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1 Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, 91054 Erlangen, Germany
2 Division of Bioinformatics, Institute of Biochemistry, University of Erlangen-Nuremberg, 91054 Erlangen, Germany
Correspondence
Manfred Marschall
manfred.marschall{at}viro.med.uni-erlangen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A supplementary figure and a supplementary table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). As is characteristic for most DNA viruses, HCMV replicates its genome in the nucleus of the host cell. Thereafter, preformed viral capsids are packaged with genomic DNA and have to be transported to the cytoplasm for final maturation and viral release. Due to the large size of cytomegaloviral capsids (
130 nm; Chen et al., 1999), these cannot be transported through the nuclear pore complex (40 nm; Panté & Kann, 2002). The most widely accepted model for nuclear egress of HCMV and other herpesviruses is based on a transient primary envelopment by budding through the inner nuclear membrane (Mettenleiter, 2004, 2006; Sanchez & Spector, 2002; Sampaio et al., 2005). However, before herpesvirus-encoded capsids gain access to the inner nuclear membrane, the rigid proteinaceous network of the nuclear lamina provides a major obstacle. Thus, the locally restricted destabilization of the nuclear lamina is required during the viral replication process (Buser et al., 2007).
A basic principle of the reorganization of the nuclear lamina is the recruitment of cellular and/or virus-encoded protein kinases to increase the site-specific phosphorylation of nuclear lamins and lamin-binding proteins (Dechat et al., 2008). Cellular protein kinases, i.e. protein kinase C (PKC) and cdc2 (Cdk1), are known to phosphorylate the nuclear lamins during prometaphase of the cell cycle, leading to destabilization of the nuclear lamina (Peter et al. 1990; Collas et al., 1997). Recruitment of PKC to the nuclear membrane occurs during infection with HCMV, murine cytomegalovirus (MCMV) and herpes simplex virus type-1 (HSV-1) (Muranyi et al., 2002; Park & Baines, 2006; Milbradt et al., 2007). A conserved group of herpesvirus-encoded lamina-associated proteins have been implicated in the recruitment of PKC, the rearrangement of nuclear lamina components and viral nuclear egress (Reynolds et al., 2004; Muranyi et al., 2002; Leach et al., 2007; Morris et al., 2007; Fuchs et al., 2002; Farina et al., 2005; Milbradt et al., 2007; Camozzi et al., 2008). This group comprises pUL50 and pUL53 in HCMV and their homologues in other herpesviruses. Recently, we have demonstrated that pUL50 and pUL53 interact directly with and localize to the nuclear rim. Importantly, pUL50 is able to recruit PKC to this site by direct protein–protein interaction (Milbradt et al., 2007). A recent study by Camozzi et al. (2008) supported these findings and further characterized the interaction between pUL50 and pUL53. The authors also observed rearrangement of the nuclear lamina during HCMV-infection and proposed that pUL50 and pUL53 have a role in this process (Camozzi et al., 2008).
The HCMV protein kinase pUL97 is also recruited to the nuclear lamina by a pUL50/pUL53-independent mechanism (Marschall et al., 2005). A direct interaction between the cellular multi-functional protein p32 and pUL97 recruits pUL97 to the lamin B receptor. Moreover, overexpression of p32 in HCMV-infected cells leads to an increased efficiency of viral replication and release of viral particles. The cellular protein kinase, PKC, and the viral protein kinase, pUL97, could potentially act in combination at the nuclear lamina to mediate the reorganization of the nuclear lamina in HCMV-infected cells.
In this study, we focussed on defining components involved in HCMV nuclear egress and characterizing their interaction. Deletion studies and yeast two-hybrid analyses were used to define interaction domains. Based on the combined results, a model for the postulated HCMV-specific nuclear egress complex (NEC) was postulated.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) using the discrimination of secondary structure class (DSC; King & Sternberg, 1996), multivariate linear regression combination (MLRC; Guermeur et al., 1999) and profile network from Heidelberg (PHD; Rost & Sander, 1993) methods. Transmembrane regions were identified using the transmembrane hidden Markov model (TMHMM; Käll et al., 2004) and protein disorder was predicted by GlobPlot (Linding et al., 2003) using the Deleage/Roux scale.
Plasmid constructs.
Expression constructs were generated by PCR amplification of the pUL50 or PKC
open reading frame (ORF). N-terminal (i.e. encoded amino acids 20–397, 40–397, 70–397, 100–397 or 150–397) and C-terminal (i.e. encoded amino acids 1–340, 1–310, 1–280 or 1–250) deletion mutants of pUL50 were generated by cloning of PCR products. Standard PCR amplification, using template pcDNA-UL50-HA or peGFP-N1-GFP (Milbradt et al., 2007), with primers carrying tag sequences, resulted in a fusion of the ORFs to a C-terminal haemagglutinin (HA) or Flag tag. After cleavage with EcoRI/XhoI, PCR products were inserted into the vector pcDNA3.1 (Invitrogen). The expression constructs, pcDNA-UL50-HA, pcDNA-UL50(1-358)-HA, pcDNA-UL53-HA, pcDNA-UL97-HA, pcDNA-UL97-F, pcDNA-UL97(231-707)-HA, pcDNA5/FRT-p32-F, pcDNA5/FRT-p32(50-282)-F, have been described previously (Marschall et al., 2001, 2003, 2005; Schregel et al., 2007; Milbradt et al., 2007) and all yeast two-hybrid constructs are shown in Fig. 3.
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Cell culture and transfections.
293T and HeLa cells were cultivated in Dulbecco's modified Eagle's medium containing 10 % fetal calf serum and 100 µg gentamicin ml–1. Transient transfections in 293T and HeLa cells were performed by the use of Lipofectamine or polyethylenimine–DNA complexes. Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's protocol. HeLa cells were transfected at a confluency of 70–90 % using a seeding cell number of 3.5x105 for six-well plates. Transfection of 293T cells with polyethylenimine reagent (Sigma) was performed at a cell confluency of approximately 80 %, using a seeding cell number of 5x106 for 10 cm dishes as described previously (Schregel et al., 2007).
Indirect immmunofluorescence double-staining.
HeLa cells were grown on coverslips for transfection. Two days post-transfection, cells were fixed with 4 % paraformaldehyde (10 min, room temperature) and permeabilized using PBS/0.2 % Triton X-100 (20 min, 4 °C). Indirect immunofluorescence staining was performed as described previously (Milbradt et al., 2007). Data for immunofluorescence were collected using an Axiovert-135 microscope (Zeiss) at magnifications of x400 and x630. Confocal laser-scanning microscopy was performed with a TCS SP5 microscope (Leica).
Coimmunoprecipitation (CoIP) assay.
293T cells were transfected in 10 cm dishes. Two days post-transfection, cells were lysed in 500 µl CoIP buffer under previously described conditions (Milbradt et al., 2007), using 1–2 µl polyclonal immune serum antibody (pAb) (rabbit; pAb-HA.11, HISS Diagnostics; pAb-Flag, Sigma; preimmune rabbit antiserum), 4 µl monoclonal antibody (mAb) (mouse; mAb-Myc 1-9E10.2, ATCC) or 3 µl mAb mix (mouse; mAb-HA plus mAb-Flag; Roche Diagnostics, Sigma, respectively). CoIP samples were subjected to standard Western blot analysis using mAb-HA (Roche Diagnostics), pAb-HA (HISS Diagnostics), mAb-Flag (M2, Sigma), mAb-green fluorescent protein (GFP) (clones 7.1/13.1, Roche), pAb-UL53 (received from P. Dal Monte, University of Bologna) or mAb-lamin B receptor (LBR) (rabbit monoclonal; clone E398L, Epitomics).
Yeast two-hybrid analysis.
Protein interactions were analysed using GAL4 fusion proteins in a yeast two-hybrid system (Fields & Song, 1989). Saccharomyces cerevisiae strain Y153 expressing several viral- or cellular-derived proteins (fused with GAL4-BD as bait) was used for interaction analysis with selected expression clones (putative interactors fused with GAL4-AD or vice versa) (Durfee et al., 1993). Selection for the presence of bait and interactor plasmids was achieved by cultivation on medium that restricted growth to combined tryptophan/leucine prototrophy. Transformants were analysed for β-galactosidase (β-gal) activity by filter lift tests.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), consistently predicted that the N terminus is rich in regular secondary structure and adopts a globular fold, comprising at least residues 45–180 (Fig. 1). This part of pUL50 corresponds closely to the region found to be highly conserved amongst the UL50 proteins of human, murine and other cytomegaloviruses (Rupp et al., 2007). Computational analyses predict that residues 1–45 and 180–358 of pUL50 would not be expected to adopt a defined 3-D structure and are either entirely disordered or form only short local elements of secondary structure (Fig. 1 and Supplementary Fig. S1, available in JGV Online). Such regions are less constrained and are potentially free to evolve quickly to establish short linear sequence motifs mediating specific protein–protein interactions. These findings suggest that the highly conserved globular core of pUL50 represents the major interaction site for the binding partners, while the less-conserved flanking regions mainly play a role in increasing binding affinity and specificity for distinct interaction partners.
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Regions of pUL50 important for interaction with three viral and cellular proteins
In order to determine whether the individual interactions between pUL50 and each of its three identified binding partners (pUL53, p32 and PKC) influence each other, a mapping analysis of the interaction domains was performed by constructing a series of deletion mutants of pUL50 for coexpression and CoIP experiments (Fig. 2). The specificity of the CoIP was supported by results with rabbit preimmune serum in parallel controls (Fig. 2a–f, lanes 9–14) and the expression of all recombinant proteins was monitored by the use of expression control samples taken prior to the addition of the CoIP antibody (Fig. 2a–f, central and lower panels). None of the N-terminally deleted constructs was capable of coimmunoprecipitating pUL53 (Fig. 2b, lanes 4–8), whereas C-terminal deletions up to aa 250 supported this interaction (Fig. 2a, lanes 4–8). Thus, the region required for interaction with pUL53 was suggested to be aa 1–250 of pUL50. Additionally, since only full-length pUL50 is able to coimmunoprecipitate pUL53 and an N-terminal deletion of aa 1–20 leads to a loss of interaction, the data indicate that there is an essential stretch of amino acids in this region. For the interaction of pUL50 with PKC and p32, the essential region was narrowed down to aa 100–280 (Fig. 2c–d) and 100–358 (Fig. 2e–f), respectively. Interestingly, the interaction of p32 with pUL50 constructs lacking the N-terminal 20–40 amino acids was much weaker (Fig. 2f, lanes 4 and 5). This may indicate an additional important region for p32 binding located between aa 20 and 70 or, alternatively, a defect in protein folding of these constructs. A summary of CoIP data are presented schematically (Fig. 2g), illustrating that the pUL50 interacting regions for pUL53, PKC and p32 are partly overlapping and partly distinct.
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). The N-terminal 20 residues of pUL50 were important for binding pUL53 but not for binding p32 and PKC. This region is predicted to form an -helix (Fig. 1
), which is probably not part of the globular domain itself but rather forms an independent structural element required for a subset of the interactions. The region from aa 250 to 280, which was found to be required for the interaction with PKC, is located outside the globular part of pUL50. This region is predicted to be at least partially disordered and is particularly rich in cysteines from 255 to 266 (Fig. 1). These cysteines represent candidates for ion-mediated interactions with the cysteines of PKC. This type of interaction has been observed previously between the tyrosine kinase Lck and the co-receptors CD4 and CD8 (Kim et al., 2003). Residues 340–358 were required for the interaction with p32, but not with pUL53 or PKC (Fig. 2). This sequence is predicted to be disordered and to lack any regular secondary structure. Closer inspection showed that this stretch is particularly rich in tryptophan and proline residues, both of which are generally found to be significantly enriched in linear sequence motifs mediating protein–protein interactions (Neduva & Russell, 2006). Thus, it is likely that this region of pUL50 comprises a novel sequence motif mediating the interaction with p32. In addition to the tryptophans and prolines, the presence of several arginines might be important in mediating charge complementarity with the highly acidic p32 protein (Jiang et al., 1999). Although further studies are required to clarify the role of individual residues within the 1–20, 250–280 and 340–358 sequence regions, the present data suggest that these regions do not adopt a globular 3-D structure, but contain short sequence motifs important for the binding of pUL50 to its interaction partners.
Demonstration of an interaction network by yeast two-hybrid and CoIP analyses: putative components of the NEC
A comparative analysis of the interaction of pUL50 with protein components of the postulated NEC was performed by using the yeast two-hybrid system (Fig. 3). Interestingly, pUL50 does not interact directly with the N-terminal cytoplasmic interaction platform of the LBR (aa 1–208; Gruenbaum et al., 2005). pUL53 and pUL97 were also negative for LBR interaction, which has been demonstrated previously (Mylonis et al., 2004; Marschall et al., 2005; Milbradt et al., 2007). The only protein that showed a positive reaction with LBR in this system was p32 (Fig. 3a). The vector controls of all analysed expression constructs illustrated the specificity of the reaction (Fig. 3b). In the next step, the interaction between p32 and LBR was compared with a panel of other potential interaction pairs. As shown previously, p32 is able to interact strongly with the cytomegaloviral protein kinase pUL97 (Marschall et al., 2005) and with itself (Jiang et al., 1999; Jha et al., 2002; Marschall et al., 2005). A point mutant of p32 (L243H) showed a loss of interaction with pUL97 and with the LBR but not with p32 (Fig. 3c), indicating that leucine 243 appears to be crucial for binding pUL97 and LBR. Alternatively, the point mutation might induce conformational changes in the p32 structure that impact on some, but not all, interaction properties of p32. Thus, the panel of mutants illustrates the specificity of p32 interaction with binding partners of the NEC. A series of other cytomegaloviral proteins (most probably not contained within the putative NEC) were analysed for interaction with p32 in the yeast two-hybrid system. No additional interaction was identified by this assay (Fig. 3d). Overall, the data is consistent with p32 recruiting pUL50 and pUL97 to a functional complex.
To investigate NEC formation further, 293T cells were cotransfected with plasmids encoding a Flag-tagged construct for p32, together with one of a series of HA-tagged viral proteins for analysis by CoIP assays. Positive CoIP signals for p32 interaction were obtained with pUL50 (a full-length version and a C-terminally truncated version lacking the transmembrane domain), pUL97 and pUL53, but not for N-terminally truncated pUL97 (lacking the p32 interaction region; Marschall et al., 2005) and for the HSV-1 protein kinase UL13 (Fig. 4a, lanes 3–8). The interaction between p32 and pUL53 was unexpected due to the fact that it was not detected by the yeast two-hybrid analysis (Fig. 3d). Therefore, a reverse CoIP assay was performed, in which Flag-tagged p32 was immunoprecipitated (Fig. 4b). Again, a clear coimmunoprecipitation of pUL53 with p32 was detected (Fig. 4b, upper panel, lane 3). The discrepancy between the results from these two methods may arise from some degree of irregular folding of one or both of the proteins produced in yeast cells. On the other hand, the yeast two-hybrid technique is able to discriminate between direct and indirect interaction between two proteins in most cases. Therefore, the difference may also be because a cellular adaptor protein is required for the interaction between pUL53 and p32. This currently unidentified adaptor (Fig. 7) may be present in human but not yeast cells.
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(Fig. 4c, upper panel, lanes 1–8). The specificity controls with mAb-Myc (Fig. 4c, upper panel, lanes 9–14) and the expression controls (Fig. 4c, central and lower panels) confirmed the reliability of this experiment.
Together, CoIP and yeast two-hybrid studies indicate a tight interaction network of cellular and viral proteins that are predicted to be involved in cytomegaloviral nuclear capsid egress. Previous studies have shown that pUL50 and pUL53 interact with each other and form a complex at the nuclear rim, with pUL50 appearing to direct the complex to this location (Milbradt et al., 2007; Camozzi et al., 2008). Similar results were obtained for their homologues in other herpesviruses (Reynolds et al., 2001; Fuchs et al., 2002; Farina et al., 2005). Furthermore, the interaction network comprises multiple interactions between pUL50, pUL53 and p32, as well as a viral and a cellular protein kinase (i.e. pUL97 and PKC). Importantly, this complex is not only connected to the nuclear membrane by pUL50 but also directly to the nuclear lamina by the interaction of p32 with the LBR.
Direct and indirect interactions between NEC proteins
These data provide evidence for several points of direct interaction between NEC proteins, such as the interaction between pUL50 and pUL53 and between pUL50 and PKC. Thus, those proteins of the postulated NEC that do not interact directly (e.g. pUL53 and PKC) are very likely to be linked in a multi-protein complex through indirect interactions. To address this, we performed a triple-transfection with the constructs expressing pUL50, pUL53 and PKC each carrying an individual tag for CoIP analysis (Fig. 5a). We clearly showed that PKC was indirectly coimmunoprecipitated by a pUL53-directed antibody (mAb-Flag; Fig. 5a, lane 8), but not a non-specific antibody (mAb-Myc; Fig. 5a, lane 9). Of note, all immunoprecipitates obtained from single- or double-transfected controls, particularly pUL53 and PKC in the absence of pUL50 (Fig. 5a, lane 6), were negative for PKC. Moreover, the partial overlap of binding sites on pUL50 apparently does not generally prevent simultaneous direct interactions of pUL50. At least in this CoIP experiment, the interactions of pUL50 with pUL53 and PKC were not mutually exclusive.
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by CoIP with proteins from HCMV-infected fibroblasts (Fig. 5b). Lysates were prepared at a late time point of infection (3 days) and were subjected to CoIP with mAb-PKC (or mAb-Calreticulin as a control). pUL53 was specifically detectable, following inoculation with an m.o.i. of 1.0, in the immunoprecipitate obtained with mAb-PKC (but not with mAb-Calreticulin; Fig. 5b, lanes 3 and 6, respectively). Due to the fact that a direct interaction between pUL53 and PKC was not identified in various assay systems, this finding is consistent with the model of a multiply linked protein complex which includes pUL50, pUL53 and PKC as major components.
Imaging of protein complex formation at the nuclear lamina
A series of single transfections and cotransfections was performed with HeLa cells to visualize the intranuclear localization of putative NEC components. There was a rim-like accumulation of these proteins in the area of the nuclear lamina. Endogenous LBR was stained which illustrated its colocalization with pUL50 (Fig. 6a, i–v). Interestingly, coexpressed p32 (N-terminally truncated 50–282, similar to its processed endogenous form) also showed a partial rim-like accumulation (Fig. 6a, vi–x). Similarly, a partial relocalization was detected for pUL97 (Fig. 6a, xi–xv) and PKC (Fig. 6a, xvi–xx) when coexpressed with pUL50. In contrast with full-length pUL50, no relocalization of PKC was obtained when the truncated version of pUL50 that lacks the transmembrane domain was used (Fig. 6a, xxi–xxv). Since the truncated version of pUL50 still interacts with PKC in CoIP assays (Fig. 2c, lane 4), it appears that the transmembrane domain and consequently the nuclear rim localization of pUL50 is necessary to enable it to relocate PKC.
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). Notably, this colocalization between pUL97 and endogenous p32 is pronounced in those cells that carry a large amount of nuclear p32, dependent on the phosphorylation-mediated nuclear translocation of otherwise mitochondrial p32 (Brokstad et al., 2001). In the present study, confocal microscopy was used to assess the tendency of pUL97 to accumulate at the nuclear rim (Fig. 6b, i, ii). The expression of pUL97 alone was sufficient to induce a reorganization of the nuclear lamina [Fig. 6b, iii, iv; note the difference between the pUL97-F-positive (large arrowheads) and -negative (small arrowheads) cells]. The lamina-reorganizing activity of pUL97 had been demonstrated previously by conventional fluorescence microscopy (Marschall et al., 2005). Notably, the coexpression of pUL97 with pUL50 intensified the nuclear rim localization of pUL97 (Fig. 6b, vi, vii) and revealed a speckled accumulation of both proteins in some locations (Fig. 6b, viii, xii, small arrows; compare with Fig. 6a, xiv). Importantly, a very strong, locally restricted reorganization of lamin A/C was found in pUL50/pUL97-cotransfected cells (Fig. 6b, xi, large arrows), which was reminiscent of the lamina-free sites of the restructured nuclear envelope described for HCMV-infected fibroblasts (Buser et al., 2007). In summary, the immunofluorescence experiments are consistent with the CoIP and yeast two-hybrid analyses, suggesting a complex formation among these viral and cellular proteins. In this context, p32 appears to be an important factor for establishing the NEC due to its various protein–protein interactions with pUL50, pUL53, pUL97, PKC and LBR (Milbradt et al., 2007; Marschall et al., 2005; Mylonis et al., 2004; Storz et al., 2000; Robles-Flores et al., 2002). Additionally, the recruitment of cellular and viral protein kinases to the nuclear rim is essential for the reorganization of the nuclear lamina (Muranyi et al., 2002; Park & Baines, 2006). In contrast with the observed pUL50-mediated relocalization of PKC, even in the absence of pUL53, an association of the HSV-1 homologues of pUL50 and pUL53 appears to be required for a similar nuclear rim recruitment of PKC in HSV-1-infected cells (Park & Baines, 2006). Our data suggest that pUL50 is sufficient to induce recruitment of PKC to the nuclear lamina, at least in transfected cells. In the case of pUL97, pUL50 appears to reinforce the recruitment of pUL97, which can alternatively be mediated by p32 in a pUL50-independent fashion.
Conclusion: model of a postulated viral–cellular multi-protein complex
The data derived from this study and from the cited studies has led to the development of a model of protein interactions at the nuclear envelope of HCMV-infected cells (Fig. 7). Interestingly, interactions between the putative NEC components are multifold and, for some proteins, involve several interaction regions. This suggests that there is a tight network of interacting proteins. Although some of the regions identified as being responsible for protein interactions are overlapping while others are distinct, it is not clear at this stage whether individual interactions influence each other or whether they are mutually exclusive. It is possible that the interactions are ordered in a timely fashion during the course of viral replication and egress. Thus, it will be interesting to see whether a native NEC can be isolated from HCMV-infected fibroblasts. Another main finding is the recruitment of at least two protein kinases, i.e. PKC and pUL97. There is accumulating evidence that the involvement of both cellular and viral protein kinases (some of which possess direct lamin-phosphorylating activity) is important for the regulation of nuclear egress of other herpesviruses as well (Klupp et al., 2001; Reynolds et al., 2002; Marschall et al., 2005; Bjerke & Roller, 2006; Park & Baines, 2006; Kato et al., 2006; Leach et al., 2007). It will be crucial to determine the exact phosphorylation target sites on lamins and lamina-associated proteins of these protein kinases for a detailed understanding of the cytomegaloviral nuclear egress.
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ACKNOWLEDGEMENTS |
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, T. Arimura (University of Tokyo, Japan) for clone pADT7-PKC
, Thomas Stamminger and his research group for helpful methodical collaboration and Sabine Rechter for scientific discussions. This work was supported by the Deutsche Forschungsgemeinschaft (grant MA 1289/4-1). |
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Received 1 July 2008;
accepted 7 November 2008.
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]’. Two protein kinases, pUL97 and PKC, are recruited by the NEC to the nuclear lamina. Important substrate protein phosphorylations resulting from the activity of these kinases, particularly the phosphorylation of lamins, are indicated (P). Further as-yet unidentified proteins/protein kinases may additionally be involved ‘X’, e.g. a putative cellular adaptor between pUL53 and p32. This model was constructed using data from the present study and previously published studies (Muranyi et al., 2002
