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The 86-kDa IE2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a potent transactivator of viral as well as cellular promoters. Several lines of evidence indicate that this broad transactivation spectrum is mediated by protein-protein interactions. To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the isolation of two ubiquitin-homologous proteins, SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2. The polypeptides SUMO-1 and hSMT3b have previously been shown to be covalently coupled to a subset of nuclear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By Western blot analysis, we were able to show that the IE2-p86 protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1. Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling. By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant. This suggests a functional relevance of covalent modification by ubiquitin-homologous proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.
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, 3). 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, 47, 68, 69). 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, 64, 65). 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, 50, 59). 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, 52, 59). 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 (30,50).
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, 51), 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 (38,40). 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, 35, 43). 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, 56) and TFIIB (10) and with distinct cellular transcription factors such as CREB, AP-1, Egr-1, or Spi-1/PU.1 (37, 55, 67, 74), 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, 57).
Experimental data indicated that IE2-p86 interacts with several additional, as yet unidentified cellular proteins (18). Therefore, we decided to perform a yeast two-hybrid screen using a N-terminally truncated IE2-p86 as a bait in order to identify novel binding partners of this important regulatory protein. In this study, we report the isolation of two human ubiquitin-homologous (UbH) polypeptides, SUMO-1 and hSMT3b, and their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as interaction partners of IE2. We demonstrate that IE2-p86 can be modified by covalent coupling to either SUMO-1 or hSMT3b, resulting in a 105-kDa isoform of IE2 in transient transfection experiments as well as in infected human foreskin fibroblasts (HFFs). Furthermore, we were able to precisely define the modification sites within IE2-p86. Concerning the functional relevance of IE2-p86 conjugation by SUMO-1 or hSMT3b, we demonstrate that modification does not affect the subnuclear distribution of IE2-p86 within nuclear domain 10 (ND10). However, transfection experiments using an IE2 mutant that can no longer by modified by SUMO-1 and hSMT3b suggest that a covalent attachment of UbH proteins to IE2-p86 plays a role for IE2-mediated transactivation.
Oligonucleotides.Oligonucleotides were obtained from Eurogentec (Seraing, Belgium) and ARK (Darmstadt, Germany). The following oligonucleotides (5′ to 3′ sequences) were used for cloning and in vitro mutagenesis: IE2pACT5 (AGCTGGATCCTGGGGCATCCGCTACTCCCGA), IE2pACT3 (AGTCGGATCCGAGACTTGTTCCTCAGGTCC), pSC5 (ACCCAAGCTTGGTACCGAGCTCCGCACC), pSC3 (CTAGTCTAGACTGACTAACTAGATGCATGC), SUMOflag5 (AGTCGGATCCTCTGACCAGGAGGCAAAACCT), SUMOflag3 (AGTCTCTAGACTAAACTGTTGAATGACCCCC), SMT3flag5 (AGTCGGATCCGCCGAAAAGCCCAAG), SMT3flag3 (AGTCTCTAGATCAGTAGACACCTCCCGTCTG), IE2-N1 (AGCGAAGCTTGTCGACCTCGAGCCACCAT), IE2-N2 (AGCGAAGCTTGTCGACCTGCGGCCATCAGAGC), IE2-N3 (AGCGAAGCTTGTCGACCCGCATCTCCGAGTTG), C-IE2 (AGCTGAATTCAAGCTTCCCCACGGGTAGGC), 5IE2mut175 (ATGCTGCCCCTCATCCGACAGGAAGACATCAAG), 3IE2mut175 (CTTGATGTCTTCCTGTCGGATGAGGGGCAGCAT), 5IE2mut180 (AAACAGGAAGACATCCGACCCGAGCCCGACTTT), 3IE2mut180 (AAAGTCGGGCTCGGGTCGGATGTCTTCCTGTTT), IE2-180mut175-5 (ATGCTGCCCCTCATCCGACAGGAAGACATCCGA), IE2-180mut175-3 (ACGGATGTCTTCCTGTCGGATGAGGGGCAGCAT), IE2-pQE16A (AGTCGGATCCATGGAGTCCTCTGCCAAGAG), IE2fImut3 (AGTCGAATTCGAGGGGCAGCATGATAGG), IE2fIImut5 (AGTCGAATTCGAGCCCGACTTTACCATC), IE2fIImut3 (AGTCTCTAGATTACTGAGACTTGTTCCTCAG), PR-ul84A (GATCGCTAGCGGTGGCCAACCTAGCCTCGG), and PR-ul84B (CAGCCTCGAGTTGGTGTCTGCGGGCGCGAG).
Plasmid constructions and in vitro mutagenesis.The bait plasmid pHM247 for the yeast two-hybrid screen was constructed by isolation of a SmaI/SalI fragment from plasmid pHM134 containing the IE2-p86 cDNA (37) followed by Klenow treatment and insertion into the NcoI restriction site of the GAL4 DNA-binding domain vector pAS1 (15), which had also been filled in by treatment with Klenow enzyme. The yeast vector pHM704 expressing the same IE2-p86 sequence as fusion with the GAL4 activation domain was constructed by PCR amplification using oligonucleotides IE2pACT5 and IE2pACT3, followed by cleavage with BamHI and ligation with the pACT vector (15). The resulting GAL4 fusion proteins comprise amino acids 135 to 579 of IE2-p86. An expression vector for a GAL4 activation domain fusion protein containing the viral open reading frame UL84 was created by cleavage of vector pcDNAUL84 (19) with XbaI followed by Klenow treatment and thereafter release of the UL84 fragment by cleavage with EcoRI. The GAL4 activation domain vector pGAD424 (Clontech, Palo Alto, Calif.) was prepared by cleavage withSalI followed by Klenow treatment and restriction withEcoRI. Vector and insert were then ligated, resulting in plasmid pHM379.
To construct a eucaryotic expression vector for epitope-tagged proteins, the coding sequence for the FLAG epitope was amplified by PCR using primers pSC5 and pSC3 and plasmid pSuperCATCH (20) as the template. Conditions for PCR were as described previously (19). The amplified fragment was cleaved withHindIII/XbaI followed by insertion into the pcDNA3 vector (Invitrogen Corp., San Diego, Calif.), resulting in plasmid FLAG-pcDNA3. The SUMO-1 and hSMT3b cDNAs were amplified by PCR from one of the library clones obtained in the yeast two-hybrid screen, using either oligonucleotides SUMOflag5 and SUMOflag3 or oligonucleotides SMT3flag5 and SMT3flag3 as primers. The resulting fragments were inserted into the FLAG-pcDNA3 vector viaBamHI/XbaI sites.
For mapping of sites modified by SUMO-1 (i.e., SUMOylation sites), the IE2 deletion mutants IE2(290-548), IE2(310-548), and IE2(329-548) were generated by PCR amplification using plasmid pHM134 as a template and oligonucleotides C-IE2 plus IE2-N1, IE2-N2, and IE2-N3, respectively. The PCR products were cleaved with SalI and EcoRI and ligated with the pSuperCATCH-NLS vector (20). Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit as instructed by the manufacturer (Stratagene, Heidelberg, Germany). The IE2-p86 single amino acid mutants were constructed using oligonucleotides 5IE2mut175 and 3IE2mut175 (resulting in the mutant IE2mut175 carrying a K-R substitution at amino acid 175 of IE2-p86) or 5IE2mut180 and 3IE2mut180 (resulting in the mutant IE2mut180 with a K-R substitution at amino acid 180 in IE2-p86). The IE2 double amino acid mutant IE2mut175+180 was created by using the IE2mut180 plasmid as the template and oligonucleotides IE2-180mut175-5 and IE2-180mut175-3. The internal IE2 deletion mutant IE2del174-181 was constructed by PCR amplification of an N-terminal IE2 fragment (nucleotides 139 to 658) using the oligonucleotides IE2-pQE16A and IE2fImut3 and a C-terminal IE2 fragment (nucleotides 679 to 1953) using the oligonucleotides IE2fIImut5 and IE2fIImut3. Thereafter, the N-terminal fragment was inserted into the pcDNA3 vector using the BamHI and EcoRI restriction sites, followed by ligation with the C-terminal fragment via EcoRI/XbaI, thus resulting in an internal deletion of amino acids 174 to 181 in IE2-p86. The internal IE2 deletion mutant IE86ΔSX was obtained from D. Spector (San Diego, Calif.) (56). Construction of a vector expressing IE2 in fusion with the green fluorescent protein (GFP) will be described elsewhere. The IE1 expression vector pHM494 was created by isolation of the IE1 cDNA from plasmid pHM124 followed by insertion into the pcDNA3 vector via HindIII/EcoRI (52). The luciferase expression plasmids pHM142 containing the UL112/113 promoter and pHIV-luc containing the HIV LTR have been described previously (5, 19). The luciferase expression plasmid pHM571 containing the UL84 promoter was constructed by PCR amplification of the UL84 promoter sequence (nucleotides 123072 to 123404) using oligonucleotides PR-ul84A and PR-ul84B and cosmid pCM1106 (17) as the template followed by ligation with luciferase expression vector pGL3basic (Promega, Mannheim, Germany) viaNheI/XhoI. The DNA sequence of each plasmid construct was confirmed by automated sequence analysis (ABI, Weiterstadt, Germany).
Yeast two-hybrid screening.Yeast two-hybrid screening was performed using GAL4 fusion proteins as described previously (16). Saccharomyces cerevisiae Y153 was transformed by the lithium acetate method using the bait plasmid pHM247 (21). The presence of pHM247 in the yeast cells was stably maintained by selection for tryptophan prototrophy. Expression of the GAL4-IE2 fusion protein was confirmed by Western blot analysis using the IE2-p86 antiserum. Y153 yeast cells transfected with pHM247 alone or in combination with pACT were then tested for activation of the reporter genes HIS3 and lacZ. In neither case was activation of the reporter genes by GAL4-IE2 observed. Yeast strain Y153 containing pHM247 was subsequently transformed with a cDNA library derived from human B lymphocytes fused to the GAL4 activation domain in the pACT vector (15). Primary transformants (107) were selected for growth on histidine dropout plates containing 25 mM 3-aminotriazole. His+ colonies were subsequently analyzed for β-galactosidase activity by filter test experiments (9). The interaction was then quantified byo-nitrophenyl galactopyranoside (ONPG) assays as described elsewhere (23). Interactor plasmids from clones positive in both tests were rescued by transformation of competentEscherichia coli KC8 with total yeast DNA (31). Interactor plasmids derived from double-positive clones were tested for bait specificity by retransformation with the GAL4 plasmid pAS1. The nucleotide sequences of the cDNA inserts were determined by automated sequence analysis (ABI).
Cell culture, transfection, and reporter assays.HFFs were cultured as described previously (5). U373MG and 293 cells were obtained from the American Type Culture Collection (Manassas, Va.) and maintained in Dulbecco's minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 8 and 10% fetal calf serum, respectively. The day before transfection, HFFs were plated onto 100-mm-diameter plastic dishes at 1.3 × 106 cells per dish. 293 cells were seeded onto six-well plates at 3 × 105 cells per well 2 days before transfection. Plasmid transfections were performed by the calcium phosphate coprecipitation procedure usingN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid as described elsewhere (19) and a total of 10 μg of DNA for 100-mm-diameter dishes or 1 to 2 μg of DNA for six-well plates. Cells were harvested 48 h after transfection and used for Western blotting or immunoprecipitation. For indirect immunofluorescence analysis, HFFs grown on coverslips were transfected with 2 μg of plasmid DNA using the FuGENE transfection reagent as specified by the manufacturer (Boehringer, Mannheim, Germany).
For luciferase assays, U373MG cells were plated onto six-well dishes at 2.8 × 105 cells per well the day before transfection. Plasmid transfection was performed by the DEAE-dextran method as described previously (5). Routinely, 1 μg of luciferase target and 2.3 μg of the cotransfected transactivator plasmid were used. The total amount of transfected DNA was kept constant by using the cloning vector pCB6 in order to replace the missing transactivator plasmid. At 48 h after transfection, cells were harvested and luciferase assays were performed using a lysis buffer containing 50 mM Tris-H3PO4 (pH 7.8), 50 mM trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 2% Triton X-100, 4 mM dithiothreitol, and 20% glycerol. Luciferase activity in the supernatant was determined using a luminometer (Bertholt, Freiburg, Germany). Each transfection was performed in triplicate and was repeated at least three times.
Antibodies.The polyclonal antisera against IE2-p86 (19), exon 5 of IE2-p86 (referred to as anti-pHM178), or pUL84 of HCMV were generated by immunizing rabbits with the respective procaryotically expressed proteins. Monoclonal antibody (MAb) 810, which recognizes an epitope contained within exon 2 of both IE1-p72 and IE2-p86 (46), was obtained from Chemicon (Hofheim, Germany). The anti-FLAG MAb M2, which is directed against the synthetic FLAG octapeptide N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C, was purchased from INTEGRA Bioscience (Fernwald, Germany). MAb p63-27 directed against IE1-p72 and the anti-Sp26 antiserum directed against Sp100 (kindly provided by T. Sternsdorf, Hamburg, Germany) were described previously (4, 61). MAb 21C7 directed against SUMO-1 (45) was purchased from Zymed Laboratories (Labor Diagnostika, Heiden, Germany). Anti-mouse and anti-rabbit horseradish peroxidase- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained from Dianova (Hamburg, Germany).
Western blotting and immunoprecipitation analysis.Initially, transfected cells were lysed in NP-40 lysis buffer as described previously (6). For inhibition of deSUMOylation, transfected or infected cells were lysed in 120 μl of a 1:3 dilution of buffer I (5% sodium dodecyl sulfate [SDS], 0.15 M Tris [pH 6.7], 30% glycerol) and buffer II (25 mM Tris [pH 8.2], 50 mM NaCl, 0.5% NP-40, 0.5% deoxycholate, 0.1% SDS, 0.1% azide, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg each of aprotinin, leupeptin, and pepstatin per ml) essentially as described elsewhere (13). After a brief sonification, lysates were diluted in SDS Laemmli buffer and boiled at 94°C for 10 min. Samples were subjected to polyacrylamide gel electrophoresis (PAGE) on SDS–8 to 10% polyacrylamide gels, and the proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Western blotting and enhanced chemiluminescence (ECL) detection were performed according to the manufacturer's protocol (ECL Western detection kit; Amersham Pharmacia Biotech Europe, Freiburg, Germany). Coimmunoprecipitation analysis for detection of noncovalent protein interactions was performed as described elsewhere (6). For detection of covalent modifications by SUMO-1 or hSMT3b, total cell lysates were prepared essentially as described elsewhere (13). Transfected or infected cells were lysed as described above. After a brief sonification, the lysates were diluted 1:10 in phosphate-buffered saline (PBS)–0.5% NP-40 followed by high-speed centrifugation at room temperature. The supernatant was incubated with the appropriate antibody for 1 h at room temperature; then a 50% protein A-Sepharose suspension was added for another 2 h at room temperature. The Sepharose beads were collected and washed three times in PBS–0.5% NP-40. Antigen-antibody complexes were recovered by boiling in SDS sample buffer and analyzed by Western blotting.
Indirect immunofluorescence analysis.For indirect immunofluorescence analysis, HFF cells on coverslips were washed two times with PBS followed by fixation with 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized in PBS–0.2% Triton X-100 on ice for 20 min. Thereafter, the cells were incubated for 30 min at 37°C with a 1:2,500 dilution of MAb 810 (directed against IE1-p72 and IE2-p86) and a 1:200 dilution of anti-Sp26 (directed against Sp100) in PBS, followed by incubation with anti-mouse FITC and anti-rabbit tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies. Cells were mounted using Vectashield mounting medium including 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Calif.) and analyzed using a Zeiss Axiovert-135 microscope. Images were recorded with a cooled MicroMax charge-coupled device camera (Princeton Instruments, Trenton, N.J.) and processed by using IPLab Spectrum and Adobe Photoshop package (Scanalytics Inc., Fairfax, Va.; Adobe Systems Incorporated).
Identification of SUMO-1, hSMT3b, and hUBC9 as cellular interaction partners of the HCMV IE2 protein by yeast two-hybrid experiments.To identify novel cellular interaction partners of the IE2 protein of HCMV, a yeast two-hybrid screen was carried out. Since the full-length IE2-p86 activated the reporter genes HIS3 andlacZ in yeast by itself, an N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 was used as a bait in fusion with the DNA-binding domain of the yeast factor GAL4 in the pAS1 vector (pHM247). The presence of the GAL4-IE2 expression plasmid pHM247 was maintained stably in yeast strain Y153 by selection in liquid dropout culture medium lacking tryptophan, and expression of the relevant fusion protein was confirmed by Western blot analysis (data not shown). To determine whether the bait protein was able to interact with already known binding partners in yeast, the pHM247-containing yeast strain Y153 was transformed with plasmid pHM379 or pHM704, containing either the UL84 protein of HCMV or an N-terminally truncated IE2 protein (amino acids 135 to 579) in fusion with the GAL4 activation domain, respectively. In either case, the reporter genes HIS3 andlacZ were activated, indicating that the IE2 deletion mutant was able to interact with pUL84 and was also able to dimerize as reported in previous publications (data not shown) (11, 19).
The yeast two-hybrid screen was performed by transformation of yeast strain Y153 containing plasmid pHM247 with a cDNA library derived from B lymphocytes in the vector pACT (15). This particular library was chosen since it is highly complex and has previously been used by several groups for the successful isolation of interacting proteins (15). Moreover, since IE2-p86 is able to transactivate in a broad spectrum of different cell types, our main interest was to select ubiquitously expressed proteins with a role in IE2 protein function. By using this screening procedure, 1.25 × 107 primary transformants were tested for interaction with IE2 by selection for histidine prototrophy on dropout plates supplemented with 25 mM 3-aminotriazole and by expression of β-galactosidase as determined by filter lift assays. Plasmids encoding putative interactors of IE2 were isolated from double-positive clones and retransformed into yeast strain Y153/pHM247 in order to confirm the interaction. Positive clones after this retransformation were characterized by automated sequencing and search for homologies in the National Center for Biotechnology Information databases. In total, we were able to identify 15 cellular proteins that showed an interaction with IE2 in yeast. Here, we report the identification of SUMO-1, hSMT3b, and hUBC9 as specific interaction partners of the IE2 protein (Fig. 1). For these interaction partners, more than one copy was found in the yeast two-hybrid screen, indicating a sufficient complexity of the cDNA library and the specificity of the interaction with IE2. By cotransformation experiments of the individual interactor clones and the empty pAS1 vector, it was excluded that SUMO-1, hSMT3b, and hUBC9 were able to activate the reporter genes in yeast in the absence of a bait protein (Fig. 1A). Additionally, liquid β-galactosidase assays (ONPG assays) were performed to quantify the strength of interaction with IE2 (Fig.1B). Interestingly, the interaction between IE2 and SUMO-1 turned out to be even stronger than the interaction between p53 and the simian virus 40 (SV40) T antigen, which served as a positive control.
The interactors SUMO-1 and hSMT3b belong to the family of UbH proteins (7, 44). They were shown to be covalently attached to a variety of nuclear targets in a manner analogous to protein ubiquitinylation (32, 34), which involves hUBC9 (32,54). Thus, the simultaneous identification of SUMO-1, hSMT3b, and hUBC9 as interacting proteins of IE2 suggested that IE2-p86 might be a target for posttranslational modification by coupling to SUMO-1 or hSMT3b.
Evidence for a covalent modification of IE2 by SUMO-1 and hSMT3b.Having identified the ubiquitin-like proteins SUMO-1 and hSMT3b as potential interaction partners of HCMV IE2 in the yeast two-hybrid screen, we searched for physiological situations in mammalian cells in which IE2-p86 is covalently modified by SUMO-1 or hSMT3b. For this purpose, both the SUMO-1 and the hSMT3b cDNA were expressed as FLAG-tagged proteins using the FLAG-pcDNA3 expression vector. 293 cells were transfected with the FLAG-hSMT3b or, as shown in Fig. 2, with the FLAG-SUMO-1 expression vector either alone or in combination with the IE2-p86 expression plasmid pHM134. After lysis of cells using NP-40 buffer, proteins were analyzed by Western blotting. The IE2 protein was detected using the anti-IE86 rabbit serum (19) (Fig. 2A). In lysates from cells expressing IE2-p86 and increasing amounts of FLAG-SUMO-1, the IE2 86-kDa protein was detected as well as an additional 105-kDa isoform which is fairly consistent with a 20-kDa covalent modification of IE2-p86 by FLAG-SUMO-1. After removal of the IE2-specific antibodies, the same membrane was incubated with MAb M2 directed against the FLAG epitope in order to investigate whether this 105-kDa band contained FLAG-SUMO-1 (Fig. 2B). In lanes 2 and 4 to 6 we observed a smear toward higher-molecular-weight proteins representing FLAG-SUMO-1-conjugated cellular proteins as described previously (42, 45). The 90-kDa band which could be detected in the absence as well as in the presence of cotransfected IE2 most probably represents the p90 form of RanGAP1 (45). Only in the presence of IE2-p86 did we detect an additional immunoreactive band of 105 kDa which perfectly overlapped with the IE2-immunoreactive 105-kDa band shown in Fig. 2A.
Referring to experiments as described by Desterro et al. (13), in which IκBα–SUMO-1 conjugates were rarely detectable after cell lysis using NP-40 lysis buffer, we performed analogous transfection experiments but lysed the cells in 1.7% SDS buffer (13). Again, Western blot analyses were performed with the IE2-p86 antiserum (Fig. 2C). Under conditions of SDS lysis, a 105-kDa IE2 isoform was already observable in the absence of FLAG-SUMO-1, whereas after cotransfection with FLAG-SUMO-1 we detected a second band with slightly slower electrophoretic mobility, representing the IE2/FLAG-SUMO-1 conjugate because of additional amino acids derived from vector sequences and the FLAG epitope (Fig. 2C and D, lanes 2). To exclude artifacts from the antiserum, we repeated this experiment using the same lysates and MAb 810, which is directed against an amino-terminal epitope contained within both IE1-p72 and IE2-p86 (45) (Fig. 2D). Similarly, an endogenous modification of IE2 was visible after transfection of the IE2-p86 expression vector pHM134 alone, which resolved in a double band after additional cotransfection of FLAG-SUMO-1. Analogous cotransfection experiments with IE2-p86 and FLAG-hSMT3b followed by Western blot analysis showed comparable results (data not shown). Taken together, these experiments indicate that IE2-p86 can be covalently modified after coexpression of FLAG-SUMO-1 or FLAG-hSMT3b. Furthermore, an IE2 isoform of similar molecular weight can be observed when IE2 is expressed alone in 293 cells.
As the major IE proteins IE1-p72 and IE2-p86 of HCMV have the first 85 amino acids in common, we wished to determine whether IE1-p72 is also modified by SUMO-1 or hSMT3b. Therefore, analogous cotransfection experiments were performed with the IE1-p72 expression vector pHM494 and FLAG-SUMO-1 or FLAG-hSMT3b in 293 cells. Both HCMV IE proteins were detected using MAb 810. As is evident from Fig. 3A, lanes 2 and 3, IE1-p72 could likewise be covalently modified by both FLAG-SUMO-1 and FLAG-hSMT3b. However, in contrast to IE2-p86 (Fig. 3A, lane 7), no endogenous modification of IE1-p72 was detectable in the absence of the FLAG-UbH proteins under these conditions.
This result led us to question whether SUMOylation after overexpression is a general feature of viral regulatory proteins. Therefore, we tested if modification of three additional viral proteins could be observed in the presence of FLAG-SUMO-1 or FLAG-hSMT3b. However, in contrast to IE1-p72 and IE2-p86, no conjugation was detectable for the viral protein pUL26, pUL69 (data not shown), or pUL84 (Fig.3B), indicating that SUMOylation is restricted to the major IE proteins of HCMV.
Immunoprecipitation of IE2-UbH conjugates under denaturing conditions confirms the covalent nature of the interaction.To further strengthen the evidence for a covalent modification of IE2-p86 by SUMO-1 and hSMT3b, we performed cotransfection experiments in 293 cells followed by immunoprecipitation with the IE2-p86 antiserum. Conjugates between IE2-p86 and either FLAG-SUMO-1 or hSMT3b were then detected by Western blot analyses of the precipitates using the anti-FLAG MAb (Fig. 4). Here as well as in the following immunoprecipitation experiments, an aliquot of each sample was analyzed by Western blotting prior to immunoprecipitation in order to confirm that equal amounts of protein had been expressed (data not shown). The well-characterized interaction between IE2 and the UL84-protein of HCMV (19) served as a positive control (Fig.4A and B, lanes 1). As the binding of IE2-p86 to pUL84 is of noncovalent nature, cells were lysed using NP-40 lysis buffer, whereas cells expressing IE2 and FLAG-SUMO-1 or FLAG-hSMT3b were lysed essentially as described by Desterro et al. (13). As shown in Fig. 4A, lane 2, the noncovalent interaction between IE2 and pUL84 was not observed under those denaturing conditions. On the other hand, the attachment of FLAG-hSMT3b to IE2 was detected after cotransfection of a constant amount of the IE2-p86 expression vector in combination with increasing amounts of the FLAG-hSMT3b construct (Fig. 4A, lanes 5 to 7). Surprisingly, two additional bands with calculated molecular masses of about 130 and 150 kDa could be observed apart from the 105-kDa IE2-hSMT3b conjugate in Western blot analysis (Fig. 4A, lanes 5 to 7).
Analogous experiments were performed after cotransfection of IE2-p86 and FLAG-SUMO-1 (Fig. 4B). Again, the 105-kDa IE2-SUMO-1 conjugate was detected by the anti-FLAG antibody in Western blot analysis (Fig. 4B, lane 3); two additional bands with calculated molecular masses of 130 and 150 kDa were also present (Fig. 4B, lane 3, indicated by asterisks). These two bands seem to be specific because they were observed only in the presence of both IE2-p86 and FLAG-SUMO-1 or FLAG-hSMT3b and were not observed in cell lysates either expressing IE2-p86 or the FLAG-UbH protein alone. Surprisingly, however, these bands could not be detected when Western blots were performed with IE2-specific MAbs or sera, suggesting that they do not represent additional IE2 conjugates (data not shown; Fig.5C). In summary, these results confirm a covalent conjugation of FLAG-SUMO-1 and FLAG-hSMT3b to the HCMV IE2-p86 protein.
Covalent modification of IE2-p86 by SUMO-1/hSMT3b occurs also during the replicative cycle of HCMV.Having demonstrated that HCMV IE2-p86 is conjugated by UbH proteins in 293 cells, we wanted to investigate whether a similar modification also occurs in infected primary HFFs. Therefore, we analyzed lysates from infected HFFs 6, 20, 27, 48, and 72 h after infection by Western blotting using the anti-pHM178 rabbit antiserum which is directed against amino acid sequences encoded by exon 5 of IE2-p86. As shown in Fig. 5A, a 105-kDa IE2 subform was also observed during the whole time course of lytic infection, detectable already at 6 h after infection. This subform is similar in electrophoretic mobility to extracts from IE2-expressing 293 cells (Fig. 5A, lanes 8 and 9).
To further determine whether SUMO-1 or hSMT3b modification of IE2-p86 occurs in infected cells, HFFs were transfected with FLAG-SUMO-1 or FLAG-hSMT3b. The day after transfection, cells were either mock infected or infected with HCMV for 72 h. Thereafter, immunoprecipitations were performed with MAb 810. In Western blot analysis with the anti-FLAG MAb, a 105-kDa FLAG-reactive band was detected in precipitates containing either FLAG-SUMO-1 or FLAG-hSMT3b; as already observed after immunoprecipitation analysis of 293 cell lysates, the two higher-molecular-mass forms of 130 and 150 kDa could also be detected (Fig. 5B, lanes 2 and 3). To determine which bands as detected by the anti-FLAG antibody contained the IE2 protein, an identical immunoprecipitation with MAb 810 was performed followed by Western blotting using the IE2-p86 specific rabbit antiserum. As can be seen in Fig. 5C, lanes 2 and 3, the 86- and 105-kDa IE2 isoform, but no higher-molecular-mass bands of 130 or 150 kDa, were detected with the IE2-specific antiserum, indicating that the higher-molecular-mass anti-FLAG reactive bands do not represent additional IE2 isoforms. In contrast, no modification of IE1-p72 could be observed during viral infection using the same immunoprecipitation conditions followed by Western blot analysis with an IE1-specific MAb (Fig. 5D, lanes 2 and 3). These experiments confirm that IE2-p86 modification by SUMO-1 or hSMT3b can also occur in infected human fibroblasts. Furthermore, the modification of the two major IE proteins of HCMV seems to be differentially regulated, as IE2-p86 is modified during the complete replication cycle whereas IE1-p72 conjugates are not detectable at 72 h after infection under those conditions.
Mapping of the SUMO-1/hSMT3b conjugation site within the HCMV IE2 protein identifies lysine residues 175 and 180 as two independent acceptor sites for modification.Having shown that IE2-p86 can be modified by both SUMO-1 and hSMT3b in 293 cells as well as in infected HFFs, we were interested in mapping the precise conjugation site within IE2-p86. For this, a series of N- and C-terminal IE2 deletion mutants was created (Fig. 6A). The C-terminal deletion mutants IE2(290-548), IE2(310-548), and IE2(329-548) were cloned into the pSuperCATCH-NLS vector in order to ensure a nuclear localization of the encoded proteins. This was done since a recent study suggested that a nuclear localization of a given target protein is necessary for SUMOylation (62). As determined by indirect immunofluorescence analysis, all constructs exhibited a strictly nuclear localization (data not shown). Then, each of the constructs was cotransfected with FLAG-SUMO-1 or FLAG-hSMT3b in 293 cells followed by Western blot analysis with the IE2-p86 antiserum in order to examine whether the mutants were still conjugated (data not shown). Thus, we were able to map the conjugation domain in IE2 between amino acid 173 and 290 (Fig. 6A).
Peptide sequence similarity analysis within this domain showed a striking homology between amino acids 169 to 182 of IE2-p86 and the already published SUMO-1 conjugation site in RanGAP1 (42). In addition, this sequence also exhibits similarities to the respective SUMOylation sites within IκBα, Sp100, and PML (Fig. 6B) (13,33, 62). Sternsdorf et al. (62) proposed the consensus sequence (I/L)KxE for SUMOylation in which the lysine residue is linked to the ubiquitin-like moiety (62). Interestingly, two (I/L)KxE motifs could be found in IE2-p86 within amino acids 169 to 182, in which both lysine 175 and lysine 180 might serve as acceptors for conjugation independently. To further investigate this amino acid sequence, we performed a PCR mutagenesis by which we replaced either lysine 175 (IE2mut175) or lysine 185 (IE2mut180) separately or both simultaneously (IE2mut175+180) by arginine. The replacement of lysine by arginine was chosen in order to maintain a basic charged residue at each amino acid position. Additionally, we created an IE2 mutant with an internal deletion of amino acid residues 174 to 181 (IE2del174-181) (Fig. 7A). All mutants were transfected into 293 cells in the absence or presence of FLAG-SUMO-1, and cell lysates were analyzed by Western blotting using the IE2-p86 antiserum (Fig. 7B). As observed for wild-type IE2, each of the lysine single point mutants was still modified by an endogenous moiety in the absence of FLAG-SUMO-1 (Fig. 7B, lanes 2, 4, and 6) and could also be conjugated with FLAG-SUMO-1, which again resulted in a doublet at 105 kDa (lanes 3, 5, and 7). In contrast, neither the internal deletion mutant IE2del174-181 nor the double amino acid point mutant IE2mut175+180 was modified (lanes 8 to 11).
To further confirm these results, we performed immunoprecipitations after cotransfection of the individual mutants in combination with FLAG-SUMO-1 or FLAG-hSMT3b. Consistent with the observations of Western blot experiments, the single amino acid mutants IE2mut175 and IE2mut180 were still conjugated with FLAG-SUMO-1 or FLAG-hSMT3b (Fig.8A, lanes 7, 8, 11, and 12), whereas the internal deletion mutant as well as the double amino acid mutant IE2mut175+180 were not modified by either of the two UbH proteins (lanes 14, 15, 18, and 19). In contrast, all IE2 mutants were still able to interact with FLAG-UL84 under native lysis conditions (lanes 5, 9, 13, and 17), indicating that the conformation of the IE2-protein was not severely affected by the introduced mutations. This was further strengthened by coimmunoprecipitation experiments after transfection of the IE2 mutants in combination with a FLAG-tagged, N-terminally truncated IE2 mutant lacking the epitope as recognized by the IE2 MAb 810. After precipitation with MAb 810, this FLAG-tagged IE2 could be detected in Western blot analysis using the anti-FLAG antibody, indicating that all mutants were still able to dimerize (Fig. 8B).
These experiments demonstrate that the SUMO-1/hSMT3b conjugation site within IE2 is located between amino acids 174 and 181. Within this sequence, two separate modification sites (K175 and K180) could be identified.
SUMOylation is not necessary for colocalization of IE2 with ND10.As published previously, both IE1 and IE2 proteins of HCMV are targeted to ND10 domains within a few hours after infection (1, 36, 71). For PML, the first identified ND10-associated protein, it was observed that SUMOylation is required for its accumulation in these subnuclear domains (49). Having generated an IE2 mutant which is no longer conjugated by SUMO-1 and hSMT3b, we addressed the question of whether SUMOylation affects the targeting of IE2-p86 to ND10 structures. For this purpose, wild-type IE2-p86 fused to GFP (IE2-GFP) and the IE2 mutants described before were transfected into HFFs. Immunofluorescence analysis was carried out using the Sp26 antiserum recognizing Sp100 in order to detect endogenous ND10 structures and MAb 810 to detect the IE2 mutants. Wild-type IE2 was visible through its GFP moiety. As evident from Fig.9, wild-type IE2-p86 and the single amino acid mutants exhibited a diffuse nuclear staining with some brighter speckles correlating with Sp100 localization (Fig. 9A to F). Transfection of the double amino acid mutant that is defective for SUMO-1 or hSMT3b conjugation resulted in a predominantly speckled nuclear staining that showed a perfect colocalization with Sp100 nuclear dots. Thus, modification by either SUMO-1 or hSMT3b is not a determinant for accumulation of IE2-p86 within ND10 structures.
Influence of the SUMO-1/hSMT3b modification on IE2-mediated transactivation.Since IE2-p86 is known as a strong transactivator of viral as well as cellular promoters, we addressed the question of whether the IE2 mutants described above showed any difference in transcriptional activity compared with wild-type IE2-p86. For this purpose, permissive U373MG cells were transiently transfected with reporter plasmids containing the luciferase gene under the control of two IE2-responsive early promoters of HCMV. The first reporter construct, termed pHM142, contained the UL112/113 promoter which had previously been described as a highly IE2-responsive element (5,56). The second plasmid, pHM571, contained the promoter driving expression of the UL84 gene of HCMV, which is also strongly transactivated by IE2-p86 (S. Flöss and T. Stamminger, unpublished data). After cotransfection of these reporter plasmids with either the wild-type IE2-p86 expression vector pHM134 or the IE2 mutants IE2mut175, IE2mut180, and IE2mut175+180, luciferase activity was determined in cell extracts. Each experiment was performed in triplicate and repeated at least three times. Transcriptional activation was determined by calculating fold activation based on the activity of the reporter plasmids after cotransfection with the expression vector pCB6 containing the HCMV IE1/2 enhancer-promoter without a coding sequence. As shown in Fig.10, both reporter constructs were activated by wild-type IE2 approximately 230- to 300-fold. There was no significant difference of activation mediated by IE2mut175 and IE2mut180 compared to wild-type IE2. In contrast, a strong decrease in activation mediated by IE2mut175+180 was observed for both promoters that were tested. Sommer et al. (56) had shown in a previous study using the UL112/113 promoter in U373MG cells that an internal deletion of amino acids 135 to 290 (in mutant IE86ΔSX) within IE2-p86 reduced the transactivation potential about 50% (56). We therefore included this mutant, kindly provided by D. Spector (San Diego, Calif.) in our luciferase experiments. As is evident from Fig.10, there is no significant difference in stimulation of the UL112/113-promoter mediated by IE2mut175+180 compared to the IE86ΔSX mutant or the internal deletion mutant IE2del174-181. A similar reduction in transactivation was observed when we used the HIV LTR as a heterologous promoter in combination with the SUMOylation-defective mutants of IE2-p86 (Fig. 10C). Western blot experiments excluded that a reduced expression level of the mutant proteins was responsible for their loss of transactivation capacity (Fig. 10D). In summary, these results suggest that a covalent modification of IE2-p86 by UbH proteins is critical for the transactivation capacity of IE2-p86.
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, 25), several transcription factors (e.g., AP-1, Egr-1, and CREB) (37, 55, 74), and the cell cycle regulators pRb and p53 (24, 57). 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, 24, 56). 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 (7,44, 45). 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 (32,34). 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, 33,42, 62). 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, 45). 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, 49, 63). 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, 28, 66, 72). 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, 53). 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, 50, 56,60). 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.
We thank Michael Winkler for assistance with the yeast two-hybrid system and Donatella de Gaspero-Hoops and Regina Kupfer for excellent technical assistance. We also thank D. Spector (San Diego, Calif.) and T. Sternsdorf (Hamburg, Germany) for reagents, H. Fickenscher for critical reading of the manuscript, F. Melchior for helpful discussions, and B. Fleckenstein for continuous support.
This work was supported by the Deutsche Forschungsgemeinschaft (grants Sta 357/3-1 and SFB 473) and the Bundesministerium für Forschung und Technologie (IZKF Erlangen).
- Received 4 October 1999.
- Accepted 22 December 1999.
↵* Corresponding author. Mailing address: Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany. Phone: 9131/8526783. Fax: 9131/8522101. E-mail:tsstammi{at}viro.med.uni-erlangen.de.
- Copyright © 2000 American Society for Microbiology


text:
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subset: commoncrawl


text: On 22nd June 2010, three of our responders, Damon Galliers, Rebecca Drury and David Wiles ran the Penkridge Fun Run on our behalf. We would like to thank all those that sponsored one of runners. Money raised will be put towards keeping us on the road and regularly responding. Thank you very much indeed particularly to the Priors Dental Practice for running on our behalf.


text:
Idmee Handover
We are currently phasing in handover for the echo system. So what is handover ?
Handover are those situations where your client is not mobile first in the context where you require to perform identification.
There may be several reasons for this where the most prominent reason is real estate, in many situation having a 27 inches real estate for presentation is really required, very convenient or mandated in a corporate scenario.
The process can be depicted as illustrated.
OOur improved transparent handover is demonstrated below. This will be available from August to end customers.
A demo of the current available version is shown below.
This can also be described as an aggregator model. The rationale is that the provider of mobile app can be seen as the landlord providing the service for others. This landlord would then configure for each of the tenant where to provide the result of the process when complete.
The task of getting your app on the handset and providing maintenance can be hard for even the bigger actors so viewing this from an ecosystem perspective it makes sense to forge allianses with others unless you would like to capture the aggregator niche yourself.
- This is the process of determining that identification should be performed. Based on this you would in your channel (using javascript) or in your backend execute an extremely simple rest call. The result of the rest call would be a reference where we also provide the artwork to display a QR code. This QR code will be scanned and the user will be presented a confirmation screen in the mobile app to ensure that the correct context is the cause for the subsequent steps.
- This is the normal identification process for document validation.
- This is the normal process for matching in a tamper secure way of the person presenting the document to the holder of the identity document.
- The result is transferred to the preferred address and preferred method of the tenant.
- When receiving the result the tenant can proceed the process. This can be done using a push or the channel client can perform regular polls to determine when to proceed.
- The client can proceed performing whatever business logic which required identification.
A very simple video can be found at https://www.youtube.com/watch?v=qXqO16My84I&vq=hd1080
Complexity of implementation
It is still 3 boilerplate lines of code in the app and a similar amount of boilerplate code in the tenant channel.
In addition the tenant would need to be capable of accepting the files and to trigger further progress in the channel based on these files. To aid the tenant channel can set a reference with will be reflected in all naming, including the received file names, which will make it simple to correlate the reception to the instance initiating the process.
OK so how can I do a test spin
First you need our demo app from the play store. https://play.google.com/store/apps/details?id=no.idmee.android.demo
Go to https://www.bankofdad.dev:7443/sbm/
Please notice that full protocol specifier must be entered, i.e https:// must also be entered due to non standard ports used.
Choose «Using identity document» and please notice that these are simple pages to show the concept.
You can always watch the demo video if in doubt or if this is unclear https://www.youtube.com/watch?v=qXqO16My84I&vq=hd1080
It's still privacy oriented. Everything is wiped when your session is gone, the only thing we keep is your email address used for authentication.
Limitations and roadmap
The current version requires the app to be installed and scan of the QR or manual entry of the reference to be performed.
The short term roadmap has the following functionality defined.
- Automatic invocation on the device if the action was started on an eligible device.
- Using universal links.
- If the QR code is scanned on a supporting device which will allow the opening of an URL the process can be started outside the app.
- If the app is installed on the device it will be started and proceed directly to the confirmation screen.
- If the app has not been installed the user will be taken to the app store.
- After installing the app and opening it the app will automatically proceed to the confirmation screen. This requires a minor, simple change in existing apps on start to handle this aspect.
- Dynamic branding. This would use the tenant preferred branding during the process. This implies that the individual tenants of one landlord can present their own branding during the process by agreement with the landlord but where we will do all of the lifting and shifting required.
So basically the message would be is that the current mechanism of QR codes is a simple but not friction less method suited for easy implementation (for you) to cover the desktop or any channel capable of user dialogue and the display of QR codes which can even be done on non graphic interfaces.
The methods on the roadmap will be frictionless and with low cost implementations for other channels. Should you wish less friction on the desktop or similar you may achieve thus today but that would require push notifications. We are providing a detailed description but you as the app owner, or with the app owner, must to the lifting to implement the actual push required.


text:
Samsung Smart Switch download for Windows, Mac.
General information
- Category: Other multimedia apps
- Version: 4.2.20013.12
- Last update: Feb 2, 2020
- OS: Windows, Mac
- License: freeware
- File size: 38 MB
- Developer: Samsung Group
- Homepage: samsung.com
- Downloads: 5377
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What is Samsung Smart Switch?
Samsung Smart Switch makes it easy to transfer contacts, photos, messages, phone history, movies, music, notes, apps, videos, settings and more from your old phone to your new Galaxy.
The application can be also used to create and restore backups, migrate all data from iOS, Android and BlackBerry devices to Samsung Galaxy. Samsung Smart Switch is really easy to use, it is simple and with few clicks, you can get access to every option. You can also synchronize data with Outlook.
You can restore iPhone backup from iCloud to your Samsung Galaxy using Smart Switch. Photos, Contacts, Text Messages, Events, Wi-Fi settings, Alarms, Browser bookmarks and Call history are supported for transfer via Smart Switch from iCloud.


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University of Baltimore School of Law
Phone: 410-837-4468
Dean: Ronald Weich
Alumni Relations Contact: Laurie TerBeek
Alumni Selected by Super Lawyers
Class of 2016
Kuwamura Law Group, P.A.
Class of 1997
Stacey E. Andersen, LLC
Class of 2015
Meiselman Helfant & Wills, LLC
Class of 1975
BaldwinLaw LLC
Class of 1991
Beach-Oswald Immigration Law Associates, PC
Class of 1994
Love, Fleming, Bearsch & Hurff, LLC
Class of 1991
Belsky, Weinberg & Horowitz
Class of 2003
Hardwick Benfer, LLC
Class of 1993
Clara Martone Bereston, P.A.
Class of 1975
Gary S. Bernstein, P.A.
Class of 1991
Alan L. Billian PA
Class of 2012
JM Blattner, LLC
Class of 2016
Davis, Agnor, Rapaport & Skalny
Class of 1968
Bours & Lucero, LLC
Class of 2007
Gormley Jarashow Bowman, LLC
Class of 1982
Clifford Debelius Boynton & Hyatt Chtd.
Class of 1978
Attorney at Law
Class of 1985
Brown & Barron, LLC
Class of 1995
Duane Morris LLP
Class of 1986
Joseph Greenwald & Laake, P.A.
Class of 2008
Potter Burnett Law, LLC
Class of 2010
Potter Burnett Law, LLC
Class of 2009
The Law Office of Laura E. Burrows, LLC
Class of 1981
Law Offices of Kathleen Cahill LLC


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'Raindrops on roses and whiskers on kittens
Bright copper kettles and warm woollen mittens
These are a few of my favourite things'
Here's a few things I've been loving recently.
Firstly, Rimmel Lasting Finish 25 hour foundation. I love this foundation, I would describe it as medium to full coverage with a satin finish. It definitely wouldn't last 25 hours but it does a good job at staying in place for most of the day, which to be honest is all you can really ask from a foundation. I use the lightest shade (010 Light Porcelain) and it's a perfect match for my skin tone and that's quite a rarity because I'm a pasty gal!
Recently I've been getting into wearing liquid liner, I love Seventeen's Tattoo Me liner. I mean I will say that it's an absolute pain in the arse to get off but it doesn't smudge, goes on opaque and doesn't flake at all. The little brush also means you can be quite precise with application, which is good when you're in a rush and trying to even up your wings!
Benetint is great as a natural looking blush on lighter skin tones, all you really need on each cheek is one quick swipe of the brush and then just blend it in with your fingers. Also one little bottle of it will last ages, this is good because at the moment it's the only blusher I use and I really don't want it to run out!
How could I not mention the Naked palette? This is hand-on-heart is my holy grail product. The colours are so versatile, you can do a neutral daytime look to a full on night out smokey eye in just a few shades. At the moment I'm really loving Sin all over the eye, Naked in the crease and then Virgin on my browbone. If you want to see the shades closer up then check out my post devoted to this palette here.
I'm also loving Benefit Roller Lash, I won't bore you by talking about it in this post if you have already seen my last post, if you didn't see it then here it is! 🙂
And last but not least is my current favourite lipstick, MAC Persistance. Normally it's not a colour I would go for as it's a nudey-brown but as a birthday present my mum and sister paid for a make up lesson at MAC and the woman who did it used this lipstick. It looks quite good with a gold shimmery eye but really I think you could wear it with anything. I also like that fact that it's matte, it gives a slightly more sophisticated look and it stays on even after eating and drinking which is always a bonus!
Olivia x


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O'Hare International Airport – Chicago – ORDVisit ORD website – Airport Phone: 1.800.832 6352
Midway International Airport – Chicago – MDWVisit MDW website – Airport Phone: 1.800.832.6352
Click here for general travel & driving information courtesy of Embassy Suites.
Please note: The hotel does not provide shuttle service.
Click here for the Chicago Transit Authority Train Schedules and information from Chicago O'Hare and Midway Airports.
To enter your Hilton Honors #, select the "Add special request" at the bottom of the Guest Information Page.
Embassy Suites by HiltonLombard / Oak Brook
707 Butterfield Rd.Lombard, IL 60148


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Ways to Give
Donate Now!
There are a number of ways to you can support Healthy Mothers, Healthy Babies Coalition of Broward County and our mission to save babies lives and improve the health of our community.
Please call us at 954-765-0550 or contact our Special Events and Volunteer Coordinator,
Anarid Adriano at firstname.lastname@example.com to learn more.


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Husband: Eli Barnum BENNETT 1
Born: 24 JUN 1792 at: Fairfield, Fairfield, Connecticut Married: at: Died: 5 SEP 1883 at: 1 Father:Stephen BENNETT Mother:Lucy BARNUM Other Spouses: NOTES
Wife: Auah EVERSLEY 1
Born: at: Died: 6 MAR 1883 at: Father: Mother: Other Spouses:
SOURCES 1) Research by Day, Russell Bennett "Ben"
A Research Guide to the Genealogy of the Barnum/Barnam/Barnham Family Worldwide
AncientFaces - Family History Photos
©1998-2011. The format of this website and all original statements and narrative included on it are copyrighted and all rights are reserved. Factual information may be freely quoted for use in private genealogical research when accompanied by a full source citation, including the date of acquisition. Click here to view the format of a citation for an Internet resource. The publication of large extracts from this site in any form requires prior written consent.


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We live close to a dual carriageway. Traffic noise was not an issue until the local Council decided to re-surface both carriageways and install transverse yellow marking bars on the approach to a nearby roundabout. Noise and vibration levels increased dramatically. The local authority attempted to remove them but remnants still remain and still cause some vibration to some properties in our street. However the greatest problem is now the traffic noise. We have measured an increase in noise of up to 10 decibels where the old road surface joins the new surface. The Environmental Health Department advised that it was a Highways matter. I am in contact with our Local Councillors but progress is very slow.
Ultimately, the environmental health department can't really take action against the Council (unless they are based in the district/borough council and the highways are taken care of by the County council or another authority – such as Transport for London). However, you could try to argue that they still have a duty to investigate (even if, ultimately, they take no enforcement action) and that, as it affects wellbeing, it may be prejudicial to health.
Traffic noise is excluded (largely) from the statutory nuisance regime. Although, in this case, you might suggest that the noise is associated with a defect with the land rather than noise emitted from the vehicles themselves; that is another argument for the authority to get involved. My advice is to start the Council complaints procedure right away (as this in itself can be laboured). Keep communicating in the meantime. Ask neighbours to do the same. If you have no luck from them then you could consider taking your own action (via the courts).


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Latest Posts
NSX – Installation Guide (high level overview)
Kenny Ahnemann, 2 years ago 0 7 min read
vSphere Beta – Sign up now!
Kenny Ahnemann, 2 years ago 0 1 min read
About the Author
Kenny Ahnemann has over 10 years of experience with VMware in corporate production environments and 20 years in the IT industry. He has consulted on projects for global corporations, healthcare facilities, Las Vegas casinos, NASA, and others he's not allowed to mention. He has a passion for what he does and believes in helping others on their journey through the always changing IT landscape.


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You can call us on 01793 401830, email us at email@example.com
or visit our shop in person at 1 Spring Close, Swindon, SN1 2BF.
The Sandwich people is a social enterprise run by the Salvation Army at Booth House (1 Spring Close, Swindon, SN1 2BF, Tel. 01793 401834)
The Salvation Army is a Christian Church & registered charity. Registered charity numbers: 214779, 215174, in Scotland SC009359, SC037691, and in Ireland CHY6399
Website Designed, Developed & Managed by Silent-G Design - All Content © 2015
Come and be social with us


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Scarlet Ibis by Barbara Haviland | Barbara Haviland – Blog
|© Barbara Haviland
I found this Scarlet Ibis at Moody Gardens in Galveston a few years ago. My Grandsons liked him and I took several photos. I have finally painted him on a 20″x10″ canvas. I really like how he came out. He is available for purchase on my web site under Animals or you can email me.


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Wipeout of the Wireless Weenies
And Other Warped and Creepy TalesWeenies Stories
David Lubar
On Sale: 04/22/2014
ISBN: 9781429946629176 Pages, Ages 9-12
Welcome to the Weenie Zone! Wipeout of the Wireless Weenies is the seventh collection of scary short stories for middle grade readers by acclaimed author David Lubar.
A boy and his family narrowly escape a zombie apocalypse—or do they? A girl neglects her cat's litter box and finds herself in a heap of trouble. And a group of clicking, scrolling, tapping wireless weenies find themselves on the edge of disaster.
Here are thirty-three hilarious and harrowing stories that will scare you, make you laugh, or see the world in a whole new way. Find out where the author got the idea for each story at the end of the book.
Don't be a weenie. Read these stories. If you dare!
At the Publisher's request, this title is being sold without Digital Rights Management Software (DRM) applied.
Kansas State Reading Circle, Junior Library Guild Selection
"Zombies!" Dad screamed, pointing out the living room window.
"Where?" Fear and curiosity fought a short battle in my brain. Curiosity won. I rushed over to see for myself.
"No! It's too...
Praise for Wipeout of the Wireless Weenies
"Lubar's sixth Weenies short story collection offers plenty to amuse, spook, and entertain. With supernatural events and creatures, the short and super-short stories are fun, fast-paced reading for Weenies fans and new readers. As usual each story's genesis is appended." —Booklist on Beware the Ninja Weenies
"Whoever thinks the short story is dead, or that kids don't like short stories, hasn't talked to any real live kids and hasn't read the latest in this popular series." —School Library Journal on Attack of the Vampire Weenies
"Bullies and others get their due in this hilarious new collection of 35 warped and creepy tales by the master of the 'weenie' story." —Buffalo News on The Battle of the Red Hot Pepper Weenies
"This book will talk itself right off the shelves, and reluctant readers will devour it." —School Library Journal on The Curse of the Campfire Weenies
"Another cool collection. This would be perfect to read around a campfire--or at any sleepover. They are creepy, but also hilarious." —Detroit Free Press on The Curse of the Campfire Weenies
"Lubar strikes again. Another winning round-up." —Booklist on Invasion of the Road Weenies-


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Identify Upper Gastrointestinal Problems with Upper Endoscopy
An upper endoscopy is a procedure our board certified physicians use to examine and diagnose problems in the upper GI tract – the esophagus, stomach and duodenum (the first portion of the small intestine). Performed with an endoscope, a thin, flexible instrument, the procedure allows for the evaluation of persistent upper abdominal pain, heartburn, nausea, vomiting, or difficulty swallowing. An upper GI endoscopy is also the best method for finding the source of bleeding from the upper gastrointestinal tract, and detecting inflammation, ulcers, and tumors of the esophagus, stomach and duodenum.
The Power of Upper Endoscopy
Upper endoscopy lets us examine the lining of the upper part of your gastrointestinal tract, which includes the esophagus, stomach and duodenum (first portion of the small intestine). We will use a thin, flexible tube called an endoscope, which has its own lens and light source, and will view the images on a video monitor. You might hear us refer to upper endoscopy as upper GI endoscopy, or esophagogastroduodenoscopy (EGD).
Why is Upper Endoscopy Done?
Upper endoscopy helps us evaluate symptoms of persistent upper abdominal pain, heartburn, nausea, vomiting or difficulty swallowing. It's the best test for finding the cause of bleeding from the upper gastrointestinal tract. Upper endoscopy is more accurate than X-ray films for detecting inflammation, ulcers, and tumors of the esophagus, stomach, and duodenum.
We might use upper endoscopy to obtain a biopsy (small tissue samples). Remember, biopsies are taken for many reasons, and we might take one even if we do not suspect cancer. For example, we might use a biopsy to test for Helicobacter pylori, a bacteria that causes ulcers.
Upper endoscopy is also used to treat conditions of the upper gastrointestinal tract. We can pass instruments through the endoscope to directly treat many abnormalities with little or no discomfort. For example, we might stretch a narrowed area, remove polyps (usually benign growths) or treat bleeding.
How Should I Prepare for the Procedure?
An empty stomach allows for the best and safest examination, so you should have nothing to eat the on the day of the procedure and nothing to drink, including water, for approximately six hours before the examination. We will tell you when to start fasting.
Tell us in advance about any medications you take, especially blood thinners; you might need to adjust your usual dose, or temporarily discontinue them, for the examination. Discuss any allergies to medications as well as medical conditions, such as heart or lung disease with us.
Also, alert us if you require antibiotics prior to undergoing dental procedures, because you might need antibiotics prior to an upper endoscopy as well.
What Can I Expect During Upper Endoscopy?
First, you will meet our anesthesiology team who will administer the anesthesia during the procedure. You will then lie on your side, and we will pass the endoscope through your mouth and into the esophagus, stomach, and duodenum. The endoscope doesn't interfere with your breathing. You will fall asleep before the procedure starts, once the anesthesia is administered.
What Happens After Upper Endoscopy?
You will be monitored until most of the effects of the medication have worn off. Your throat might be a little sore, and you might feel bloated because of the air introduced into your stomach during the test. You will be able to eat after you leave unless we instruct you otherwise.
We generally can tell you your test results on the day of the procedure; however, the results of some tests might take several days.
You won't be allowed to drive after the procedure even though you might not feel tired. You should arrange for someone to accompany you home because the anesthesia might affect your judgment and reflexes for the rest of the day.
What are the Possible Complications of Upper Endoscopy?
Bleeding can occur at a biopsy site or where a polyp was removed, but it's usually minimal and rarely requires follow-up. Other potential risks include a reaction to the anesthesia used during the procedure, pneumonia, and perforation (a tear in the gastrointestinal tract lining). It's important to recognize early signs of possible complications. If, after the test, you have a fever, trouble swallowing, cough, shortness of breath, or increasing throat, chest or abdominal pain, contact your doctor immediately.
For more information about upper endoscopy, visit Long Island Digestive Disease Consultants.
Upper endoscopy may be used to obtain a small tissue sample to determine whether any tissue abnormal in appearance is benign or malignant."


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Special Feature
- The SDF's Activities in the Great East Japan Earthquake
- Minister of Defense Expresses His Appreciation for the Support Provided by Each Country
Meeting with Foreign Leaders
- First Japan–U.S. Security Consultative Committee (SCC) "2+2" Ministerial Meeting in Four Years
- Attendance at the Asia Security Summit by Defense Minister Kitazawa and Bilateral Talks Among the Respective Defense Ministers
- Activities of female SDF personnel in international peace cooperation activities in Timor-Leste
- Anti-piracy measures off the Coast of Somalia and in the Gulf of Aden
- Maritime Self-Defense Force Training Squadron
- Entrance Ceremony for New Class- I and II Recruits for FY2011
- Introduction of New Equipment
- Circular Flying Object
Activities of female SDF personnel in international peace cooperation activities in
The Ministry of Defense and the SDF have currently dispatched Lieutenant Colonel Kurita and Captain Kawatani to the United Nations Integrated Mission in Timor-Leste (UNMIT) as military liaison officers. Lieutenant Colonel Kurita is the first SDF female personnel to be dispatched on an individual basis to a United Nations mission. In Timor-Leste, she visits border observation points and villages in her district of jurisdiction to gather information on the security situation and other information while making use of her unique perspectives as a woman.
The Ministry of Defense and the SDF recognized to be greatly significant to dispatch female personnel in terms of meeting the United Nations' needs of utilizing the female perspectives in missions or promoting gender equality , and thus continue to actively dispatch female personnel to international peace cooperation activities.
Anti-piracy measures off the Coast of Somalia and in the Gulf of Aden
Japanese facility for counter-piracy mission in Djibouti
On June 1, Japanese facility for counter-piracy was set up in the Republic of Djibouti in the east of Africa. The setting up of this facility would facilitate the stable operation of P3C patrol aircraft engaged in surveillance activities in the Gulf of Aden.
The new facility is situated north of the Djibouti-Ambouli International Airport, and comprises commanding headquarters, personnel quarters, hangars for the maintenance of P3C aircraft, sports gymnasiums for personnel, and other facilities. An estimated total of 4.7 billion yen has been invested into the construction of the facility. Previously, the squadrons had shared the use of U.S. Forces air bases. However, on top of the fact that the runway for patrol aircraft had been far away, the lack of hangars for the maintenance of aircraft had meant that personnel sometimes had to work on the aircraft under daytime temperatures exceeding 50 degrees Celcius. In contrast, the new facility is not only located close to the runway; it is also equipped with hangars for aircraft maintenance. In addition, living conditions have improved significantly for personnel–from receiving food supplies from the U.S. Forces previously, to having its own dining hall and medical office at the new base.
Overview of anti-piracy operations (March to June)
Escort operations conducted by surface units off the coast of Somalia and in the Gulf of Aden
Operational Activity from March to June 2011
- Number of escort operations: 34 (The 225th to the 258th escort operations) (The 184th to the 217th escort operations under Japan's Anti-Piracy Measures Law)
- Number of escorted vessels: 303 (Accumulated total of 1,858 since the commencement of escort operations based on the Anti-Piracy Measures Law)
Japanese - registered ships: 2 (Accumulated total of 12)
Foreign ships operated by Japanese shipping companies: 57 (Accumulated total of 424) (Out of the 57 ships, Japanese citizens were on board 2 ships)
Foreign ships other than the above: 244 (Accumulated total of 1,422) (Out of 244 foreign ships, Japanese citizens were on board 2 ships)
- *The above record does not include the 1st-41st escort operations performed as Maritime Security Activities, which were performed before the establishment of the Anti-Piracy Measures Law.
- *Of those listed above, 235 ships (235 other foreign ships) were considered not to be within the context of escort operations for Maritime Security Activities.
P-3C patrol operations off the coast of Somalia and in the Gulf of Aden
On April 22, the Sixth Air Unit (commanded by Captain Masaaki Moritake and consisting of approx. 180 personnel), which is conducting surveillance flights from Djibouti in East Africa, flew its 450th mission, starting with when operations started with the First Air Unit.
Operational Activity from March to June
- Number of flights: 70 (Accumulated total of 488)
- Flight hours: Approximately 570 (Accumulated total of approximately 3,790 hours)
- Number of commercial ships confirmed: Approximately 5,740 (Accumulated total of approximately 36,370)
- Number of times information was provided to escort ships, foreign vessels, and civilian commercial ships: Approximately 670 (Accumulated total of approximately 4,450)
One example of the MSDF's information provision activities was on April 12 when it received information that a commercial ship was under attack by an unidentified ship. A German naval P-3C had been monitoring the unidentified ship, however, due to engine trouble an MSDF P-3C that was conducting surveillance operations in the Gulf of Aden at the time took over for the German aircraft and continued to monitor the unidentified ship. DD Sazanami, an MSDF destroyer in the vicinity, dispatched one of its helicopters to confirm the situation. The helicopter detected the unidentified ship (seven passengers and large numbers of ladders and fuel tanks onboard) and visually confirmed that ladders had been disposed of in the sea. The unidentified ship turned around and distanced itself from the commercial ship, and the DD Sazanami helicopter turned over response to the Turkish Navy.
In addition, on June 15 a P-3C conducting surveillance flights discovered a suspicious looking skiff (one ladder, many polyethylene tanks, and no fishing equipment). The P-3C notified a nearby U.S. vessel. Upon reconfirmation, the P-3C discovered that the skiff had five people aboard and that the ladders were now missing. The P-3C continued to confirm the situation based on a request received from the U.S. vessel, which had now arrived at the scene, then turned over response to the U.S. vessel and resumed its warning and surveillance mission.
Maritime Self-Defense Force Training Squadron
The FY2011 Training Squadron of the Maritime Self-Defense Force departed from Tokyo on May 24 for a fivemonth voyage to North, Central, and South America.
The Squadron, commanded by Rear Admiral Umio Otsuka, comprised training vessels "Kashima" and "Asagiri," as well as destroyer "Mineyuki." A crew of about 730 personnel , including about 180 who have completed the Basic Officer ' s Course, took up the challenge of the 55,000- kilometer voyage.
After it departed from Tokyo, the Squadron first visited Anchorage in the United States. It then dispersed and docked separately in Seattle and Vancouver, followed by San Francisco and San Diego. After meeting at sea again, the Squadron sailed through the Panama Canal and followed the Caribbean Sea route through Veracruz (Mexico), Tampa and Norfolk (United States), Halifax (Canada), Valparaiso (Chile), Callao (Peru), and Mansaniyo (Mexico), and docked at Pearl Harbor. The Squadron is scheduled to return to Harumi in Tokyo on 27 October.
Entrance Ceremony for New Class- I and II Recruits for FY2011
On 1 April, a combined entrance ceremony for 123 new Class- I and II recruits joining the Ministry in FY2011 was held at the Ministry of Defense. The ceremony was attended by Defense Minister Kitazawa, as well as senior officials including Parliamentary Senior Vice-Defense Minister Ogawa, Parliamentary Vice-Defense Ministers Matsumoto and Hirota, Administrative Vice-Defense Minister Nakae, and Director General of the Minister's Secretariat Kanazawa.
The ceremony commenced with a moment of silence for the victims of the Great East Japan Earthquake, followed by a speech from the Defense Minister providing advice and guidance to the new recruits. Thereafter, the recruits were presented with their letters of appointment and sworn-in.
In his speech to the new recruits, Defense Minister Kitazawa said, "As the Government works as one to deal with an earthquake of such unprecedented scale, the Ministry of Defense and Self-Defense Forces have gained a heightened awareness of their role as the last stronghold protecting the citizens. As such, your predecessors are working hard in the undertaking of various activities. For all of you who have become a part of the Ministry of Defense, I hope that you will carry out your duties with a sense of mission in order to meet the expectations of the citizens. This is the first year in which we take steps to realize the goals laid out in the new National Defense Program Guidelines, and you are the ones who will become the core of a group of outstanding personnel armed with great knowledge and insight. You have joined the Ministry at a time when the true value of the Ministry of Defense and the Self-Defense Forces is being questioned, and I hope that you will treasure what you are feeling now."
After the recruits had received their letters of appointment, Rie Endo represented the new recruits in taking the oath. Following that, Administrative Vice-Defense Minister Nakae gave the new recruits a word of encouragement, saying, "You have all joined the Ministry of Defense just as the Ministry of Defense and Self-Defense Forces are working as one in response to this unprecedented situation. You will be able to learn much by closely observing your predecessors who battle against this national crisis."
Introduction of New Equipment
GSDF Training Helicopter TH-480B
On February 25, the TH-480B (manufactured by US company Enstrom Helicopter Corporation) that will serve as the training helicopter for the GSDF was delivered to the Ministry of Defense, and a delivery ceremony was held at the JAMCO Corporation's maintenance facility in Sendai, Miyagi Prefecture.
On February 27, the first unit was ferried to Camp Akeno by a three-person crew led by Major Hiromichi Irifune. The next day, on February 28, an event was held to commemorate the receipt of the helicopter by about 300 SDF personnel under Major General Yuichi Fukumori, Principal of the Aviation School and Commander of Camp Akeno.
30 units of the TH-480B, the next-generation training helicopter that holds up to four people, are scheduled to be introduced into Camp Kita-Utunomiya in the near future.
Helicopter Destroyer "Ise"
On March 16, the helicopter destroyer "Ise" projected in 2006 was delivered to MDSF and the MSDF flag ceremony was held at the IHI Marine United Yokohama Shipyard in Yokohama City.
"Ise" is the second destroyer of "Hyuga" class that is the largest class with 13,950 tons at standard. It has the flight deck with enough length to arrange 4 landing spots for helicopters of GSDF, ASDF, Japan Coast Guard, police,fire department etc. other than MSDF. Thus it is possible to respond rapidly and flexibly to disasters and emergencies.
At the ceremony, the MSDF Chief of Staff Sugimoto handed over the Self-Defense Fleet flag to the first Commanding Officer, Captain Ryoichi Hoshiyama, and the destroyer was commissioned. It was deployed to the 4th MSDF Escort Flotilla at Kure.
Next-generation transport aircraft XC-2 (second test aircraft)
Forty years have passed since the existing transport aircraft C-1 owned by the ASDF commenced operation. With a view to replacing this aircraft in order to enhance military transportation capability and participate actively in peacekeeping activities, a budget for has been set aside for the introduction of two next-generation transport aircraft XC-2 in the budget for this fiscal year. This aircraft is the largest transport aircraft ever developed by Japan, and has a transportation capacity of 30 tons–three times that of the C-1. It also has a flight range of 6,500km when fully loadedapproximately four times that of the C-1. It is scheduled to be deployed to Miho Air Base in future. For now, the Ministry of Defense's Technical Research and Development Institute, in cooperation with the ASDF, is conducting test flights.
Circular Flying Object
The world's first circular flying object, which garnered much attention at the Defense Technology Symposium 2010 held in Tokyo in November 2010 as well a s at other events, was invented by Fumiyuki Sato at the Technical Research and Development Institute's Advanced Defense Technology Center. He has also put much effort into conducting research and development on the flying object. The sphere, with a diameter of 42cm, is able to hover, fly horizontally, and make transitional flights. It is also able to fly at low altitudes in the city, forests, or indoors, and can move in an agile manner, including tumbling forward on the ground. The flying sphere is able to approach close to the target that it is observing and capture detailed images of the target through the small-sized camera that has been built into the sphere. As such, there are also high expectations for the active utility of the object in areas such as disaster relief and counterterrorism activities.


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We go for a lovely muddy Boxing Day walk, I get home and my bed is occupied. No room at the inn – now where have I heard that story before?
Absolutely no shame.
We go for a lovely muddy Boxing Day walk, I get home and my bed is occupied. No room at the inn – now where have I heard that story before?
Absolutely no shame.
Snuggling up to my mum last night then realised what was on her knee – Marley Cat! I thought I was a safe distance but then I felt a sensation on my shoulder. He touched me!
Look at the fear in my eyes. Too close for comfort!
Woe, woe and thrice woe! That cheeky Marley has not given up possession of my bed, MY bed. Not happy having it all to himself for the weekend, he's still in residence now I'm home. I had a brief nap in it after the beach but I went to see what my mum was doing in the kitchen (making bread, marzipanning a Christmas cake, no snacks for dogs…) and when I come back – there he is, IN MY BED.
My mum does not see the seriousness of it and stands there taking photos and sniggering instead of helping me take back ownership. Says I'm bigger than him and could either shift him or get in beside. Cuddle up with Marley? Me? I'm frit!
Suspicious goings-on back home while I'm at bootcamp this weekend. I hear that Marley has taken over my bed, claiming possession is nine-tenths of the paw! Mum has warned him I'll be back tomorrow and will be most upset if I'm homeless.
So, get out of my bed, Marley Cat! You have the free run of house; are allowed on chairs, sofas and human beds so shift your silvery butt!
Look at me, look at me – I got a waxed coat! Only the poshest dogs get a waxed rain mac. It was pelting down this morning so we got to try it for the first time. My mum thinks she got the wrong size because it doesn't fully cover my bum, and the collar is a bit of an apology, but it really kept the rain off my back so we were both happy.


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Illumination of the Virtues of the Family of the Most Favored Prophet
Nūr al-ibṣār fi manāqib Āl bayt al-Nabī al-mukhtār (Illumination of the virtues of the family of the Most Favored Prophet) is a collection of biographies of major personalities in Islamic history. It begins with narration of critical incidents in the life of the Prophet Muhammad, followed by biographies of Ahl al-Bayt (the Prophet's family), the four earliest caliphs, and other significant figures of Islamic history, such as the founders of the four canonical legal traditions and the major Sufi orders. The author, Mu'min ibn Ḥasan Mu'min al-Shablanji (circa 1836−circa 1890), was born and educated in Egypt and was well-known as a teacher, Sunni judge, and Sufi. Although the date of printing is not given, a note mentions that the author completed the book on September 9, 1873. The printer notes that the published text was authenticated against the author's manuscript. On the margins is printed a second work, Is'af al-Raghibin (Assistance for the desirous), a similar biographical compilation by the 18th-century Egyptian scholar Muhammad al-Sabban. Nur al-Absar has received much attention from Shia scholars. Shia Muslims emphasize their unique devotion to the descendants of the Prophet. Books treating the Prophet's grandson, Husayn, and other relatives tend to be carefully scrutinized by Shia readers for what they see as possible misinterpretations. In general, Shia scholars view Nur al-Absar and Is'af al-Raghibin favorably for their treatment of such topics as Husayn's death at the Battle of Karbala (680) and the hadith entitled Ghadir Khamm which, according the Shiite interpretation, established the succession of leadership after the Prophet's death.
Date Created
Subject Date
Publication Information
al-Maktabah al-Tijariyah al-Kubra Press, Cairo
Title in Original Language
نور الأبصار في مناقب آل بيت النبي المختار
Additional Subjects
Type of Item
Physical Description
248 pages ; 27 centimeters
- Hassan al-Amin, Islamic Shi'ite Encyclopaedia (Beirut: Slim Press, 1968−73).
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Last updated: January 16, 2015