Human cytomegalovirus (HCMV), a member of the beta subgroup of herpesviruses, is characterized by its narrow host range and prolonged replicative cycle in cell culture as well as in the infected human host. Generally, HCMV possesses low pathogenicity when infecting healthy individuals. However, it is of considerable clinical importance in immunocompromised patients like transplant recipients or patients suffering from AIDS as well as in prenatally infected newborns (2
). As found for other herpesviruses, the lytic cycle gene expression of HCMV occurs in a sequential fashion. Initially after infection, the immediate-early (IE) gene products are the first to be synthesized, followed by the early and late gene products (12
). IE gene expression, which does not require any prior viral protein synthesis, can be detected from the UL36-38, US3, TRS1, and major IE gene regions (58
). The latter encodes two predominant proteins during the IE phase, the 72-kDa IE1 polypeptide (also called IE1-p72 or ppUL123) and the 86-kDa IE2 protein (also called IE2-p86 or ppUL122a) (30
). Several additional isoforms of IE2 that arise either via differential splicing or via the usage of a late promoter within the IE2 gene region have been described (50
). Both IE1-p72 and IE2-p86 have regulatory functions and have been proposed to play a pivotal role in the discrimination between replication and latency. In particular, IE2-p86 appears to play a master role in triggering the lytic replicative cycle of HCMV (30
Two main functions of IE2-p86 have been well characterized during the last years. First, this protein is able to repress transcription of its own promoter (29
), the potent major IE enhancer-promoter of HCMV (8
), thus antagonizing its own expression. This negative autoregulation is mediated by a direct DNA contact of IE2-p86 with a sequence element located between the TATA box and the transcriptional start site of the enhancer-promoter (38
). DNA binding of IE2-p86 at this specific position of the promoter has been shown to block the association of RNA polymerase II with the preinitiation complex (39
). Second, IE2-p86 is a strong transactivator of viral early promoters and of several heterologous promoters, including the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) (26
). The transactivating function of IE2-p86 is thought to be required for progression of the replicative cycle from the IE to the early phase. The mechanism of transactivation has not been defined entirely. However, since IE2-p86 interacts with the basal transcription factors TATA-binding protein (26
) and TFIIB (10
) and with distinct cellular transcription factors such as CREB, AP-1, Egr-1, or Spi-1/PU.1 (37
), protein contacts are believed to be essential for transactivation. In addition to the well-characterized functions of IE2 in transactivation and autorepression, the demonstration of interactions with the cell cycle regulatory proteins pRb and p53 suggested that IE2-p86 could also have an influence on cell cycle regulation (24
The IE2-p86 protein of HCMV is a multifunctional regulator of viral as well as cellular gene expression. Protein-protein interactions are thought to play a major role in IE2-mediated regulation of this broad spectrum of promoters, and it has been reported that IE2-p86 is able to interact with at least 15 cellular factors (18
), some of which have been identified. Known interaction partners include members of the basal transcription machinery such as TATA-binding protein and TFIIB (10
), several transcription factors (e.g., AP-1, Egr-1, and CREB) (37
), and the cell cycle regulators pRb and p53 (24
). However, since the majority of those binding partners are still unknown, we performed a yeast two-hybrid screen using the carboxy terminus of IE2 as a bait. An N-terminally truncated protein was chosen since this IE2 version lacking the N-terminal acidic transcriptional activation domain did not activate the reporter genes in yeast when fused to the GAL4 DNA-binding domain. In addition, published data indicate that the C terminus of IE2 as used in our yeast two-hybrid screen contains important protein interaction motifs (10
). Furthermore, we were able to observe an interaction of this bait construct after coexpression with the viral UL84 protein fused to the GAL4 activation domain as well as a dimerization with an IE2-GAL4 activation domain fusion protein. This suggests a native conformation of the bait protein in fusion with the GAL4 DNA-binding domain which allows for an interaction with other proteins.
Using the yeast two-hybrid screen, we were able to isolate several copies of two polypeptides termed SUMO-1 and hSMT3b as well as the hUBC9 enzyme as specific interaction partners of IE2 in yeast. The proteins SUMO-1 and hSMT3b are highly homologous, being 46% identical on the amino acid level. Since they exhibit a low but significant homology to ubiquitin, they are referred to as UbH proteins (7
). Similar to ubiquitin, these two UbH polypeptides can be covalently attached to a variety of mostly nuclear target proteins by an enzymatic process with analogy to protein ubiquitinylation (32
). This covalent linkage involves the hUBC9 enzyme, a member of the E2 ubiquitin-conjugating enzyme family with a strict specificity for the substrates SUMO-1 and hSMT3b (54
). Hereby, hUBC9 mediates the formation of an isopeptide bond between the carboxy terminus of the respective UbH and the amino group of a lysine residue in the target protein (42
). However, in contrast to ubiquitinylation, there is currently no evidence that the proteins modified by SUMO-1 or hSMT3b are targeted for destruction via the proteasome (70
Having identified UbH polypeptides and hUBC9 as specific interaction partners of IE2, we wondered whether IE2-p86 could be modified by covalent linkage to SUMO-1 or hSMT3b. By Western blot experiments using lysates derived from transfected 293 cells, we were able to demonstrate a modification of IE2-p86 after coexpression with a FLAG-tagged SUMO-1 or hSMT3b resulting in a 105-kDa IE2 isoform, which could be detected using two different IE2-specific antibodies. To reliably detect this isoform, it turned out to be critical to lyse cells under strictly denaturing conditions, since SUMO-1 or hSMT3b conjugates are highly unstable in NP-40 lysis buffer most probably due to a deSUMOylation activity in cell lysates (13
). Under denaturing lysis conditions, a 105-kDa IE2 isoform could even be detected after expression of IE2-p86 in the absence of a FLAG-tagged SUMO-1, most probably due to a modification by an endogenous UbH moiety.
Furthermore, we were able to immunoprecipitate the conjugated 105-kDa IE2 isoform from lysates of transfected 293 cells under denaturing conditions, thus further confirming the covalent nature of the interaction. Interestingly, two additional FLAG-reactive bands of 130 and 150 kDa could reproducibly be observed after immunoprecipitation with IE2-specific antibodies. Since these signals were detectable only in reactions containing both IE2-p86 and the respective FLAG-UbH polypeptide, it is highly unlikely that they are the result of nonspecific binding of FLAG-tagged proteins to primary antibodies or to the Sepharose beads. Surprisingly, these bands were not detected in Western blot analyses using various IE2-specific antibodies, suggesting that they do not represent additional IE2 isoforms. A similar observation of a higher-molecular-weight protein species has also been described for IκBα/SUMO-1 conjugates (13
). Whether these signals are due to cellular UbH-conjugated proteins that strongly interact with IE2-p86 is not known.
After having demonstrated that IE2-p86 can be covalently modified by SUMO-1 or hSMT3b after transient expression of each protein, we wanted to know whether this conjugation could also occur in HCMV-infected human fibroblasts. By Western blot analysis, we were able to show that a 105-kDa IE2 isoform is detectable during the whole time course of the HCMV replicative cycle, with the most prominent signal occurring at late times after infection. Moreover, we could immunoprecipitate the 105-kDa IE2 isoform after HCMV infection of fibroblasts that had been transfected with expression vectors for either the FLAG-tagged SUMO-1 or hSMT3b, demonstrating the stability of these isoforms under conditions of viral infection.
In summary, these experiments show that IE2-p86 can be covalently coupled to SUMO-1 or hSMT3b both after cotransfection and during viral infection. At present, we are not able to definitely demonstrate whether the endogenous moiety that is coupled to IE2-p86 corresponds to SUMO-1 or hSMT3b. This is due to several reasons. First, since no specific antibody against hSMT3b is available as yet, it is impossible to detect the endogenous protein. Second, the only available antibody against SUMO-1, MAb 21C7 (45
), does not react with SUMO-1-conjugated IE2 even in experiments where IE2-p86 was cotransfected with FLAG-tagged SUMO-1, which allowed for a reliable monitoring of the conjugation by using an anti-FLAG MAb (data not shown). This may be due to the fact that this antibody has been generated against RanGAP1-conjugated SUMO-1 and may require an epitope shared by SUMO-1 and RanGAP1 for high-affinity binding, thus making the detection of other SUMO-1-conjugated proteins difficult. Alternatively, the epitope recognized by MAb 21C7 may not be accessible in the IE2-p86-SUMO-1 conjugate. Therefore, the final evidence for the nature of the endogenous UbH moiety conjugated to IE2-p86 awaits the availability of novel reagents allowing the detection of distinct species of UbH polypeptides.
IE2-p86 is not the only protein encoded by HCMV that can be modified by covalent coupling to SUMO-1 or hSMT3b. Consistent with a recent publication by Müller and Dejean (48
), we observed a higher-molecular-weight species of the IE1-p72 transactivator after cotransfection of a vector encoding FLAG-tagged SUMO-1. This does not reflect a general, nonspecific modification after overexpression of UbH molecules since several other viral proteins (e.g., pUL84, pUL69, and pUL26) were not coupled under those conditions. However, we noticed differences in the modification of IE1-p72 in comparison to IE2-p86. First, no endogenously modified protein species could be detected for IE1-p72 after transfection of an IE1 expression vector alone. Second, we were not able to immunoprecipitate an IE1-p72/SUMO-1 conjugate after HCMV infection of cells that had been transfected with a vector for FLAG-tagged SUMO-1/hSMT3b. At present, we do not know whether these differences are due to a higher instability of the respective IE1 conjugate or reflect a differential regulation of SUMOylation during the HCMV replicative cycle.
To identify the lysine residue within IE2-p86 which serves as an acceptor for covalent coupling by UbH moieties, we used a panel of N- and C-terminal IE2 deletion mutants. Hereby, we were able to localize a domain within IE2-p86 that shares a high similarity to already published SUMOylation sites of other cellular proteins (13
). By PCR mutagenesis we could show that this domain contains two SUMOylation motifs involving amino acids 175 and 180; only a double mutation of both lysine residues abolished modification by SUMO-1 or hSMT3b. The loss of the endogenous 105 kDa IE2-isoform after mutation of both lysine residues can serve as an additional strong argument that a UbH-like protein is responsible for this modification. As we observed that mutation of either conjugation site alone still results in a 105-kDa IE2 isoform, we believe that both lysine residues can serve as acceptors for modification. However, we never detected a 130-kDa isoform in Western blot analysis which would be consistent with a simultaneous linkage of two UbH moieties to IE2-p86. Therefore, we favor the hypothesis that these sites are used alternatively, probably because of steric hindrance: once a modification has taken place, the second site would no longer be accessible for coupling. However, it remains to be determined why IE2-p86 possesses these two motifs in an immediate vicinity.
Finally, we were interested in the function of IE2 SUMOylation. For RanGAP1, a protein involved in nuclear transport, it was shown that only the SUMO-1-modified isoform p90 is localized at the nuclear pore complex whereas the nonmodified form p70 has a cytoplasmic distribution (41
). As HCMV IE2-p86 is strictly nuclear and as the conjugation sites are not contained within the nuclear localization signals of IE2 (73
), a targeting to the nuclear pore complex as a consequence of modification is probably not the function of IE2 SUMOylation. For the inhibitor of NFκB, IκBα, it was shown that covalent attachment of SUMO-1 protected against signal-induced degradation (13
). However, since protein steady-state levels of the SUMOylation-defective IE2 double mutant did not differ significantly from those of the single amino acid mutants or the wild-type protein, a major influence of SUMOylation on the half-life of IE2 appears to be unlikely. Recently, two proteins of ND10 (also termed PML oncogenic domain or Kr bodies), PML and Sp100, have turned out to be SUMO-1 conjugated (33
). For PML, the first identified ND10-associated protein, it was observed that SUMOylation is necessary for its accumulation within these domains (49
). In contrast, the Sp100 protein is present in ND10 even when SUMOylation is abolished, indicating that covalent coupling with SUMO-1 does not necessarily refer to a localization within nuclear bodies (62
). Having generated an IE2 mutant which is no longer conjugated, we showed that SUMOylation of IE2-p86 is not required for its accumulation within ND10. This suggests that SUMOylation is not a common pathway for protein targeting to ND10 domains.
Apart from the known cellular target proteins for SUMOylation as mentioned above, several publications have described an interaction of the hUBC9 enzyme with various bait proteins in yeast two-hybrid screens (27
). Since the only function of hUBC9 known so far is the attachment of UbH moieties to target factors, one might speculate that at least some of those bait proteins are likewise SUMOylated. Interestingly, several of those bait proteins, e.g., Ets-1 (27
), WT-1 (66
), and viral regulatory proteins like the adenovirus E1a protein (28
), exhibit transcriptional activity. For some of these factors it was reported that cotransfection of a hUBC9 expression vector resulted in increased transactivation of a given reporter construct (27
). This implicates a potential role of modification by UbH polypeptides for transcriptional regulation and raised the hypothesis that SUMOylation might be of a more general importance for transcription factors. Consistent with this, two recent publications demonstrated that a modification of p53 by SUMO-1 resulted in enhanced transactivation by p53 (22
). Therefore, we investigated whether the IE2 mutants described above showed differences in transactivation potential from wild-type IE2-p86. Hereby, we observed a drastic reduction of transactivation mediated by the double amino acid mutant which is negative for SUMOylation, whereas the single amino acid mutants that are still coupled to UbH proteins were not defective. This suggests that SUMOylation is of importance for IE2-mediated transactivation. At present, we cannot totally exclude that the simultaneous mutation of two lysine residues within this region results in an alteration of IE2 protein conformation leading to a reduction in transactivation levels, although each of the single amino acid mutants was fully functional. However, we performed several different approaches to confirm the integrity of the double mutant protein structure. First, we substituted each lysine residue against another basic amino acid, arginine, in order to maintain the basic charge within the mutated sequence in IE2-p86. Second, we demonstrated that all IE2 mutants could still interact with the viral pUL84 protein, as confirmed by coimmunoprecipitation analysis. Previous experiments in our laboratory suggested that an extended domain within IE2-p86 is required for binding to pUL84 (T. Stamminger, unpublished observation), indicating that the structure of the individual mutants still allows for an interaction. Third, the individual mutants are still able to dimerize, as shown by coimmunoprecipitation experiments. Last, we were able to confirm the interaction between the individual mutants and the hUBC9 enzyme in yeast (H. Hofmann and T. Stamminger, unpublished data). This is of particular importance since it proves that mutation of lysine residues which abolishes SUMOylation does not interfere with hUBC9 interaction, which in turn would prevent modification.
Extensive investigations by other laboratories have characterized domains within IE2-p86 that are necessary for transactivation. Hereby, it was shown that both an amino-terminal domain comprising amino acids 1 to 98 as well as the carboxy-terminal half of the IE2-p86 protein are required for stimulation of early HCMV promoters (43
). Additionally, Yeung et al. reported that amino acids 169 to 194 are necessary for IE2-p86 mediated transactivation of the HIV LTR (73
). Consistent with this, our data show that amino acids 175 and 180 are important for stimulation of the early HCMV promoters of the UL112/113 and UL84 gene regions as well as for stimulation of the HIV LTR. The UL112/113 promoter had also been the subject of studies by Sommer et al. who showed that an internal deletion of amino acids 135 to 290 within IE2-p86 (in deletion mutant IE86ΔSX) resulted in an approximately 50% reduction in the transactivation potential compared to wild-type IE2-p86 (56
). Luciferase experiments using a reporter construct under control of the UL112/113 promoter in combination with either the IE86ΔSX mutant or the internal deletion mutant IE2del174-181 showed a transactivation potential similar to that observed with the SUMOylation negative-double mutant IE2mut175+180.
Our current model concerning the function of IE2 SUMOylation includes the UbH residue as an additional protein interaction motif for other cellular cofactors, which in turn might also be modified by UbHs (7
). To back up this hypothesis, it would be necessary to identify cellular cofactors which preferentially interact with wild-type IE2 but not with the double amino acid mutant. To determine the importance of IE2 SUMOylation for the lytic replicative cycle of HCMV, the construction of a recombinant virus carrying the double amino acid mutation in the IE2 open reading frame is in progress.