contain a stabily integrated copy of ICP0, and the osteosarcoma line U2OS were cultured as previously described (63, 65, 84).

Growth factors and kinase inhibitors. Viral infections were conducted in PC12 cells in the presence and absence of NGF or other growth factors with and without serine/threonine kinase inhibitors. Briefly, PC12 cell monolayers (10⁶) were seeded in 35-mm-diameter dishes and incubated at 37°C in 10% CO₂ for 12 to 18 h. Three hours prior to infection at 0.01 PFU/cell, culture medium was removed and replaced with fresh medium containing NGF 2.5S (100 ng/ml; Collaborative Biochemical, Bedford, Mass.) and FGF 100 ng/ml; Collaborative Biochemical). Serine/threonine kinase inhibitors K252a, KT5720, PD98059, and calphostin C were purchased from Calbiochem (La Jolla, Calif.) and were added 30 min prior to NGF addition. Inhibitors were added at concentrations 10-fold higher than the published 50% inhibitory concentration for each inhibitor (5, 38, 56). The concentrations of the inhibitors were as follows: K252a, 0.25 µM; KT5720, 0.5 µM; PD98059, 20 µM; and calphostin C, 0.5 µM. The specificity of each inhibitor for cellular kinases has been described elsewhere (5, 38, 40, 56, 71, 76). Replicate cultures were harvested at 24 h postinfection (hpi), and virus yields were determined by standard plaque assays on Vero (KOS) or L7 or U2OS (n212) cell monolayers as described previously (37).

PMA and second messenger analogs. PC12 cells were treated with 16 nM phorbol 12-myristate 13-acetate (PMA), 50 µM dibutyryl cyclic AMP (dbcAMP), 1 µM ionomycin, or 50 µM forskolin, alone or in combinations, in the presence and absence of NGF for 3 h prior to infection. Following the 3-h incubation, the cultures were infected with 0.01 PFU of n212 or KOS per cell, and viral yields were measured at 24 hpi as described above.

Viral entry. PC12 cells (5 × 10⁶) in 60-mm-diameter dishes were treated with 100 ng of NGF per ml or mock treated 3 h prior to infection with 1.0 PFU of KOS or n212 per cell. At 3 hpi, the cells were scraped into the medium and pelleted by centrifugation at 1,000 × g for 4 min at 4°C. The cell pellet was washed once with 1× Tris-buffered saline (25 mM Tris-HCl [pH 7.4], 140 mM sodium chloride, 5 mM potassium chloride), and the cells were repelleted by centrifugation at 1,000 × g for 4 min at 4°C. The cell pellet was resuspended in 0.375 ml of lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM potassium acetate, 5 mM magnesium acetate, 0.5% Nonidet P-40, 1 mM dithiothreitol) and incubated on ice for 5 min to lyse the cells. The nuclei were centrifuged at 14,000 × g for 5 min at 4°C through a 0.5-ml 20% sucrose cushion prepared in lysis buffer lacking Nonidet P-40. The nuclear DNA from this preparation was isolated by using minicolumns as recommended by the manufacturer (Qiagen, Chatsworth, Calif.).

Northern blot analysis. PC12 cell cultures (5 × 10⁶) in 60-mm-diameter dishes were treated with 100 ng of NGF per ml for 0, 15, 30, and 60 min or infected with KOS and n212 at 5.0 PFU/cell for 30, 60, 90, or 120 min. Cytoplasmic RNA was isolated at each time point as described previously (37). The RNA was size fractionated by denaturing gel electrophoresis and transferred to nylon membranes by Northern blotting. The Northern blot was probed with 100 ng of ³²P-labeled antisense RNA probe (8 × 10⁵ cpM/ng) specific for the mouse c-fos message. The blot was reprobed with 150 ng of ³²P-labeled antisense RNA probe (8.8 × 10⁵ cpM/ng) specific for the rat GAPDH message. The blot was visualized by PhosphorImager analysis. The range values for the image display were set at 0 to 500 counts.

RNA isolation and RNase protection. Cytoplasmic RNA isolation and quantitative RNase protection assays were performed as described previously (37).

	RESULTS

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NGF and FGF stimulate replication of the ICP0 null mutant, n212, in PC12 cells. Reactivation of latent HSV-1 correlates with changes in levels of stress-induced growth factors and neurotrophins (32, 79). NGF, FGF, and epidermal growth factor (EGF) are among the many growth factors induced by stress that have a wide range of biological effects in vivo and in cell culture. In PC12 cells, physiological concentrations of NGF and FGF activate cellular signaling cascades leading to morphological and biochemical differentiation (28). Differentiated PC12 cells resemble sympathetic neurons in that they develop neurites, become electrically excitable, and express a number of neuron-specific genes (27, 28). In contrast, physiological concentrations of EGF stimulate mitogenesis in PC12 cells, even though many of the same signaling molecules are activated by all three factors (47). To determine the effects of growth factor-induced cellular activities on viral replication, PC12 cells were treated with NGF, FGF, or EGF or were mock treated for 3 h prior to infection with 0.01 PFU of wild-type HSV-1 strain KOS or the ICP0 null mutant n212 per cell. At 3 and 24 hpi, virus yields were measured by plaque assay.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Effects of growth factors on replication of n212 and KOS. PC12 cells (106/35-mm-diameter dish) were treated with 100 ng of NGF, FGF, or EGF per ml or 50 mM KCl in the presence and absence of EGF for 3 h prior to infection with 0.01 PFU of KOS or n212 per cell. At 24 hpi, the cultures were harvested and viral yields were measured. Titers of KOS and n212 were determined on Vero cells and U2OS cells, respectively. The data are expressed as fold difference in titer relative to mock treatment.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Growth of KOS and n212 in PC12 cells in the presence and absence of NGF. PC12 cells (106/35-mm-diameter dish) were mock treated or treated with NGF at 100 ng/ml for 3 h prior to infection with KOS or n212 at 0.02 or at 2.5 PFU/cell. At the times indicated, cultures were harvested and viral yields were measured by plaque assay. Titers of KOS and n212 were determined on Vero cells and U2OS cells, respectively.

NGF treatment does not affect viral entry. It is conceivable that NGF treatment of PC12 cells may affect viral entry, even though the dramatic morphological changes associated with NGF-dependent differentiation require 24 h or more to develop. To test whether NGF treatment affects viral infectivity, PC12 cells were treated with NGF 3 h prior to infection with KOS or n212. At 3 hpi, nuclear DNA was isolated and immobilized on a nylon membrane by slot blot hybridization. The membrane was probed with radiolabeled KOS DNA. The blot was then stripped and reprobed with a radiolabeled RNA probe specific for the cellular gene, GAPDH. The results of these tests show that NGF treatment had no significant effect on the amount of nuclear KOS or n212 DNA recovered from PC12 cell nuclei (Fig. 3). These results indicate that ICP0 and NGF treatment do not effect viral entry or transport of viral DNA to the nucleus. Similar results were obtained when entry was measured by indirect immunofluorescence (i.e., by counting the number of ICP4-expressing cells in NGF-treated or mock- treated cells infected with KOS or n212 [data not shown]).

View larger version (29K): [in this window] [in a new window]   FIG. 3.   NGF does not affect viral entry. PC12 cells were mock treated or treated with NGF (100 ng/ml) for 3 h prior to infection with KOS or n212. Nuclear DNA was harvested prior to the onset of viral DNA replication at 3 hpi and applied to a nylon membrane by slot blot hybridization (inf.). Cesium chloride-purified KOS DNA was applied to the membrane as indicated (std.). The blot was probed for viral DNA by using 32P-labeled nick-translated KOS DNA. The blot was stripped and reprobed for cellular DNA by using a 32P-labeled antisense RNA probe specific for the cellular gene GAPDH. The image was visualized by PhosphorImager analysis. The range values for the image display were set at 0 to 791 counts for the KOS probe and 0 to 258 counts for the GAPDH probe.

The NGF-induced n212-complementing activity requires activation of multiple NGF-dependent signal transduction pathways. (i) Inhibitor studies. NGF activates multiple signal transduction pathways, including ras-dependent signaling pathways, whose downstream endpoint is the expression of differentiation-specific genes (16). The PC12-derived cell line MM17-26 constitutively expresses a dominant negative ras allele that blocks downstream functions of ras (69). Consequently, MM17- 26 cells fail to differentiate in response to NGF or FGF treatment. To test whether ras is required for the NGF- or FGF-dependent stimulation of n212 replication, MM17-26 cells or PC12 cells were pretreated with NGF or FGF 3 h prior to infection with 0.01 PFU of n212 per cell. At 24 hpi, n212 yields were measured and compared to those on NGF-treated PC12 cells. As shown in Fig. 4, the replication efficiency of n212 in NGF- and FGF-treated MM17-26 cells was only 18% of that observed in NGF- and FGF-treated PC12 cells. These results indicate that the NGF/FGF-dependent stimulation of n212 replication is partially ras dependent.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Serine/threonine kinase inhibitors block NGF-dependent replication of n212. PC12 cells (106/35-mm-diameter dish) were incubated for 30 min prior to NGF addition with serine/threonine kinase inhibitors at the following concentrations: K252a, 0.25 µM; KT5720, 0.5 µM; PD98059, 20 µM; and calphostin C, 0.5 µM. At 3 h posttreatment, the cultures were infected with n212 (0.01 PFU/cell) in the presence and absence of inhibitors and NGF. At 24 hpi, the cultures were harvested and viral yields were measured. The data are expressed as percentage of NGF-induced n212 replication, which was set at 100% to show all of the data in a single figure. MAPKinase, mitogen-activated kinase.

(ii) Activator studies. Many of the intracellular signaling pathways activated by NGF can be stimulated indirectly by activators of cellular protein kinases or second messenger analogs. To test whether direct stimulation of protein kinases or second messenger pathways result in n212-complementing activity, and to test whether NGF can synergize with these activities, PC12 cells were treated alone or in combination with PMA, forskolin, dbcAMP, and ionomycin in the presence and absence of NGF for 3 h prior to infection with 0.01 PFU of KOS or n212 per cell. At 24 hpi, virus yields were measured (Table 1).

The NGF-induced ICP0-complementing activity is expressed transiently. To determine the optimal time of addition and length of NGF treatment relative to the time of infection required to produce maximal expression of the NGF-induced n212-complementing activity, PC12 cells were treated with NGF 3 h prior to infection, at the time of infection, or 15 min, 30 min, 1 h, and 3 h after infection with 0.01 PFU of KOS or n212 per cell. Twenty-four hours after infection, virus yields were measured by plaque assay. Addition of NGF to cultures 3 h prior to infection and at the time of infection produced six- and ninefold increases, respectively, in the 24-h yield of n212 (Fig. 5A). However, addition of NGF 15 min, 1 h, or 3 h after infection produced a progressive decrease in the 24-h yield of n212 (Fig. 5A). These treatments had only minor effects (approximately twofold) on the 24-h yield of KOS (Fig. 5A). The results of these tests suggest either that (i) the NGF-induced activity which is able to substitute for ICP0 is required within the first 3 h prior to infection and the 30 min immediately after infection or (ii) by 3 hpi, infected PC12 cells no longer respond to stimulation by NGF.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Time course of the NGF-induced n212-complementing activity. (A) PC12 cells (106/35-mm-diameter dish) were treated with NGF (100 ng/ml) 3 h prior to infection, at the time of infection, or 15 min, 30 min, 1 h, and 3 h after infection with 0.01 PFU of KOS and n212 per cell. Virus yields were measured at 24 hpi and expressed as fold increase in viral replication relative to mock-treated samples. (B) PC12 cells (106/35-mm-diameter dish) were treated with NGF (100 ng/ml) for 144, 72, 48, 24, 12, 6, and 3 h prior to infection, at the time of infection, or 3 h after infection with 0.01 PFU of n212 or KOS per cell. Virus yields were measured at 24 hpi. NGF-induced n212-complementing activity is expressed as fold increase in viral titer relative to mock-treated samples.

NGF treatment of PC12 cells increases the steady-state levels of viral mRNA. ICP0 increases the steady-state levels of viral E and L mRNAs during low-multiplicity infection by increasing the rate of initiation of mRNA synthesis (37). To test whether the NGF-induced n212-complementing activity functions at the same level as ICP0 to increase the steady-state level of viral mRNA, PC12 cells were treated with NGF or mock treated for 12 h prior to infection with 0.1 PFU of KOS or n212 per cell. At 0, 4, 7, and 10 hpi, cytoplasmic RNA was isolated and levels of ICP4, thymidine kinase (TK), and gC mRNAs were measured by quantitative RNase protection assay. The results of these tests showed that at all times tested after infection, the levels of TK and gC but not ICP4 mRNAs in n212-infected cells were markedly higher in the presence than in the absence of NGF, whereas the level of cellular GAPDH mRNA remained relatively constant in the presence and absence of NGF (Fig. 6). A similar result was observed for these viral mRNAs in KOS-infected cells; however, the NGF-induced enhancement of viral mRNA accumulation was greater in n212-infected cells. This result indicates that the NGF-induced n212-complementing activity functions to increase the accumulation of E and L but not IE viral mRNAs. Notably, although levels of viral mRNAs in KOS-infected cells were also enhanced, this NGF-induced enhancement was less evident at the level of viral replication (Fig. 2A and 5). The results of these tests indicate that the NGF-induced activity that complements n212 functions at the level of mRNA accumulation and, like ICP0, serves to increase E and L gene expression.

View larger version (37K): [in this window] [in a new window]   FIG. 6.   NGF induces viral mRNA accumulation in infected PC12 cells. PC12 cells (5 × 106/60-mm-diameter dish) were mock treated or treated with NGF (100 ng/ml) for 12 h prior to infection with 0.1 PFU of KOS or n212 per cell. At 0, 4, 7, and 10 hpi, cytoplasmic RNA was isolated and levels of ICP4, TK, and gC mRNAs were measured by quantitative RNase protection assay. The range values for the image display were set at 0 to 2,000 (ICP4), 0 to 1,000 (TK), and 0 to 200 (gC).

ICP0 does not affect the induction of mRNAs of cellular primary response genes. Activation of growth factor-dependent signal transduction pathways leads to the transient expression of cellular primary response genes such as c-fos, c-jun, junD, and krox24 (30). These genes encode transcription factors which regulate secondary response genes. In PC12 cells, the secondary response genes induced by NGF treatment are responsible for the morphological and biochemical changes associated with differentiation (66). In addition, herpesvirus infection also induces selected cellular primary response genes (1, 4). The biological consequences of this induction are not well understood. To determine whether ICP0, like the NGF-induced n212-complementing activity, is involved in the herpesvirus infection-specific induction of cellular primary response genes, PC12 cells were infected with KOS or n212 for 60, 90, or 120 min or treated with NGF for 0, 30, 60, 90 or 120 min. At the times indicated (Fig. 7) cytoplasmic RNA was isolated. The RNA was separated according to molecular weight by denaturing gel electrophoresis and transferred to a nylon membrane by Northern blotting. The membrane was probed with radiolabeled RNA probes specific for the human c-fos message and the rat GAPDH mRNA. As shown in Fig. 7, the kinetics and extent of activation of c-fos mRNA were similar in cells treated with NGF or infected with KOS or n212, suggesting that ICP0 is not involved in the herpesvirus-induced activation of cellular primary response genes. Similar results were observed when northern blots were probed with radiolabeled c-jun, junD, and krox24 probes (data not shown). Moreover, as a control, addition of media in the absence of NGF did not induce cellular primary response gene expression. The results of these tests indicate that ICP0 is not involved in the herpesvirus infection-specific induction of cellular primary response gene expression.

View larger version (66K): [in this window] [in a new window]   FIG. 7.   ICP0 does not induce c-fos expression. PC12 cells (5 × 106/60-mm-diameter dish) were treated with NGF (100 ng/ml) for 0, 15, 30, and 60 min or infected with 5.0 PFU of KOS and n212 per cell for 30, 60, 90, and 120 min. Cytoplasmic RNA was isolated at each time point as described in the text. The RNA was size fractionated by denaturing gel electrophoresis and transferred to a nylon membrane by Northern blotting. The Northern blot was probed with 100 ng of 32P-labeled antisense RNA probe (8 × 105 cpM/ng) specific for the mouse c-fos message. The blot was reprobed with 150 ng of 32P-labeled antisense RNA probe (8.8 × 105 cpM/ng) specific for the rat GAPDH message. The image was visualized by PhosphorImager analysis. The range values for the image display were set at 0 to 500 counts.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The balance between productive and latent infection is ultimately determined by the transcriptional permissivity of the infected cell. Growth factors and extracellular signals influence the transcriptional permissivity of the cell by activating intracellular signaling cascades. Viral IE proteins circumvent the need for extracellular signaling by increasing the transcriptional permissivity of the infected cell, thereby promoting viral gene expression. During reactivation, in the absence of viral regulatory proteins, activation of cellular signaling cascades likely changes the transcriptional permissivity of the infected cell and stimulates viral gene expression. In support of this hypothesis, Tal-Singer et al. have reported that viral E and L genes may be induced prior to IE gene expression during reactivation in mice, suggesting that cellular functions can substitute for viral IE genes during reactivation (70). In addition, the levels of cyclic nucleotides, which function as second messengers during signal transduction, influence the maintenance and reactivation of latent virus in mice (23, 64). Moreover, activators of PKA and PKC as well as second messenger analogs stimulate reactivation of latent HSV-1 in primary rat neurons latently infected in vitro (68). While activators of PKA and PKC induce reactivation in this system, they fail to induce the ICP0-like activity, suggesting that activation of these enzymes alone is not sufficient to complement replication of n212. Taken together, these observations suggest that intracellular signaling plays a major role in regulating viral gene expression during reactivation.

NGF and FGF are two of the many neurotropic factors whose concentrations change during the host stress response and may contribute to the signals that induce HSV reactivation (45). NGF induces expression of the LAT promoter in a ras-dependent manner in PC12 cells, suggesting that during latency, LAT expression may be regulated by NGF or NGF-like extracellular signals (26). Changes in the levels of stress-induced growth factors like NGF may well lead to activation of cellular signaling pathways which in turn induce activities that stimulate viral gene expression.

We have identified and characterized an NGF/FGF-inducible cellular activity that stimulates replication of the ICP0 null mutant, n212. The NGF-induced cellular activity is partially ras dependent and requires activation of multiple NGF-dependent signaling pathways. This activity is transiently expressed, with peak complementing activity observed within the first 12 h of NGF treatment. Like ICP0, the NGF-dependent n212-complementing activity functions to stimulate viral E and L mRNA accumulation.

The NGF-induced complementing activity is cell type specific. The NGF/FGF-induced n212-complementing activity is specific to PC12 cells. NGF treatment of a human neuroblastoma cell line, SY5Y, had little effect on viral growth (data not shown). Notably, even clonal isolates of SY5Y constitutively expressing the trkA gene, which encodes the NGF receptor, failed to induce the n212-complementing activity in response to NGF. Furthermore, treatment of Vero cells with NGF, FGF, or EGF produced little difference in virus yields at 3 and 24 hpi relative to mock treatment. Although Vero cells do not express NGF receptors, they do express FGF and EGF receptors (12, 19). Notably, FGF and EGF treatment of Vero cells stimulates mitogenesis, indicating that these receptors are biologically active (12). These data imply that NGF/FGF signaling in conjunction with some other factor(s) in PC12 cells is required for complementation of n212 and that NGF/FGF signaling alone is not sufficient for induction of the n212-complementing activity.

Cellular signaling, phosphorylation, and transcription factor activation. How do NGF and FGF complement n212? A well-recognized endpoint of NGF/FGF-induced signal transduction is the phosphorylation of nuclear transcription factors (58, 66, 82). Activation of transcription factors by phosphorylation is a common mechanism by which growth factors initiate changes in cellular transcription (35). NGF and FGF activate multiple serine/threonine kinase cascades including the ras/raf-1/ MEK/ERK pathway and cyclin-dependent kinase activities (16, 69). Once activated, these kinases phosphorylate transcription factors such as cAMP response element binding protein and serum response factor, thereby stimulating their transcriptional activating activities (43, 58, 66). NGF also induces phosphorylation of SP1 and c-Fos and stimulates NF-B DNA binding activity (72, 81, 83). The NGF/FGF-induced n212-complementing activity may require several of these activated kinase signaling pathways to phosphorylate and activate transcription factors which are then used by the virus in the absence of ICP0 to stimulate viral gene expression. The observation that serine/threonine kinase inhibitors partially block the NGF-dependent stimulation of n212 replication (Fig. 4) is consistent with this hypothesis.

A comparison of ICP0-like cellular activities. A comparison of the known ICP0-like cellular activities is shown in Table 2. Like ICP0, both the NGF-induced activity in PC12 cells and the cell cycle-regulated activity in Vero cells stimulate viral E and L but not IE gene expression. In contrast, the activity expressed constitutively in U2OS cells and the cell cycle-regulated activity in NB41A3 cells preferentially stimulate basal IE gene expression in transient expression assays. These differences may be due to the differential effects of the cellular activity on DNA delivered by transfection versus infection. In addition, it is unknown whether increased basal IE gene expression is sufficient to complement ICP0 null mutant replication. The U20S cell activity also stimulates IE gene expression during infection; however, it is unknown whether this effect occurs throughout infection or only at later times postinfection.