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Short Communication |
Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
Correspondence
Thomas Stamminger
thomas.stamminger{at}viro.med.uni-erlangen.de
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ABSTRACT |
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MAIN TEXT |
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; Sandri-Goldin, 2004; Boyne & Whitehouse, 2006; Lischka & Stamminger, 2006). Furthermore, all these proteins are able to interact directly with RNA (Mears & Rice, 1996; Sandri-Goldin, 1998; Hiriart et al., 2003; Toth et al., 2006). The UL69 protein and its counterparts share a conserved homology region that is localized at the C terminus of the 
- and 
-herpesvirus factors, whereas, due to a unique C-terminal protein domain, this region can be found in the central part of pUL69 (Fig. 1a). Interestingly, most of the protein domains with importance for mRNA export stimulation (e.g. interaction site for cellular mRNA export factors, nuclear export signal) have been mapped to regions of the herpesviral mRNA export factors that show either no or low sequence conservation (Fig. 1a). In contrast, no common functional domains shared by all protein family members have yet been assigned to the homology region. Nevertheless, several functions can be linked to this region that seem to be specific for either of the herpesviral mRNA export factors. For instance, the C-terminal half of ICP27 contains three putative KH-like domains that are postulated to contribute to the RNA-binding specificity of ICP27 (Soliman & Silverstein, 2000); furthermore, the C terminus is required for binding to splicing factors and to the cellular mRNA export receptor TAP (Sciabica et al., 2003; Chen et al., 2005). The homology region within pUL69 has been identified as a binding domain for the transcription elongation factor hSPT6 (Winkler et al., 2000); in ORF57 of KSHV the respective region has been shown to be required for binding to CK2 kinase (Malik & Clements, 2004). Thus, as a common theme, the conserved homology region appears to be involved in protein–protein interactions.
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; Wadd et al., 1999; Malik & Clements, 2004). Due to these findings we examined the possibility of pUL69 self-association. To be able to uncover a putative interaction by co-immunoprecipitation experiments, we first constructed myc-tagged eukaryotic expression vectors encoding either pUL69 (m-UL69) or pUL84 (m-UL84) (Lischka et al., 2003). These constructs were cotransfected into HEK293T cells together with the reciprocal plasmids expressing pUL69 and pUL84 as in-frame fusions with the FLAG epitope (F-UL69 and F-UL84; Fig. 1b
, input) (Lischka et al., 2003, 2006). Subsequently, co-immunoprecipitation experiments were performed using either a monoclonal antibody directed against the FLAG (M2) or the myc (9E10) epitope (IP-FLAG, IP-myc). Co-immunoprecipitated proteins were analysed by Western blotting. As shown in Fig. 1(b), lower panel, lanes 1 and 5, m-UL69 was efficiently co-immunoprecipitated with F-UL69 and vice versa. This interaction was specific because (i) pUL69 did not co-precipitate with the control protein pUL84 (lanes 2 and 6) and (ii) no signal was present when a non-specific control antibody was used for precipitation (lanes 4 and 8). Since pUL69 is an RNA-binding protein, we also carried out identical experiments after digestion with RNase A. As shown in Fig. 1(b), lanes 3 and 7, treatment with 100 µg RNase A ml–1 did not affect pUL69 co-precipitation, indicating that pUL69 self-association did not occur through RNA-bridging. To address the question whether pUL69 forms dimers or multimers, the effect of glutaraldehyde (GA) as an inducer of covalent cross-links between interacting proteins was assessed. For this, we first purified pUL69 as a FLAG-fusion protein from transfected HEK293T cells using an anti-FLAG affinity gel (Toth et al., 2006). As shown by Coomassie blue staining and Western blotting, three characteristic pUL69 isoforms were purified that correspond to differentially phosphorylated protein isoforms (Fig. 1c) (Winkler & Stamminger, 1996). GA efficiently forms covalent bonds between lysine residues and thus stabilizes multimeric forms of proteins that can be subsequently visualized by SDS-PAGE (Mische et al., 2005). As shown in Fig. 1(d), no higher-order form of pUL69 was evident in the absence of cross-linker (0 min), whereas exposure of purified protein to 0.1 % GA resulted in the progressive formation of SDS-resistant multimers in a time-dependent manner (2–20 min). Interestingly, apart from very high molecular mass entities, no band consistent with a pUL69 dimer (
220 kDa) could be detected. This result supports the idea that pUL69 is engaged more in the formation of multimers than dimers. To prove this, we subjected pUL69 to gel filtration analyses (Fig. 1e). For this, pUL69, purified from transfected mammalian cells, was fractionated on a Pharmacia Superdex 200 column in 150 mM NaCl, 50 mM Tris/HCl (pH 7.5), 10 % glycerol. Fractions (0.5 ml) were collected and analysed by SDS-PAGE and Western blotting using a polyclonal pUL69-specific rabbit serum. Protein molecular mass standards (HMW/LMW gel filtration calibration kits; Amersham Biosciences) were used to calibrate the column. Examination of pUL69 in the fractions showed that the protein elutes in a heterogeneous high molecular mass population ranging from 438 kDa up to >2 MDa. Importantly, consistent with the cross-linking experiments, no pUL69 monomers (110 kDa) or dimers (220 kDa) could be detected. RNase treatment of the purified protein prior to gel filtration did not induce a significant change of the elution profile (Fig. 1e, lower panel). Taken together, these experiments strongly suggest that (i) pUL69 has the capacity to self-interact, (ii) pUL69 self-interaction is not bridged by RNA and (iii) pUL69 forms multimeric complexes in mammalian cells.
To further evaluate the self-association of pUL69 in vivo, we next performed interaction studies using the yeast two-hybrid system exactly as described by Winkler et al. (2000). Saccharomyces cerevisiae strain Y153 was co-transformed with expression plasmids encoding pUL69 fused either to the GAL4 activation domain (AD-UL69) or to the GAL4 DNA-binding domain (BD-UL69). Subsequently, the activation of the 
-galactosidase reporter gene was monitored by filter lift experiments. As shown in Fig. 2(a), an in vivo interaction of pUL69 with itself could be observed (Fig. 2a, lane 5). This interaction was considered specific since control transformations of AD-UL69 or BD-UL69 with the appropriate empty GAL4-plasmids excluded self-activation of the reporter gene by pUL69 (Fig. 2a, lanes 2 and 3). Next, we sought to identify the interaction motif within pUL69. To uncover this domain, a series of N- or C-terminal pUL69 deletion mutants fused to the GAL4 DNA-binding domain was constructed. These mutants were analysed in the yeast two-hybrid assay for their interaction with AD-UL69. Fig. 2(b) and (c) summarize the results of these experiments. They show that a central domain within pUL69, spanning residues 269–574, is both required and sufficient for efficient self-interaction. Interestingly, this region exactly corresponds to a domain within pUL69 that mediates binding to the cellular transcription elongation factor hSPT6 (Winkler et al., 2000). In addition, an internal deletion of a cysteine-rich region between amino acids 478 and 527, a region that is conserved within the homologous proteins of other herpesviruses, or a mutation of cysteine-495, which has previously been shown to abrogate the interaction with hSPT6, also abrogated pUL69 self-interaction (Winkler et al., 2000). These findings suggest that the integrity of the identified pUL69 domain is either essential for proper folding and protein stability and/or that pUL69 self-interaction is a prerequisite for hSPT6 binding.
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). The respective proteins were still able to self-interact as analysed by co-immunoprecipitation experiments (see Fig. 3b). Next, we determined the subcellular localization of each protein in transfected HeLa cells by indirect immunofluorescence analyses. As depicted in Fig. 3(c), panels 1–3, GFP-UL69 was detected exclusively in the nucleus, whereas UL69mNLS2, due to the replacement of four NLS-determining basic residues by alanine, displayed a partial cytoplasmic localization (Fig. 3c, panels d–f). However, as described above (Fig. 2), the N-terminal part of pUL69, including the NLS, is not required for pUL69 self-interaction. Taking this into consideration, we reasoned that co-expression of UL69mNLS2 and the strictly nuclear-localized GFP-UL69 would increase the nuclear localization of the mutant protein by heterodimer formation. To verify this, HeLa cells were cotransfected with GFP-UL69 and UL69mNLS-2. Subsequently, the localization of co-expressed proteins was assessed by indirect immunofluorescence. Due to the presence of two different tags (GFP and FLAG, see Fig. 3a), the wild-type protein could be easily distinguished from the mutant protein within the same cell. For GFP-UL69, nuclear staining was detected, as expected (Fig. 3c, panel h). However, when the subcellular localization of UL69mNLS-2 was assessed, the majority of cells were found to have a predominant nuclear fluorescence with no or only a slight cytoplasmic staining (Fig. 3c, panel i). Since this staining pattern was never observed with UL69mNLS-2 alone, these data strongly suggest heterodimer/multimer formation between GFP-UL69 and the pUL69 mutant in the context of mammalian cells.
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), since the respective regions could be deleted without affecting pUL69 self-interaction. This is also supported by our previous notion that C-terminal pUL69 deletion fragments lacking the self-interaction domain still demonstrate RNA binding and UAP56/URH49 interaction (Lischka et al., 2006; Toth et al., 2006). Thus, we present strong evidence to suggest that self-association is a shared property of all members of the ICP27 protein family that appears to be mediated by the conserved homology region. A comparison of the predicted secondary structure of the respective proteins using the PSIPRED method (Jones, 1999) supports the assumption that the homology region may form a globular, highly structured core domain (Fig. 1a). One may speculate that this structure forms a central scaffold for both self-interaction and binding to other specific proteins. Due to structural constraints, this domain may have been less amenable to divergence during co-evolution of the different herpesviruses, whereas other regions of these proteins were able to adapt to the specific requirements that these proteins have to fulfil during the life cycle of the individual viruses. Further studies aimed at the determination of the three-dimensional structure of these proteins will be required to ascertain this hypothesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Lischka, P., Rosorius, O., Trommer, E. & Stamminger, T. (2001). A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69. EMBO J 20, 7271–7283.[CrossRef][Medline]
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Received 16 August 2006;
accepted 9 October 2006.
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478-527 and mutant C495A of pUL69, generated as in-frame fusions to the GAL4 DNA-binding domain, are indicated. The location of the NLS and NES, the binding sites for UAP56 (UAP56-BD) and hSPT6 (hSPT6-BD), and the RNA-binding domain (RNA-BD) containing arginine-rich sequences (R1, R2, RS) is depicted (Winkler et al., 2000