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Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany
Correspondence
Manfred Marschall
manfred.marschall{at}viro.med.uni-erlangen.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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). Previous studies demonstrated that deletion of the UL97 region from the viral genome or pharmacological inhibition of the pUL97 kinase activity drastically reduced virus replication (Prichard et al., 1999; Marschall et al., 2002, 2005; Biron et al., 2002; Krosky et al., 2003a). Furthermore, pUL97 plays an important role in pro-drug activation during chemotherapy of HCMV-associated diseases, which is mediated via its phosphorylation of distinct nucleoside analogues, such as ganciclovir. Although pUL97 has been studied intensively at the molecular level for more than a decade, several properties of its activity and structure remain speculative so far.
As described in several previous reports, pUL97 possesses a strong autophosphorylation activity (He et al., 1997; van Zeijl et al., 1997; Wolf et al., 1998; Michel et al., 1999; Marschall et al., 2001; Baek et al., 2002). The regulatory consequences of autophosphorylation for the kinase activity of pUL97, however, are discussed controversially (Michel et al., 1999; Baek et al., 2002; Marschall et al., 2002). So far, it is not clear whether autophosphorylation is an absolute requirement for the kinase activity of pUL97. However, a very likely consequence of autophosphorylation is autoactivation. It is tempting to speculate that autoactivation is based on a mechanism of trans-autophosphorylation by an intermolecular interaction of pUL97 with itself. In fact, under the experimental conditions of an in vitro kinase assay, we were able to demonstrate trans-autophosphorylation (see results described for Fig. 6). This points to a possible self-interaction of pUL97 and raises the question whether this property may be linked with protein kinase activity.
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; Williams, 1999). Another example for dimerization-mediated activation is the transmembrane receptor tyrosine kinase EGFR (epidermal growth factor receptor), which self-associates upon binding of EGF to its extracellular ectodomain and autophosphorylates in trans. Subsequently, autophosphorylated EGFR is recognized at specific phosphotyrosine residues by downstream protein kinases which, as a consequence, gain activity and stimulate cellular signalling cascades (Honegger et al., 1990; Jorissen et al., 2003). Another interesting example is the cAMP-dependent protein kinase PKA. Unstimulated PKA exists as a heterotetramer (holoenzyme), consisting of two regulatory subunits (R) which sequester two catalytic subunits (C). Upon binding of the second messenger cAMP to the R subunits, the holoenzyme dissociates and active C subunits are released (Johnson et al., 2001). This illustrates that, in contrast to PKR dimerization, PKA tetramerization is associated with enzymic autoinhibition. Thus, as illustrated by these examples, the formation and dissociation of protein dimers or tetramers is an important mechanism that coregulates protein kinase activity. In this study, we addressed the question of pUL97–pUL97 interaction and its relation to catalytic activity. Based on the findings, a functional link between pUL97 self-interaction and protein kinase activity is discussed. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), pcDNA-UL44-FLAG and pcDNA-UL84-FLAG (Marschall et al., 2003), and pcDNA-UL97(1-595)-F, pcDNA-UL97(1-523)-F, pcDNA-UL97(1-459)-F, pcDNA-UL97(1-365)-F, pcDNA-UL97(366-707)-F, pcDNA-UL97(181-707)-F, pcDNA-UL97(111-707)-F and pcDNA-UL97(49-707)-F (Marschall et al., 2005). pcDNA-UL97-(M1L)-F was derived from pcDNA-UL97-F by PCR amplification using a forward primer carrying a single base exchange (ATG
CTG). PCR was performed using Vent DNA polymerase (New England BioLabs) in 35 cycles (denaturation 40 s at 95 °C, annealing 40 s at 50 °C and polymerization 120 s at 72 °C). Constructs pDsRed1-N1 and pEGFP-N1, expressing red fluorescent protein (RFP) or green fluorescent protein (GFP) (both from BD Clontech), respectively, were used as positive controls for transfections.
Cell culture and transfections.
293T cells were cultivated in Dulbecco's modified Eagle medium (DMEM) containing 10 % fetal calf serum. Transfection was performed with Transient Lipofectamine 2000 (Invitrogen) according to the protocol of the manufacturer at a cell confluency of 70–90 % using a seeding cell number of 6x105 for 6-well plates (or 3x106 for 10 cm dishes). For protein purification, transient transfection of 293T cells was performed with polyethylenimine reagent (PEI; Sigma) at a cell confluency of 60 %. For the transfection of a 10 cm dish, 12 µg DNA was mixed with 1 ml HBS (150 mM NaCl, 20 mM HEPES/NaOH, pH 7.4) and subsequently incubated with 20 µl PEI 2000 in 1 ml HBS for 20 min at room temperature. Thereafter, the solution was mixed with 36 µl PEI 25 000 in 1 ml HBS and incubated for 20 min before dropwise addition to the cell layer in a 10 cm-dish. After incubation for 4 h at 37 °C, the transfection solution was replaced by fresh medium.
Coimmunoprecipitation assay.
293T cells were transfected in 6-well plates (6x105 cells) or 10 cm dishes (3x106 cells, used for control experiments to increase sensitivity when the expression level of mutants was limited). Two days post-transfection, cells were lysed in 500 or 1000 µl coimmunoprecipitation (CoIP) buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5 % NP-40, 1 mM PMSF, 2 µg aprotinin ml–1, 2 µg leupeptin ml–1 and 2 µg pepstatin ml–1) and used for CoIP with 1 µl or 2.5 µl anti-HA rabbit antiserum (anti-HA.11; Babco), respectively, for 2 h at 4 °C under rotation. Protein A Sepharose beads were added to the CoIP reactions (2.5 mg, 2 h at 4 °C; Amersham Pharmacia Biotech). The precipitates were pelleted and washed before samples of approximately 10 µl (total volume approximately 40 µl) were separated by SDS-PAGE and subjected to Western blot analysis using ANTI-FLAG (M2; Sigma there after referred to as mAb-FLAG) for the detection of coimmunoprecipitates (ECL staining; New England BioLabs). For use as expression controls, samples of the lysates were taken prior to addition of the CoIP antibody and analysed by SDS-PAGE and Western blot (number of cell equivalents approximately 4.5x104).
In vitro kinase assay.
The kinase activities (auto- and substrate phosphorylation) of pUL97 wild-type and mutants were determined in vitro with 2.5 µCi (92.5 kBq) of [
-33P]ATP after immunoprecipitation of the kinase from transfected 293T cells as described previously (Marschall et al., 2001). Purified histone 2B (H2B; Roche) was added exogenously to the reaction at a concentration of 15 µM.
Two-step protein purification, reducing and non-reducing SDS-PAGE analysis.
293T cells were cultivated and transfected with pQE-UL97(111-707)-SH (encoding a C-terminal StrepHis-tag) in 10-cm dishes, thus leading to overexpression of pUL97 in transfected cells. Two days post-transfection, the cells were lysed and subjected to a two-step purification which was performed first with StrepTactin and then with Ni-NTA (nitrilotriacetic acid) magnetic agarose beads (Qiagen) according to the manufacturer's instructions. Aliquots of the two-step eluates were subjected to reducing and non-reducing SDS-PAGE and Western blot analysis (mAb-Strep; Qiagen). Purified protein or total lysates from transfected cells were analysed by reducing and non-reducing SDS-PAGE (Gramberg et al., 2006). For this, cells were harvested 2 days post-transfection and incubated on ice either in 90 µl non-reducing lysis buffer (NRL) or radioimmunoprecipitation (RIPA) lysis buffer for 90 min or 10 min, respectively (NRL buffer: 10 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM PMSF, 0.5 % Triton X-100 in PBS; RIPA buffer: 10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 % sodium deoxycholate, 0.1 % SDS). The lysates were centrifuged at 14 000 r.p.m. in an Eppendorf 5417 R centrifuge with the standard rotor at 4 °C for 10 min. Samples were prepared in equal volumes of SDS buffer (62.5 mM Tris–HCl pH 6.8, 1 mM EDTA, 10 % glycerol, 2 % SDS, 0.005 % bromophenol blue) containing either 5 % or no 
-mercaptoethanol (-m.). The samples under reducing conditions (5 % -m.) were denatured for 10 min at 95 °C. All samples were then subjected to SDS-PAGE and Western blot analysis [anti-HA.11 (Babco); anti--actin (Sigma)].
Affinity gel protein purification and gel filtration chromatography.
An analytical Superdex 200 10/300 GL column (GE Healthcare) was equilibrated in buffer containing 150 mM NaCl, 50 mM Tris/HCl, pH 7.5, 10 % glycerol. The column was calibrated with protein standards (aldolase, catalase, ferritin and thyroglobulin; Amersham). Mutant pUL97(M1L)-F was expressed to high levels in 293T cells (approximately 7.5x107 cells) and purified by the FLAG purification procedure according to the standard protocol of the manufacturer (Sigma; mAb-FLAG M2 affinity gel). Approximately 25 µg of the purified protein was injected onto the column in a volume of 50 µl (corresponding to 5 % of the total eluate). Fractions were evaporated, denatured in SDS buffer and loaded onto SDS-PAGE for Western blot analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, lanes 2 and 7). In these assays, pUL44, a known interactor and a phosphorylation substrate of pUL97 (Marschall et al., 2003; Krosky et al., 2003b), was used as a positive control (Fig. 1a, lane 4). Another HCMV protein, pUL84, which does not interact with pUL97, served as a specificity control (Fig. 1a, lane 3). Generally, in Western blot stainings, pUL97 appears as a double band consisting of a dominant band of 100 kDa and a less abundant band of approximately 90 kDa, possibly arising from independent translation initiation events at two of five N-terminal in-frame ATG start codons (Michel et al., 1996, 1999). Both forms were detectable in coimmunoprecipitates, indicating self-interaction (Fig. 1a, lanes 2 and 7; Fig. 1c, lane 2). A point mutant in which the first translation start codon was exchanged (M1L) exclusively expressed the 90-kDa form of pUL97 lacking the extreme N terminus (Fig. 1d, lane 3). As shown in Fig. 1(c), lane 3, this mutant was also positive in coimmunoprecipitation. The most relevant panels shown in Figs 1–3 were reproduced by the use of mAb-FLAG instead of anti-HA as the coimmunoprecipitation antibody, providing identical results (data not shown).
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). Deletion of amino acids 366–707 at the C terminus or 1–180 at the N terminus did not negatively affect pUL97 self-interaction (Fig. 2a, lanes 4–7 and lanes 10–12, respectively). Further large deletions on the N-terminal or C-terminal side resulted in a complete loss of interaction (Fig. 2a, lanes 8–9). Interestingly, kinase activity of pUL97 was not required for self-interaction, as demonstrated by the use of the catalytically inactive point mutant K355M in both versions of pUL97 (FLAG- and HA-tagged) in the coimmunoprecipitation assay (Fig. 2a, lane 13). Importantly, a protein comprising the internal region 181–365 of pUL97 showed a positive reaction (Fig. 2a, lane 16). Thus, region 181–365 is sufficient to mediate pUL97 self-interaction.
Further deletion mutants were subsequently used for fine-mapping of the interaction region (Fig. 3). Deletion of amino acids on the N-terminal side up to position 230 [i.e. pUL97(231-707)-F] did not destroy self-interaction, as demonstrated by the coimmunoprecipitation with full-length pUL97-HA (Fig. 3a, lanes 4 and 18). However, loss of self-interaction was noted for N-terminal deletions up to position 280 or 336 (Fig. 3a, lanes 5–6). Since pUL97(231-707)-F was expressed to higher levels than the latter two mutants, the experiment was confirmed by transfecting parallel setups with series of increasing concentrations of the expression plasmids. The resulting coimmunoprecipitation data were identical to those described for Fig. 3(a) (data not shown). The dispensability of region 1–230 for self-interaction was confirmed by the demonstration that two fragments with different tags, both lacking region 1–230 [i.e. clones pUL97(231-707)-F and pUL97(231-707)-HA], were still clearly positive in interacting with each other (Fig. 3a, lane 8). On the C-terminal side, deletion was tolerated to amino acid 281 but not to 231 (Fig. 3a, lanes 1–2). These findings point to a region spanning amino acids 231–280 which correlates with self-interaction. Interestingly, partial deletion of this region, i.e. deletion of 231–255 or 256–280, did not lead to a loss of reactivity (Fig. 3a, lanes 12–13). However, complete deletion of region 231–280 eliminated positive signals in coimmunoprecipitation (Fig. 3a, lane 19), underlining the importance of this region for self-interaction.
Dimerization and oligomerization are suggested modes of pUL97 self-interaction
Total lysates of transfected 293T cells expressing pUL97-HA were analysed by reducing and non-reducing SDS-PAGE followed by Western blot detection (Fig. 4a). The non-reducing SDS-PAGE method was used to demonstrate the dimeric or oligomeric nature of proteins as described previously (Gramberg et al., 2006; Liu et al., 2004; El-Battari et al., 2003). Under reducing conditions, pUL97-HA showed the characteristic double band at 90–100 kDa (Fig. 4a, lane 2). Under non-reducing conditions, which are suggested to maintain intermolecular as well as intramolecular disulfide bonds, pUL97 stained diffusely at around 90–100 kDa and, additionally, at approximately 210 kDa (Fig. 4a, lane 4). This pattern is in accordance with the formation of a dimer. Alternatively, this band could result from the formation of a heterogeneous protein complex, containing pUL97 and additional cellular proteins. Therefore, purification of pUL97 [mutant pUL97(111-707)-SH] was performed by following a two-step procedure (Fig. 4b). This purified protein showed the expected PAGE migration of approximately 90 kDa under reducing conditions (Fig. 4b, lane 3; Fig. 4c, lanes 1–2). Under non-reducing conditions, however, purified pUL97(111-707)-SH appeared as a band at approximately 210 kDa, including diffuse faster-migrating signals (Fig. 4c, lanes 3–4). Thus, these data confirm that formation of a homodimer is highly likely. In a control staining on Western blots, we addressed the question whether a known cellular interactor of pUL97, p32, is contained within these protein complexes. Importantly, using an anti-p32 antiserum, no positive signals were obtained (data not shown), which is consistent with the fact that no additional protein bands could be detected after visualization of purified pUL97 by silver staining (Fig. 4b, lane 3). This indicates that the 210 kDa form of pUL97 detected under non-reducing conditions does not include detectable amounts of p32 and generally argues against the non-specific formation of disulfide bonds with cellular proteins under these conditions.
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, lanes 3, 6 and 9). In addition, a band of approximately 60 kDa was visible that reacted in Western blot analysis with the mAb-FLAG antibody, thus indicating that this protein represents an N-terminally truncated pUL97 (Fig. 5a, lanes 6 and 9). As assessed by Western blot analyses, the recently defined cellular interaction partner of pUL97, p32, could not be detected in the eluate containing purified pUL97 (Fig. 5a, lower panel, lane 9). This excludes the possibility that the FLAG affinity purification procedure performed with cells overexpressing pUL97 leads to a stoichiometric co-purification of cellular proteins. To analyse the self-interaction of the purified pUL97, gel filtration chromatography was performed (Fig. 5b). The eluted fractions were subjected to SDS-PAGE and Western blot analysis (mAb-FLAG) to detect the presence of high-molecular-mass complexes, as indicated by the protein standards used for calibration of the gel filtration column. The first UL97-specific signals were detected at an elution volume that corresponded to a molecular mass of approximately 177 kDa, which would be consistent with the formation of homodimers (Fig. 5b, 22–23). Importantly, no signals were observed below 177 kDa, excluding the existence of monomeric pUL97 (Fig. 5b, 24–28). Furthermore, higher molecular-mass-complexes up to more than 400 kDa (
438 kDa marker; Fig. 5b, 15–21) were also present. This finding argues for oligomerization of pUL97 in addition to dimerization. A costaining of the Western blots with anti-p32 antiserum was negative, indicating that the cellular interactor p32 was not a detectable component of these pUL97 complexes (data not shown). Thus, the PAGE migration and gel filtration characteristics of pUL97 suggest the formation of homodimers and homooligomers.
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). When the catalytically inactive mutant K355M of pUL97 was coincubated with the faster migrating, kinase-active mutant M1L of pUL97, phosphorylated forms were detectable not only for mutant M1L but also for K355M (Fig. 6a, lane 3), suggesting an autophosphorylation in trans.
The region correlating with pUL97 self-interaction (231–280) is distinct from the postulated pUL97 protein kinase domain which is determined by subdomains I–XI spanning the amino acid region 337–650 (Chee et al., 1989; Michel et al., 1999; Schregel et al., 2005). Therefore, mutagenesis of the self-interaction region should not directly affect catalytic activity of pUL97. However, self-interaction of pUL97 might be a prerequisite for kinase activity, i.e. region 231–280 could be of indirect importance. To address this question, a number of pUL97 mutants were assayed for substrate phosphorylation (histone 2B) and autophosphorylation activity in vitro (Fig. 7). Wild-type pUL97 (double band 90–100 kDa) and point mutant M1L (single band 90 kDa) showed both analysed activities, i.e. substrate phosphorylation and autophosphorylation (Fig. 7a, lanes 2–3 and 6). Used as a control, point mutant K355M (lacking an essential lysine within the ATP-binding site in subdomain II) was completely negative (Fig. 7a, lanes 4–5). The internal deletion mutant pUL97(1-255/281-707), which still conferred self-interaction (see Fig. 3a), was kinase-positive (Fig. 7a, lane 7). Also parallelling the findings of self-interaction, deletion of amino acids 1–180 or 1–230 (Fig. 7a, lanes 8–9) did not affect catalytic activity, while deletion of amino acids 1–280 or 1–336 (lanes 10–11) fully destroyed catalytic acitivity. The mutant shown in lane 12 carries a partial deletion of the ATP-binding site (subdomains I and II) and was used as another catalytically inactive control. Thus, the described deletions causing a loss of self-interaction resulted in catalytic inactivation. These results suggest that pUL97 self-interaction may be important for its catalytic activity. It should be mentioned that, in some cases, however, we also obtained mutants which were intact in both the postulated self-interaction region and the kinase domain, but were nevertheless kinase-inactive [e.g. deletion mutants in the C terminus including amino acids 596–707 (Marschall et al., 2005), and internal deletion mutant 231–255, data not shown]. This might be due to the disruption of additional structural requirements by mutagenesis which are essential for kinase activity. Finally, we analysed the mutant carrying the smallest deletion that was negative in coimmunoprecipitation [pUL97(1-230/281-707)-F] for autophosphorylation activity. Interestingly, this mutant, although not completely negative in activity, was strongly impaired in autophosphorylation and histone 2B phosphorylation (Fig. 7c, lane 3). This underlines the hypothesis that self-interaction may be important for kinase activity, although other interpretations are possible and deletion mapping is generally linked with the risk of unwarranted structural effects. Nevertheless, the sum of our data suggests a structure–activity correlation, i.e. although mutants lacking self-interaction may still contain some basal activity due to an intact kinase domain, self-interaction seems to be a prerequisite for optimal catalytic activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The interaction of pUL97 with viral and cellular proteins has been described previously. The domains of pUL97 responsible for interaction with viral pUL44 and cellular p32 were attributed to amino acids 366–459 (Marschall et al., 2003) and 181–365 (Marschall et al., 2005), respectively. This indicates that the pUL44 interaction region is distinct, while the p32 interaction region (181–365) overlaps with the self-interaction region (231–280). Therefore, p32 might in principle be involved in the self-interaction process of pUL97. This possibility cannot be ruled out when considering that the coimmunoprecipitation experiments in this study were performed with total cell lysates containing endogenous p32. However, specific pUL97 mutants lacking p32 interaction were still positive for self-interaction. In particular, mutant pUL97(231-707)-F, which is negative for p32 interaction in the yeast two-hybrid system (Marschall et al., 2005), clearly reacts positively for pUL97 self-interaction in coimmunoprecipitation experiments (Fig. 3a). A second point that argues against the requirement for p32 (or other cellular proteins) for pUL97 self-interaction is the formation of pUL97 dimers/oligomers after protein purification by two different affinity purification procedures (see Figs 4 and 5). In these samples, pUL97 was purified to near homogeneity as indicated by Coomassie blue and silver staining of polyacrylamide gels. The lack of additional bands excludes the stoichiometric co-purification of cellular proteins (Figs 4b and 5a). Furthermore, since p32, which binds with high affinity to pUL97, could not be detected in purified pUL97 preparations by Western blot analysis (Fig. 5a), this argues strongly against the presence of contaminating cellular proteins after affinity purification of pUL97 from transfected 293T cells that overexpressed the viral protein. Thus, we conclude that pUL97 self-interaction occurs independently of cellular proteins.
The question was addressed whether pUL97 homologues of related other herpesviruses show a similar propensity for self-interaction. Our current data provide evidence for self-interaction of the rat cytomegalovirus (RCMV) protein kinase, pR97–pR97 (M. Marschall, unpublished data). Moreover, a heterologous mode of interaction was investigated, e.g. the interaction between HA-tagged pUL97 and FLAG-tagged homologues of other herpesviruses. Coimmunoprecipitation was positive for pUL97–pR97 but negative for pUL97–BGLF4 (Epstein–Barr virus) as well as pUL97–UL13 (herpes simplex virus type 1) (Romaker et al., 2004). This finding illustrates the close structural relationship between the two cytomegalovirus-encoded protein kinases pUL97 and pR97.
The presented data point to homodimerization or homooligomerization as the mechanism of self-interaction of pUL97. The findings that argue for dimerization are the detection of a 210 kDa form under non-reducing conditions, the enrichment of this form by steps of protein purification and the lack of detection of other proteins in these samples. Non-reducing conditions were chosen to maintain intermolecular as well as intramolecular disulfide bonds. Two cysteine residues are positioned within the mapped self-interaction domain of amino acids 231–280, i.e. C272 and C274. Intermolecular disulfide bridges might be formed by these residues, but their maintenance in a nuclear protein would be surprising. It seems more probable that intramolecular disulfide bridges may be important for pUL97 self-interaction, e.g. by the stabilization of a structural conformation such as a binding pocket responsible for self-interaction. These structural requirements might be mediated by cysteine residues lying within or even outside the self-interaction region. Concerning the latter aspect, we noticed that the introduction of an additional cysteine residue at position 296 neighbouring the self-interaction domain partially interfered with self-interaction (randomly selected mutant R296C). This was documented by the finding that mutant R296C, although showing a positive reaction in the coimmunoprecipitation assay (interaction of mutant R296C with wild-type pUL97), did not show self-interaction in SDS-PAGE analysis under non-reducing conditions (interaction of mutant R296C with itself; V. Schregel and M. Marschall, unpublished data). Combined, the data suggest the importance of correctly formed intramolecular disulfide bridges for pUL97 self-interaction.
An alternative mechanism of self-interaction is the formation of pUL97 oligomers. An example for a protein kinase forming hexamers (i.e. the dimer of a trimer) was reported for bacterial HPr kinase (Allen et al., 2003). Dimerization, on the other hand, was described for several eukaryotic protein kinases such as PKR. In the case of PKR, dimerization is directly linked with autophosphorylation and autoactivation (Williams, 1999). The data described by Fig. 6 suggest a similar situation for pUL97 and, even if not absolutely essential, self-interaction seems to be important for an increased level of catalytic activity.
Recent results from our laboratory contributed to define structural properties and essential motifs by performing structure predictions of the pUL97 kinase domain, i.e. sequence alignment analyses and computer-based structural modelling (Romaker et al., 2006). Hereby, the predicted kinase domain, composed of subdomains I–V (putative ATP-binding site) and VI–XI (putative catalytic region), could be modelled and depicted in a three-dimensional design. Unfortunately, modelling of a putative dimerization domain was not successful because of the lack of conserved motifs. However, a clear physical distinction could be made between the modelled kinase domain (337–650) and the biochemically defined self-interaction region (231–280). The two domains seem to be spatially distinct and non-overlapping. This suggests that, in addition to pUL97-specific protein kinase inhibitors, which have been intensively investigated (Marschall et al., 2002; Wang et al., 2003; Herget et al., 2004), a second type of kinase-directed inhibitors, i.e. self-interaction inhibitors, might be very helpful for developing novel antiviral strategies. Future studies, in particular the determination of the molecular structure of pUL97, should provide further details for the understanding of its structure–activity relation and specific modes of antiviral targeting.
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ACKNOWLEDGEMENTS |
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Received 19 July 2006;
accepted 2 October 2006.
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