Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany1
Axxima Pharmaceuticals AG, Martinsried, Germany2
Author for correspondence: Manfred Marschall. Fax +49 9131 852 6493. e-mail mdmarsch{at}viro.med.uni-erlangen.de
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Abstract |
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Introduction |
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To date, therapeutic antiviral compounds against HCMV are limited. Ganciclovir (GCV), cidofovir and foscarnet inhibit HCMV genome replication, either directly or indirectly, but these drugs may induce the formation of resistant virus, have low oral bioavailability and show dose-related toxicity (reviewed by Hayden, 1995 ). However, the panel of anti-HCMV compounds becoming available is expanding. Firstly, the phosphorothioate oligonucleotide fomivirsen (ISIS 2922) is a sequence-specific inhibitor of transcription of the major immediate early gene region which effectively blocks the onset of the virus replication cycle (reviewed by Perry & Balfour, 1999
). Secondly, benzimidazole riboside compounds (BDCRB, TCRB, 1263W94 and others) possess inhibitory capacities towards different stages of HCMV replication (for example, genomic DNA maturation) and represent attractive candidates for orally applicable and low-toxicity therapeutic compounds (reviewed by Chulay et al., 1999
). Thirdly, a novel compound, termed A771726 [N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotoamide], which is the active metabolite of the anti-inflammatory drug leflunomide, has recently been described as a potent inhibitor of HCMV replication, probably acting at the late stage of infection by preventing virion assembly (Waldman et al., 1999a
, b
). The general value of most of these compounds for use as antiviral drugs will have to be proven through clinical trials.
pUL97 is a well-characterized target for antiviral therapy, as the specific activity of pUL97 is required to convert GCV into its monophosphorylated derivative (Littler et al., 1992 ; Sullivan et al., 1992
). Cellular enzymes further phosphorylate GCV into its triphosphated form, which inhibits viral DNA synthesis by inhibiting the viral DNA polymerase (reviewed by Hayden, 1995
). Several reports have shown that pUL97 acts as a serine/threonine-specific protein kinase that can phosphorylate itself and other proteins (Chee et al., 1989
; He et al., 1997
; Kawaguchi et al., 1999
; Michel et al., 1998
, 1999
; van Zeijl et al., 1997
; Wolf et al., 1998
). Interestingly, functional domains for protein and GCV phosphorylation are partially distinct (Michel et al., 1998
; Wolf et al., 1998
) and natural nucleosides are not recognized substrates of the pUL97 kinase, indicating that nucleoside kinase activity is not its principal function (Michel et al., 1996
). A recent publication by Slater et al. (1999)
provided the first evidence that protein kinase inhibitors are potent and selective antagonists of HCMV replication in tissue culture and as pUL97 function is critical for efficient HCMV replication (Michel et al., 1996
; Prichard et al., 1999
), it was considered to be a promising target for antiviral therapy.
In the present work, we established a novel system for examining pUL97 kinase activity in transfected cells. By quantifying the cytotoxic effects induced by the conversion of GCV into its phosphorylated derivatives, it was possible to determine the kinase activity of pUL97. The induction of apoptosis as a side effect of intracellular GCV phosphorylation has also been described for herpes simplex virus (HSV) thymidine kinase (Beltinger et al., 1999 ; Freeman et al., 1993
; Oldfield et al., 1993
). We considered GCV phosphorylation to be a useful indicator for pUL97 protein kinase activity, since it was reported that mutations in UL97 which abrogate protein kinase function (i.e. resulting in a lack of autophosphorylation) also resulted in a complete loss of GCV phosphorylation (Michel et al., 1999
). In the second stage of our analysis, we performed conventional in vitro kinase assays to confirm the level of protein phosphorylation. We describe the identification of specific indolocarbazole compounds, which are known to act as inhibitors of serine/threonine protein kinases of the protein kinase C (PKC) family (reviewed by Goekjian & Jirousek, 1999
), that inhibit pUL97 kinase activity. In contrast, other indolocarbazole compounds and a tyrosine protein kinase inhibitor did not interfere with the activity of pUL97. Furthermore, additional information about the kinase activity and characteristics of pUL97 mutants was obtained. A screening system for antiviral agents is also presented.
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Methods |
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(2) pcDNA-UL97(K355M), pcDNA-UL97FLAG, pcDNA-UL97VSV and pcDNA-UL97HA PCR primers 2 and 5, 6, 7 or 8, respectively, were used with pcDNA3 [ORF-UL97(K355M) was generated by site-directed mutagenesis (Kunkel, 1985 ). The primary construct containing the wild-type UL97 ORF and the mutagenesis primer 9 were used to substitute the codon AAG355 (lysine) with ATG (methionine)].
(3) pSC-UL97 and pI18neo-UL97 PCR primers 1 and 4 were used with either pSuperCatch (Georgiev et al., 1996 ) or pI18neo (Marschall et al., 1999
).
(4) pLXSN-UL97 and pCmn-UL97 PCR primers 2 or 3, respectively, and 5 were used with either pLXSN (Clontech) or pCMV/myc/nuc (Invitrogen). pCmnGFP is a green fluorescent protein (GFP)-encoding expression construct of the pCMV/myc/nuc vector used as a standard transfection control.
Oligonucleotides.
Synthetic oligonucleotide primers for PCR were purchased from Sigma. UL97-specific sequences are underlined. Restriction sites are indicated in bold and tagged or mutated sequences are indicated in italics. The sequences of the forward primers used are as follows:
(1) 5-UL97-BglII (TAGTAGATCTATGTCCTCCGCACTTCGGTTCT)
(2) 5-UL97-EcoRI (CCCGAATTCATGTCCTCCGCACTTCGG)
(3) 5-UL97-NcoI (CATGCCATGGGCATGTCCTCCGCACTT).
Reverse primer sequences are as follows:
(4) 3-UL97-SalI (TAGTGTCGACTTACTCGGGGAACAGTTGGCG)
(5) 3-UL97-XhoI (CCGCTCGAGTTACTCGGGGAACAGTTG)
(6) 3-UL97-FLAG-XhoI (CCGCTCGAGTTACTTGTCGTCATCG TCTTTGTAGTCCTCGGGGAACAGTTG)
(7) 3-UL97-VSV-XhoI (CCGCTCGAGTTACTTGCCCAGCCGGT TCATCTCGATGTCGGTGTACTCGGGGAACAGTTG)
(8) 3-UL97-HA-XhoI (CCGCTCGAGTTAAGCGTAATCTGGAA CATCGTATGGGTACTCGGGGAACAGTTG)
(9) Mut355-UL97 (CTTACGCGCCACCATGACCACGCGATA).
Protein kinase inhibitors.
Staurosporine (STP), Gö6976, Gö7874 and NGIC-I are inhibitors of serine/threonine protein kinases. AG-490 (tyrphostin B42) is an inhibitor of tyrosine protein kinases (Meydan et al., 1996 ). All compounds were purchased from Calbiochem. Stock solutions were prepared in DMSO and stored at -20 °C.
Cultured cells and infection procedures.
293 cells (human embryonic kidney cells) were cultivated in Dulbeccos minimal essential medium (DMEM) containing 5% foetal calf serum (FCS) and 100 µg/ml gentamycin. Primary human foreskin fibroblast (HFF) cell cultures were grown in minimal essential medium (MEM) containing 5% FCS and 100 µg/ml gentamycin. Subconfluent monolayers were used for HCMV strain AD169 infection.
The UL97 in-cell-activity assay.
On day 0, 293 cells were seeded in 96-well plates (20000 per well) to obtain 50% confluent monolayers. On day 1, transfection was performed according to the lipofectamine procedure (Lipofectamine Plus reagents, Gibco BRL). For this, identical transfection conditions were chosen for 24 wells of the 96-well plate so that measurements over an 8-well line could be carried out in triplicate. Components A [2·510 µg plasmid DNA (UL97 expression construct or control), 300 µl FCS-free DMEM and 25 µl Plus reagent] and B (12·5 µl Lipofectamine reagent and 300 µl FCS-free DMEM) were used for each transfection assay. Both components were incubated for 15 min at room temperature. Then, A and B were combined, mixed thoroughly and incubated for 15 min at room temperature. Meanwhile, the culture media of the 96-well plate were removed with a multichannel pipette and replaced by 50 µl of fresh FCS-free DMEM per well. An aliquot of 25 µl of each transfection mixture (AB) was added per well. Plates were incubated for 5 h at 37 °C in 5% CO2. Subsequently, 125 µl DMEM supplemented with 10% FCS was added per well and incubated overnight at 37 °C in 5% CO2. On day 2, transfection media were removed from the cells. GCV was diluted in DMEM containing 5% FCS to create a gradient of appropriate GCV concentrations and added in a volume of 100 µl per well. Protein kinase inhibitors were diluted in DMEM containing 5% FCS and added in a volume of 100 µl per well immediately after the addition of GCV. Plates were incubated at 37 °C in 5% CO2.
Visual evaluation of transfected cells and quantification of the colour change in the culture media.
On day 7, visual evaluation of plates became possible by the colour change (yellow to red) in the culture medium containing Phenol Red as a pH indicator. Visual quantification was confirmed by examining cytotoxic effects under a microscope and photometric quantification of the colour change was achieved using an ELISA plate reader (OD560).
Quantification of cytotoxicity signals from cell layers.
Cytotoxicity signals were quantified in the residual cell layers using the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega), which measures lactate dehydrogenase (LDH) activity in cell lysates 5 days post-transfection. For this, culture media were removed, cells were rinsed with PBS and lysed in a 1x concentration of the kit lysis buffer (100 µl per well). After 45 min of incubation at 37 °C, cell debris was removed by centrifugation and 5 µl of each lysate was diluted in a total of 50 µl PBS to determine LDH activity. A sample of 50 µl of substrate mix was added to each well and incubated for 30 min at room temperature in the dark. Thereafter, 50 µl of stop buffer was added and the colour reaction was quantified using an ELISA plate reader (OD490).
Double-selection protocol for UL97-expressing cell clones.
293 cells were transfected with either pUL97 expression constructs or control plasmids encoding a geneticin-selectable marker and selected for geneticin resistance (750 µg/ml). Individual clones were isolated and selected (in parallel) for either geneticin resistance alone (cell stock plate) or for geneticin resistance in addition to the ability to convert GCV (100 µM) (activity test plate). Clones expressing active pUL97 kinase were cultivated from the cell stock plate and used for large-scale screening of compounds that inhibit pUL97 kinase activity.
UL97 in vitro kinase assay.
293 cells were seeded in 12-well plates in a volume of 1 ml (20000 cells per well) 24 h before transfection. Transfection was performed according to the lipofectamine procedure described above and cells were incubated for 16 h at 37 °C and 3% CO2. Cells were washed with PBS and fresh medium was added. Cells were then cultivated for a further 24 h. Indolocarbazole compounds were added to the culture media 2 h before lysis. As a control, DMSO was added to monitor the effect of the solvent. Finally, media were removed and cells were lysed in 200 µl of lysis buffer on ice for 30 min (10 mM TrisHCl, pH 7·5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0·1% SDS, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF and 10 µg/ml aprotinin). After lysis, samples were centrifuged at 4 °C for 30 min at 13000 r.p.m. Supernatants were transferred to fresh tubes and used for immunoprecipitation with UL97 antiserum (PepAs 1343; 1·5 µl/supernatant) together with 60 µl of a solution of protein ASepharose beads (Pharmacia) and an additional 500 µl of lysis buffer. After 2·5 h of incubation at 4 °C on an overhead rotary wheel, precipitates were centrifuged at 2000 r.p.m. at 4 °C for 2 min in an Eppendorf centrifuge and washed twice with 500 µl of HNTG buffer (50 mM HEPES, pH 7·5, 150 mM NaCl, 1 mM EDTA, 10% glycerine and 0·1% Triton X-100). For the in vitro kinase assay, washing was repeated with 500 µl of kinase base buffer (50 mM Ches, pH 9·5, 10 mM MgCl2 and 1 mM sodium orthovanadate) and, thereafter, samples were incubated with 40 µl of complete kinase buffer for 30 min on a shaker at 30 °C (kinase base buffer, 2 mM DTT, 2 µM ATP, 1 µCi/sample [-32P]ATP or 5 µCi/sample [33P]ATP and, optionally, 15 µM histone 2B). Finally, the reaction was stopped by the addition of boiling mix buffer (see below) before samples were separated by 15% SDSPAGE. Dried gels were exposed to autoradiography films or a phosphorimager plate.
Western blot.
Protein samples were denatured under reducing conditions and electrophoresis was performed according to standard conditions, as described previously (Marschall et al., 1999 ). Blots were incubated with UL97-specific antibodies for 2 h at room temperature. The rabbit UL97 antiserum PepAs 1343 [raised by the use of an immunization peptide, UL97/116 (MSSALRSRARSASLGT)], a rabbit UL97 antiserum raised against a bacterially produced fragment of pUL97 (Michel et al., 1996
) or a monoclonal antibody MAb-UL97 (Michel et al., 1998
) was used at a dilution of 1:10000, 1:10000 or 1:5000, respectively. After washing, peroxidase-conjugated secondary antibodies (Dianova) were added and incubated at a dilution of 1:5000 for 1 h at room temperature and developed in an enhanced chemiluminescence reaction (ECL Western Detection kit; Amersham Pharmacia), according to the manufacturers instructions.
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Results |
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Indolocarbazole compounds drastically reduce pUL97 kinase activity
Indolocarbazole compounds (derivatives of the lead compound STP), which have been recently characterized as inhibitors of HCMV replication, were analysed for their inhibitory effects on the viral protein kinase pUL97. It should be stressed that for STP and its derivatives NGIC-I, Gö6976 and Gö7874, in particular, the inhibitory effect on serine/threonine-specific kinases of the PKC family has been described in several publications (Hu et al., 1996 ; Pindur et al., 1999
; reviewed by Goekjian & Jirousek, 1999
), but to date, their effect on herpesvirus protein kinases remains in question. In the UL97 in-cell-activity assay, we identified a clear reduction in pUL97 activity (as measured by the level of GCV phosphorylation) following treatment with either NGIC-I or Gö6976. In contrast, no change in pUL97 activity was noted for another indolocarbazole compound, Gö7874, or for the compound AG-490, a tyrosine kinase-specific inhibitor that was added for comparison (Fig. 3A
). In addition to these examples, a large number of other indolocarbazole compounds was tested in the UL97 in-cell-activity assay. Taken together, the results confirmed the strong inhibitory activity of NGIC-I and Gö6976 against pUL97, which is in contrast to the low efficacy of related compounds (data not shown). To confirm the level of protein phosphorylation, we performed an in vitro kinase assay for pUL97 (Fig. 3B
, C
). pUL97 was immunoprecipitated from transfected cells incubated in the presence or absence of indolocarbazole compounds and the levels of phosphorylated histone 2B or pUL97 in the precipitates were measured. Using this assay, inhibition of pUL97 by NGIC-I and Gö6976 as well as the lack of inhibition by Gö7874 and AG-490 could be confirmed. Furthermore, the catalytically inactive mutant of pUL97 (K355M) was negative in both assays, underlining the reliability of our experimental procedures. Thus, NGIC-I and Gö6976 were identified as the most effective indolocarbazole compounds that inhibit pUL97 kinase activity, with respect to both GCV and protein targets of phosphorylation.
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Discussion |
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For human herpesviruses, the nature of viral protein kinases was first recognized in the case of HSV-1 (DeWind et al., 1992 ) and the distinct functions of these viral protein kinases have been intensively investigated (Chee et al., 1989
; Smith & Smith, 1989
; Cunningham et al., 1992
; Littler et al., 1992
; Ng & Grose, 1992
; Purves & Roizman, 1992
; Sullivan et al., 1992
; Ng et al., 1994
, 1998
; Daikoku et al., 1997
; Ogle et al., 1997
; Kawaguchi et al., 1998
; Moffat et al., 1998
; Ansari & Emery, 1999
; Cannon et al., 1999
; Chen et al., 2000
). The role of HCMV-encoded protein kinase pUL97 in the virus replication cycle is not fully understood, although interaction with cellular proteins as phosphorylation targets was described previously (Kawaguchi et al., 1999
). Furthermore, it has been demonstrated that pUL97 kinase activity is a critical factor for virus replication in tissue culture and that deletion of the UL97 gene results in severe replication deficiency (Prichard et al., 1999
). In a recent study by Slater et al. (1999)
, the inhibitory effect of protein kinase inhibitors of the indolocarbazole class was demonstrated for HCMV infection in cultured human cells. However, the question of whether pUL97 kinase activity is directly impaired by these compounds was not addressed and so the antiviral mechanism remains speculative.
Indolocarbazoles were originally described as a chemical class of inhibitors of serine/threonine protein kinases of the PKC family which act by competitively blocking the ATP-binding site (reviewed by Goekjian & Jirousek, 1999 ). In order to analyse the possible inhibitory effect of indolocarbazole compounds on pUL97 in detail, novel experimental approaches were developed and, based on these findings, four main conclusions were drawn: (i) the activity of the viral protein kinase pUL97 is easily quantified in transfected cells in the absence of virus replication, (ii) the indolocarbazole compounds Gö6976 and NGIC-I specifically block pUL97 kinase activity, (iii) the block in kinase activity is measurable by the level of GCV phosphorylation as well as protein phosphorylation, and (iv) GCV-resistant mutants of the UL97 gene (e.g. M460I), which encode a pUL97 kinase incapable of converting GCV, are not defective in expressing, at least in part, the kinase activity critical for protein phosphorylation. In conclusion, during replication of a UL97 mutant virus [e.g. virus variant AD169GFP314 encoding UL97(M460I)], it seems that mutated pUL97 sufficiently fulfils its functional requirements. Considering the latter aspect, it is important to note that a GCV-resistant variant of HCMV [encoding mutant UL97(H520Q)] is also sensitive to inhibition by indolocarbazole compounds (Zimmermann et al., 2000
). It will be interesting to see whether different virus variants and different cloned mutants of pUL97 possess different sensitivities towards indolocarbazoles, with respect to their GCV and protein phosphorylation activities (M. Marschall and others, unpublished data).
In general, the complete loss of UL97 function, achieved by genetic manipulation of the viral genome, causes a severe deficiency in HCMV replication in cell culture and virus titres of two to three orders of magnitude lower than those produced by the parental virus are attained (Prichard et al., 1999 ). In the present study, we provide evidence that specific inhibition of pUL97 kinase activity can be achieved by treatment with indolocarbazole compounds. So far, it is unknown whether inhibition of pUL97 kinase activity by indolocarbazoles is solely and directly responsible for the block in virus replication. Nevertheless, our study demonstrates that some indolocarbazole compounds possess strong activity against pUL97 in the absence of major cellular side-effects. The avoidance of interfering with cellular kinase functions might be critical for the success of antiviral treatments directed towards viral protein kinases. However, it should be mentioned that inhibition of cellular protein kinases, as investigated in the case of human immunodeficiency virus type 1 (HIV-1), can result in a strong antiviral effect. It was reported previously that the indolocarbazole compound Gö6976 acts as a potent antagonist of virus reactivation in latently infected cells (Quatsha et al., 1993
) and that interference with regulatory cellular pathways might be responsible for this block. Therefore, it was speculated that inhibition of the kinase-mediated activation of the NF-
B transcription factor was a critical step in the anti-HIV-1 mechanism and the importance of this aspect was underlined in a further study on NF-
B antagonists (Mhashilkar et al., 1997
). By analogy, the activity of NF-
B plays a critical role in the cellular regulation of herpesvirus replication, as described in detail for HCMV (Kowalik et al., 1993
; Yurochko et al., 1995
, 1997
) and HSV-1 (Hanson et al., 1998
). In contrast to the situation in HIV-1 reactivation, HSV-1 replication in cell culture shows no significant sensitivity to Gö6976 (Slater et al., 1999
; M. Marschall and others, unpublished results). Thus, the antiviral effect of Gö6976 on members of the herpesvirus family such as HCMV is selective and is based on virus-specific rather than cell-specific mechanisms. Consequently, we postulate that inhibition of HCMV protein kinase pUL97 is the predominant mechanism responsible for the strong antiviral effect of the indolocarbazole compounds described here. During the preparation of this manuscript, a study was published that underlines the inhibitory capacity of indolocarbazoles at the level of HCMV replication as well as pUL97 kinase activity, and the data are in agreement with our findings (Zimmermann et al., 2000
). Future studies, possibly performed on indolocarbazole-resistant HCMV mutants, will have to demonstrate the causative linkage of these two effects and to illuminate the value of these findings for antiviral research and novel strategies in antiviral therapy.
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Acknowledgments |
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Received 23 November 2000;
accepted 2 February 2001.