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Institute of Microbiology and Virology, University of Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany
Correspondence
Markus Rahaus
rahaus{at}uni-wh.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The ERK cascade mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, differentiation and senescence. Within this cascade, c-Raf plays a key role. The kinase activity of c-Raf is negatively controlled by phosphorylation at Ser-259 (Zimmermann & Moelling, 1999). Ser-338 and Tyr-340/341 are the positive phosphoregulatory sites recognized by Ras and Src (Diaz et al., 1997; Marais et al., 1995). c-Raf activates ERK1/2 via mitogen-activated protein kinase (MEK)1/2 at Tyr-202/204 (ERK1) and Tyr-185/187 (ERK2). Once activated, ERK1/2 regulates a variety of cytoplasmic proteins, one of which is pro-apoptotic Bad. Activated ERK1/2 is also imported into the nucleus, where it phosphorylates various transcription factors, such as c-Myc, Stat1 and -3, Ets-1 and c-Fos (Robinson & Cobb, 1997).
Bad belongs to the Bcl-2 family of proteins regulating the execution of apoptosis. The members of this family are separated functionally into two groups: apoptotic antagonists (Bcl-2, Bcl-XL, Bcl-w) and apoptotic agonists (Bad, Bix, Bax). They mediate their pro- or anti-apoptotic signals through their relative abundance, subcellular localization and post-translational modification. Pro- and anti-apoptotic family members dimerize through their Bcl-2 homology domains, neutralizing each other's function (Chittenden et al., 1995; Oltvai et al., 1993; Yin et al., 1994).
Cell-survival signals block the Bad-mediated induction of apoptosis by phosphorylation at various phosphorylation sites, of which Ser-112 and Ser-136 are the most well-known (Franke & Cantley, 1997). Bad Ser-112 is phosphorylated by ERK1/2 via the ribosomal S6 kinase (RSK) and c-Raf (Harada et al., 1999; Tan et al., 1999; Troppmair & Rapp, 2003; Wang et al., 1996). Ser-136 is phosphorylated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (del Peso et al., 1997; Scheid & Duronio, 1998). Phosphorylation of Bad at Ser-112 and Ser-136 abrogates its pro-apoptotic activity by promoting its association with 14-3-3 proteins, which sequester phosphorylated Bad and thereby prevent its localization to the mitochondria and association with Bcl-XL (Harada et al., 1999; Hsu et al., 1997; Pastorino et al., 1998; Zha et al., 1996). Additional, recently discovered phosphorylation sites of Bad are Ser-128, Ser-155 and Thr-201, which are targeted by a variety of signalling molecules such as protein kinase A (PKA), JNK/SAPK and Cdc2 [for overview, see Jin et al. (2005) and references therein].
c-Myc was originally discovered as the cellular homologue of the retroviral v-Myc oncogene (Dang, 1999). It is a transcription factor involved in a wide variety of cellular processes, including cell proliferation, growth, differentiation and apoptosis (Gregory & Hann, 2000; Menssen & Hermeking, 2002). Upon phosphorylation (Gupta et al., 1993; Noguchi et al., 1999) and dimerization with Max, c-Myc binds to the E-box motif CACGTG and activates transcription. Because of its central role as an activator of diverse cellular processes, the regulation of this transcription factor is crucial for proper cell functioning and ultimately survival.
Varicella-zoster virus (VZV), a human pathogenic alphaherpesvirus causing varicella as the primary infection, establishes latency in sensory ganglia and may reactivate as zoster (Arvin, 1996; Cohen & Straus, 1996). Little is known about virus–host interactions between VZV and the infected cell. Recently, we demonstrated transient activation of JNK/SAPK and p38/MAPK pathways and downstream transcription factors c-Jun, c-Fos and ATF-2 during infection. VZV ORF61 influences the regulation of the activities of these kinases (Rahaus et al., 2003, 2004, 2005). Moreover, it is known that VZV inhibits gamma interferon (IFN-
)-mediated induction of cell-surface major histocompatibility complex class II expression on infected human fibroblasts to promote infection (Abendroth et al., 2000). In addition, treatment of cells with IFN-
and IFN- prior to infection resulted in a strong decrease in replication. However, in untreated but infected cells, two IFN-induced and antiviral-acting systems, the protein kinase R (PKR) pathway and the RNase L pathway, were not activated, indicating that VZV developed strategies to circumvent these host defence mechanisms (Desloges et al., 2005a, c).
Here, we have shown that the ERK cascade is partially activated during VZV infection and contributes to the induction of cell-survival signals. c-Raf remained inactive, whereas its downstream kinases MEK1/2 and ERK1/2 were transiently phosphorylated. Inhibition of this signal cascade resulted in a severe reduction in virus replication and in a slightly increased apoptotic response. The cell-death regulator Bad, a cytosolic indirect target of ERK1/2, was also phosphorylated significantly following VZV infection. Inhibition of the ERK pathway prior to infection suppressed phosphorylation of Bad. Expression of the nuclear transcription factor c-Myc, which is targeted by ERK1/2 and p38/MAPK and is also involved in the induction of apoptosis, was repressed in the later phase of infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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PD98059, a highly selective inhibitor of the ERK pathway, was purchased from Calbiochem. Cells were treated with this reagent 45 min prior to infection at the concentrations given in Results.
Fast-activated cell-based ELISA (FACE).
FACE systems to monitor the levels of ERK1/2, MEK1/2 and Bad phosphorylation and accumulation were obtained from Active Motif. The procedures were performed according to the manufacturer's instructions. To calculate the amounts of total and phosphorylated protein, background results (samples without primary antibody) were subtracted from the data for each sample. Data were normalized according to cell number by calculation of A450/A595. All assays were carried out in three independent experimental series.
Preparation of nuclear and cytoplasmic protein extracts.
Nuclear and cytoplasmic protein extracts for immunoblotting were prepared by using a Nuclear Extract kit (Active Motif) according to the manufacturer's instructions.
Immunoblotting.
Twenty-five micrograms of the respective protein extract was separated by SDS-PAGE and transferred to nitrocellulose as described previously (Rahaus & Wolff, 2000). After blocking in 5 % Blotto, membranes were reacted with antibodies directed against actin, c-Raf, c-Myc pThr-58/pSer-62, poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology), c-Raf pSer-259 (Biosource), USF2, c-Myc (Santa Cruz) and VZV IE63 (kindly provided by B. Rentier, Liège, Belgium), followed by an alkaline phosphatase-conjugated secondary antibody.
Measurement of cytochrome c.
A FunctionELISA cytochrome c assay was performed according to the manufacturer's instructions (Active Motif) using 2 µg freshly prepared cytoplasmic protein extract from the samples indicated in Results.
Haematoxylin-based plaque assay.
Cells were treated/infected as indicated in Results and fixed 24 h later in 4 % paraformaldehyde for 30 min and stained with Mayer's haemalum solution and eosin Y (Merck) (Rahaus et al., 2004). Evaluation was done by counting clearly visible viral plaques in an area of 2 cm2 located precisely at the centre of each well. Absolute plaque numbers were converted into percentages. Positive controls contained at least 250 plaques inside the evaluated area. All experiments were carried out in triplicate.
RNA isolation and reverse transcription.
Confluent monolayers of VZV-infected cells were harvested at the time points indicated in Results. Total RNA was isolated by using TRIzol reagent (Life Technologies). Samples were purified from DNA contamination by DNase treatment (Promega) as recommended by the manufacturer. RT-PCR was performed as a two-step procedure as described previously (Rahaus & Wolff, 2003).
Standard and quantitative PCR analysis.
cDNA obtained by reverse transcription was used for PCR amplification of Ets, VZV ORF63 and cellular pyruvate dehydrogenase (PDH). The primers used are shown in Table 1. The reaction mixture was prepared as described previously (Rahaus et al., 2003). Thermocycling conditions for amplification of Ets were: initial denaturation for 4 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 57·2 °C and 1 min at 72 °C. Conditions for the ORF63 and PDH PCR were as described previously (Rahaus & Wolff, 2003). The resulting products of 524 bp (Ets) and 326 bp (ORF63) were separated on 1·6 % agarose gels and those of 104/180 bp (PDH) on a 3 % agarose gel. The PDH PCR was used to control the quality and purity of the RNA, as it was designed to amplify a region containing an intron that will only be amplified when contaminating DNA is present (180 bp fragment). Amplification of the intronless purified RNA resulted in a product of 104 bp (Rolfs et al., 1992).
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. Thermocycler conditions were: initial denaturation for 4 min at 94 °C, followed by 30 s at 94 °C, 30 s at 59·7 °C and 1 min at 72 °C. The resulting product was 500 bp. The internal standard was designed by using the hybrid-primer technique and resulted in a truncated fragment of 380 bp. The internal standard differed from its original gene fragment only in length and was reamplified by using the respective primer pairs shown in Table 1. PCR products were separated on 1·6 % agarose gels. Densitometric evaluation of the resulting signals was carried out by using Molecular Analyst software (version 1.5; Bio-Rad).
Statistical analyses.
Data are presented as means±SD. Statistical comparisons were performed by using Student's t-test type 3 where appropriate, with a value of P<0·05 taken to indicate significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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a).
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). These results indicated that phosphorylation of ERK1/2 increased transiently, whereas accumulation of the protein remained relatively stable during infection. A control immunoblot was carried out to monitor infection by analysing the presence of VZV IE63. To ensure that the observed effects were specific and not a result of general variations in protein concentration, cellular USF2, which is known to maintain unchanged levels of expression during the infectious cycle of VZV, was detected by immunoblotting (Rahaus et al., 2004) (Fig. 1c). To analyse variations in the activity of the ERK signalling cascade in more detail, phosphorylation of the ERK upstream kinases MEK1/2 and c-Raf, which activates MEK1/2, was investigated. We performed triplicate FACE assays to examine the levels of MEK1/2 phosphorylation and accumulation in VZV-infected MeWo cells at various time points. In the very early phase of infection, MEK1/2 phosphorylation remained relatively stable, but was followed by a steady increase, reaching a 2·2-fold level of phosphorylation at 12 h p.i. (Fig. 2a). The time point of peak MEK1/2 phosphorylation corresponded to that of ERK1/2. Between 12 and 20 h p.i., MEK1/2 phosphorylation dropped and then remained constant at a level of 1·2-fold phosphorylation compared with the basal level determined at 0 h p.i. In contrast to the declining phosphorylation of MEK1/2 in the very early phase of infection, accumulation of the protein increased rapidly and reached its maximum (1·9-fold) by 1 h p.i. Subsequently, the amount of MEK1/2 protein decreased steadily. In the late phase of infection (36–48 h p.i.), MEK1/2 accumulation dropped to a level lower than that found in uninfected cells (Fig. 2b). Infection was monitored by detection of VZV IE63 and sample loading by detection of cellular USF2 as described above (Fig. 2c)
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). c-Raf is inactivated by phosphorylation of Ser-259 and activated by phosphorylation of Ser-338 and Tyr-340/341. To investigate the phosphorylation status of c-Raf during VZV infection, immunoblots were carried out using 25 µg MeWo cell-derived cytoplasmic protein extracts prepared at different time points p.i. to detect total and phosphorylated c-Raf. c-Raf was present in VZV-infected MeWo cells for the entire period of analysis (Fig. 2d). Interestingly, we found c-Raf to be phosphorylated at Ser-259 and thus inactive over the entire course of the experiment. Phosphorylation of Ser-338 or Tyr-340/341 was not detected. Additionally, no phosphorylation of Raf-B at activating phosphorylation sites was detected (data not shown). These data showed that the ERK cascade is activated only partially after infection of MeWo cells with VZV: MEK1/2 and ERK1/2 were phosphorylated, whilst c-Raf remained inactive.
Importance of a functional ERK pathway for the replication of VZV
As members of the ERK cascade were found to be phosphorylated after infection of cells with VZV, we investigated the importance of this signalling cascade throughout infection. In triplicate experiments, MeWo cells were treated with 40, 80 or 120 µM PD98059, a selective and potent inhibitor of MEK1/2 phosphorylation (Alessi et al., 1995). At 24 h p.i. with VZV, plaque assays were performed. The number of infectious centres found in untreated infected cells was set as 100 % (corresponding to 333±20·8 plaques). After treatment with 40, 80 or 120 µM PD98059, the number of viral progeny decreased steadily to 65·2 % (217±19·6 plaques), 22·7 % (75·6±9 plaques) and 14·1 % (47±12·3 plaques), respectively (Fig. 3a). The control experiment shown in Fig. 3(b) confirmed the suppression of ERK1/2 phosphorylation by increasing concentrations of PD98059. Further controls confirmed that PD98059 used at these concentrations was not toxic to the cells (not shown). In summary, a functional ERK1/2 cascade is essential for VZV replication, as the stepwise inhibition of ERK phosphorylation resulted in a stepwise reduction in the number of infectious centres.
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), but also regulators of cell death, we investigated whether the apoptotic response was modulated in cells infected with VZV after inhibition of the ERK cascade. In previous reports, we have shown that no apoptotic response is detectable in VZV-infected MeWo cells earlier than 48 h p.i. (Rahaus et al., 2005). In triplicate experiments, we determined the cytoplasmic levels of cytochrome c. Its release from mitochondria into the cytoplasm is an early event of apoptosis. MeWo cells were treated with 80 µM PD98059. This concentration did not inhibit the pathway completely (Fig. 3) and thus VZV replication at very low levels remained detectable. At 24 h p.i., freshly prepared cytoplasmic protein extracts were introduced into a cytochrome c ELISA. Uninfected and infected MeWo cells, as well as treated but uninfected cells, were used as controls. Cytoplasmic cytochrome c levels of uninfected cells were set as 100 %. After infection of untreated cells, the cytoplasmic level of cytochrome c increased only slightly (112 %), whereas inhibition of the ERK pathway prior to infection resulted in a moderate, but nevertheless significant, increase (132·2 %). Significance was confirmed by Student's t-test (Fig. 4a). However, this increased cytochrome c release was relatively low compared with the four- to fivefold increase gained after treatment of cells with actinomycin D (not shown). We also determined PARP cleavage as a late event of apoptosis. Immunoblots were performed with nuclear protein extracts derived from samples as described above. Elevated rates of PARP cleavage were found only after infection of cells in which the ERK cascade had been blocked (Fig. 4b). Detection of VZV IE63 confirmed the replication of VZV at very low levels in cells treated with PD98059 (Fig. 4b).
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Based on the above data, the activity levels of two targets of ERK implicated in the regulation of cell survival and apoptosis were analysed: c-Myc and Bad.
Availability and activity of c-Myc in VZV-infected cells
c-Myc is a transcription factor influencing growth regulation and apoptosis. It is phosphorylated at Thr-58 or Ser-62 by nuclear-translocated phosphorylated ERK1/2 or p38/MAPK (Gupta et al., 1993; Noguchi et al., 1999).
Immunoblots were performed to detect the presence of c-Myc in VZV-infected cells at various time points p.i. We found a steady decrease in the level of total c-Myc during infection. By 48 h p.i., c-Myc was no longer detectable. The phosphorylation of c-Myc was also analysed by reacting protein lysates after SDS-PAGE with a specific phospho-antibody (Thr-58/Ser-62). In lysates derived from uninfected cells, as well as in those derived from the early phase of infection, phosphorylated c-Myc was detected. By 12 h p.i., only a very weak band indicating c-Myc phosphorylation was seen; at later time points, no signal was detectable (Fig. 5a). Actin, monitored as a control, confirmed the specificity of this effect, as its expression remained uninfluenced by infection. Infection of cells was confirmed by detection of VZV IE63 (Fig. 5a). Next, we investigated whether the observed repression of c-Myc was based on protein degradation or inhibition of transcription. A quantitative RT-PCR was performed, using total RNA isolated at time points corresponding to those given above. As an internal standard for quantification, we amplified a truncated version of the c-Myc fragment. For each time point, a triplicate co-amplification series with 5x109–1x1011 molecules of the internal standard and 1 µg cDNA from reverse-transcription reactions was performed. Densitometric evaluation of the resulting bands showed a constant abundance of c-Myc mRNA in the early phase of infection. However, between 12 and 24 h p.i., c-Myc transcription decreased dramatically and was no longer detectable by 24 h p.i. (Fig. 5b). Infection was monitored by amplifying a fragment of the reverse-transcribed IE63 mRNA (Fig. 5c). To determine the specificity of the observed repression of c-Myc transcription, we analysed the presence of Ets mRNA, which is also targeted by ERK1/2. No significant variation in the presence of the Ets transcript was detected in VZV-infected MeWo cells. To verify that all RNA samples were free of DNA contamination, cellular PDH was amplified by PCR in all samples. Only the intronless RNA (104 bp) was amplified; the 180 bp product containing an intron was not amplified (Fig. 5c). As an additional control of RNA purity/DNA contamination, all PCRs were also performed without reverse transcriptase. All samples were free of DNA contamination (not shown).
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In triplicate experiments, we determined protein-accumulation levels and phosphorylation of Bad at Ser-112 in VZV-infected MeWo cells by FACE assays. During the first hour of infection, phosphorylation of Ser-112 increased approximately 5·3-fold (Fig. 6a). From 1 to 20 h p.i., it increased steadily, reaching a maximum of a 9·5-fold at 20 h p.i. Subsequently, phosphorylation at Ser-112 decreased dramatically. The accumulation of total Bad reached a maximum of 3·3-fold at 4 h p.i. and then dropped steadily; by 36 h p.i., it had reached levels lower than the basal level at 0 h p.i. (Fig. 6b). Infection was confirmed by detection of VZV IE63 and the specificity of the reaction by detecting cellular USF2, levels of which remained unchanged (Fig. 6c).
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). Taken together, these experiments demonstrated a marked phosphorylation of Bad at an essential phosphorylation site. VZV mediated this phosphorylation of Bad at Ser-112 by activating the ERK cascade. It is known that phosphorylated Bad is impaired and unable to interact with Bcl-XL. Therefore, the induction of this apoptotic pathway is prevented and cell survival is endorsed, allowing VZV sufficient time to replicate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, c). Furthermore, we found transient activation of stress-related MAPK pathways after infection (Rahaus et al., 2004), which resulted in the provision of cellular transcription factors essential for virus replication without inducing subsequent apoptotic events (Rahaus & Wolff, 2003). VZV IE61 seems to be a critical factor in influencing the activity of JNK/SAPK (Rahaus et al., 2005). These data demonstrate that VZV modulates the activities of cellular signalling pathways to gain optimal conditions for its replication. In this report, we demonstrated that the ERK cascade is also activated throughout VZV infection. However, c-Raf remained inactive, whereas MEK1/2 and ERK1/2 showed transient increases in phosphorylation. The availability of a functional ERK cascade is essential for VZV replication. Cytoplasmic Bad, a major regulator of apoptosis, which can be phosphorylated by cytosolic ERK1/2 via RSK and thus is unable to induce cell death, was also strongly phosphorylated. Bad phosphorylation is likely to be a consequence of VZV-mediated ERK activation. Expression of the cellular transcription factor c-Myc, also an important factor in the induction of cell death after phosphorylation by ERK1/2 or p38/MAPK, was repressed during VZV replication.
The functionality of the ERK cascade is essential for VZV replication, as we observed a severe decline in viral progeny after its inhibition. One explanation for this effect could be that nuclear and cytoplasmic targets of ERK1/2, which are of importance for VZV replication, are no longer regulated in the appropriate manner. The nuclear-localized transcription factor c-Fos is such a candidate, as we have shown that its expression is strongly increased after VZV infection (Rahaus & Wolff, 2003) and that AP-1, of which c-Fos is a component, is essential for VZV gene expression (Rahaus et al., 2004, 2005).
Another major effect to which ERK1/2 contributes is the fast and efficient phosphorylation of Bad at Ser-112, which then is unable to induce apoptosis (Downward, 1999). The fact that a strong increase in Bad phosphorylation at Ser-112 was found by 1 h p.i., whereas ERK1/2 phosphorylation increased only by 5 h p.i., made us speculate that other cellular or viral factors also could be involved directly or indirectly to guarantee the inactive state of Bad. It is known that PKA, protein kinase C and Pac1 are capable of phosphorylating Bad at both Ser-155 and Ser-112 (Harada et al., 1999; Tan et al., 1999). Recent reports have shown that the phosphorylation of Bad in herpes simplex virus (HSV)-infected cells occurs throughout the PKA pathway (Benetti & Roizman, 2004). In addition to this indirect phosphorylation, evidence for direct phosphorylation of Bad by viral factors was demonstrated. HSV US3 kinase mediates post-translational modification of Bad and blocks its cleavage (Munger & Roizman, 2001). US3 is also able to block apoptosis induced by members of the Bcl-2 family (Ogg et al., 2004). Interestingly, a significant reduction in Bad transcription in HSV-infected cells, as well as a strong decrease in transcripts of PI3-K and the regulatory 14-3-3 protein in both pseudorabies virus- and HSV-infected cells, has been reported (Ray & Enquist, 2004). These data stand in contrast to our findings of increased Bad protein accumulation after infection of cells with VZV, for which a preceding increase in transcription has to be presumed. However, in the late phase of infection, total levels of Bad, as well as of MEK1/2 and ERK1/2, decrease. This observation is believed to be a consequence of VZV-mediated late host-protein shutoff (Desloges et al., 2005b; Waterboer et al., 2002).
Activated c-Raf also phosphorylates Bad at Ser-112 (Troppmair & Rapp, 2003). Evidence of a mitochondrial Raf–Bad survival pathway has been demonstrated (Wang et al., 1996). However, in our studies, phosphorylation of c-Raf at Ser-259, which is targeted by Akt, was detected throughout the entire replicative cycle, indicating the inactive state of this kinase. Akt was also found to be activated strongly during VZV infection (M. Rahaus, unpublished data). This, and our additional finding that activating phosphorylation sites of the Raf family member Raf-B also remained unphosphorylated (which is an interesting observation, as melanoma cells normally have activating mutations of Ras and Raf; reviewed by Smalley, 2003), made us conclude that direct signalling via active Raf seems to be without significance during VZV infection. This hypothesis is substantiated by the finding that inhibition of the ERK cascade by PD98059, which specifically prevents phosphorylation of MEK1/2 without affecting upstream Raf, resulted (i) in an enhanced apoptotic response after VZV infection and (ii) in remarkably reduced levels of Bad Ser-112 phosphorylation. Although ERK/RSK is not the only pathway targeting Bad at Ser-112, our results underline the importance of the ERK signalling pathway for cell survival after VZV infection and correspond to data derived from HSV: here, too, an activation of the ERK survival pathway in HSV-infected cells was found. However, the data also contrast with our results, as it was shown that Raf kinase was involved in HSV anti-apoptotic activity (Perkins et al., 2003).
Despite its cytoplasmic function to inhibit the pro-apoptotic action of Bad, ERK1/2 targets a variety of cellular transcription factors, of which some, including c-Jun, c-Fos, ATF-2 and USF, are known essentially to be involved in transregulation of VZV-encoded genes (Meier et al., 1994; Rahaus & Wolff, 1999, 2003; Rahaus et al., 2003, 2004; Yang et al., 2004). c-Myc is another transcription factor phosphorylated by nuclear-translocated active ERK1/2 (Gupta et al., 1993), as well as stress kinases (Noguchi et al., 1999), and promotes both cell proliferation and apoptosis (Alevizopoulos et al., 1997). As with phosphorylated USF, c-Myc is able to bind E-box elements, of which several are identified inside VZV promoters. However, in contrast to USF, whose expression remains stable in VZV-infected cells (Rahaus et al., 2003), c-Myc phosphorylation/activation and expression are repressed in the late phase of infection. We conclude that this repression is accomplished at both post-translational and transcriptional levels and may result from the host shutoff effect. Aside from the data presented here, little is known about the expression and function of c-Myc in herpesvirus-infected cells. It is known that c-Myc plays a critical role in the disruption of Epstein–Barr virus latency (Gao et al., 2004). Nonetheless, repression of c-Myc as well as phosphorylation of Bad following VZV infection indicates a virus-mediated key mechanism enabling cell survival during infection. These results extend our recent data showing that stress-related signalling pathways are activated after infection of cells with VZV to optimize conditions for replication without induction of subsequent cell-defence and apoptotic pathways, and underline the enormous complexity of the virus–host interaction network.
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ACKNOWLEDGEMENTS |
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Received 30 September 2005;
accepted 12 December 2005.
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