1 Laboratory of Virology, Rheumatology and Immunology Research Center, Room T1-49, CHUL Research Center and Faculty of Medicine, Laval University, 2705 Laurier Blvd, Sainte-Foy, Quebec, Canada G1V 4G2
2 Laboratory of Viral Immunology, Rheumatology and Immunology Research Center, Room T1-49, CHUL Research Center and Faculty of Medicine, Laval University, 2705 Laurier Blvd, Sainte-Foy, Quebec, Canada G1V 4G2
Correspondence
Louis Flamand
Louis.Flamand{at}crchul.ulaval.ca
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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SUMOylation assays in infected cells.
Molt-3 cells were infected with the Z29 strain of HHV-6 (m.o.i. of 0·1) for 72 h. Infected cells (1x107) were pelleted, lysed and sonicated in a 1 : 3 dilution of buffer I and II containing 5 mM N-ethylmaleimide (NEM), as described by Desterro et al. (1998) and Wu et al. (1993)
. Clarified supernatants were incubated overnight with anti-IE1 antibodies (Gravel et al., 2002
) and protein ASepharose beads, followed by three washes with lysis buffer. Beads were resuspended in Laemmli buffer and boiled for 5 min. Immunoprecipitated proteins were electrophoresed and Western blotting was carried out using anti-IE1 or anti-SUMO-1 antibodies (Zymed Laboratories).
Cloning procedures.
A GSTSUMO-1GG (processed proteins terminating at the diglycine and conjugation-ready) construct was generated by subcloning of the SUMO-1 cDNA from pcDNA3/6xHIS-SUMO-1 vector (Desterro et al., 1997) (obtained from R. T. Hay, Centre for Biomolecular Sciences, University of St Andrews, Fife, UK) into the pGEX-2T vector (Amersham Biosciences). The GSTSUMO-2GG and GSTSUMO-3GG fusion proteins were generated by in-frame ligation of RT-PCR products amplified from HeLa cell RNA into the pGEX-5X3 vector, using primers as described by Tatham et al. (2001)
. The same SUMO-2 and SUMO-3 PCR products were ligated in frame with the pCMV2N3T eukaryotic haemagglutinin (HA)-tagged vector (obtained from Didier Trouche, Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 Centre National de la Recherche Scientifique, Toulouse, France). SENP1 cDNA was obtained following RT-PCR amplification of HeLa cell RNA using the primers 5'-ATGGATGATATTGCTGATAGGATG-3' and 5'-GCGAATTCTCACAAGAGTTTTCGGTGGAGG-3'. The 1·9 kb amplicon was treated with kinase, digested with EcoRI (underlined in the primer sequence) and cloned in frame in the SmaI/EcoRI site of the pCMV2N3T vector. The GSTSENP1 fusion protein was generated by subcloning a blunted SalI/EcoRI fragment from the pCMV2N3T-SENP1 vector into the SmaI site of the pGEX-2T vector. A GSThUBC9 protein was constructed by in-frame subcloning of the hUBC9 cDNA into the pGEX-2T vector. The GST, GSThUBC9 and GSTSENP1 and various GSTSUMO proteins were produced and purified according to standard procedures. Recombinant protein purity was estimated to be >95 % as determined by gel electrophoresis and Coomassie blue staining. Protein concentration was determined using the Bradford reagent (Bio-Rad).
Mutagenesis.
Conservative mutations from K to R in the SUMO consensus sites were introduced using the Quick-change Site-directed Mutagenesis kit (Stratagene). Point mutations were confirmed by sequencing.
Transfection of 293T cells with pBK-HHV-6 IE1 expression vectors.
293T cells (3x105 cells per well) were seeded the day before transfection into a six-well plate. Cells were transfected with 2 µg pBK-HHV-6 IE1B expression vectors or pBK control vector (Stratagene) using the ExGen transfection reagent (MBI Fermentas). In some experiments, HisSUMO-1, HASUMO-2 and HASUMO-3 expression vectors were included in the transfection mixture. Forty-eight hours after transfection, cells were processed for immunoprecipitation (IP)/Western blot analysis as described previously (Gravel et al., 2002). For co-localization studies, acetone-fixed cells were first reacted with an anti-promyelocytic leukaemia protein (PML) monoclonal antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Slides were washed three times for 5 min in PBS and then incubated with Alexa 568-labelled goat anti-mouse IgG antibodies for 1 h at room temperature. After three PBS washes, samples were incubated with Alexa 488-labelled rabbit anti-IE1 IgG for 1 h. Slides were washed, mounted and examined as described previously (Gravel et al., 2002). For SUMO-1 and IE1 co-localization studies, cells were first reacted with Alexa 568-labelled rabbit anti-SUMO-1 IgG antibodies, followed by the Alexa 488-labelled rabbit anti-IE1 IgG antibodies.
In vitro SUMOylation/deSUMOylation assay.
WT and K mutants of IE1B were in vitro transcribed/translated in the presence of [35S]methionine using rabbit reticulocyte lysates (Promega). Five µl [35S]methionine-labelled in vitro-translated IE1 proteins were combined with 1 µg GSTSUMO-1, -2 or -3, 1 µg GSThUBC9 or 1 µg GST in a 20 µl reaction containing 50 mM Tris/HCl, pH 7·6, 2 mM ATP, 0·5 mM DTT, 5 mM MgCl2, 19·4 µg creatine phosphokinase ml1, 15 mM phosphocreatine and 1 U inorganic pyrophosphatase ml1 and incubated at 37 °C for 2 h. In some instances, the reaction mixtures were supplemented with 1µg GST (control) or GSTSENP1 for an extra hour before the reactions were stopped by the addition of SDS sample buffer containing mercaptoethanol. The proteins were fractionated by electrophoresis on 6 % SDS-polyacrylamide gels. The gels were dried and exposed to imaging plates (Fuji Medical Systems).
Reporter gene assay.
Molt-3 cells (1x107) were electroporated (250 V, 960 µF) with 4 µg HIV-1 LTRluc reporter construct along with 4 µg of each effector plasmid. DNA levels were kept constant at 12 µg by the addition of control pBK vector. Forty-eight hours post-transfection, cells were pelleted and resuspended in 0·25 ml cell lysis buffer (Promega). Twenty µl aliquots were tested for luciferase activity according to the manufacturer's technical guidelines (Promega) using an MLX luminometer (Dynex Technologies).
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RESULTS AND DISCUSSION |
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SUMO-1, SUMO-2 and SUMO-3 are efficiently conjugated to IE1
Three different SUMOs (1, 2 and 3) have been described so far (Kamitani et al., 1998; Lapenta et al., 1997
; Mahajan et al., 1997
; Matunis et al., 1996
; Saitoh & Hinchey, 2000
). SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 are highly homologous to one another (95 % identity) and differ from SUMO-1 by 50 %. Previous studies have indicated that SUMO-2 and SUMO-3 can be conjugated to proteins via mechanisms similar to that of SUMO-1 (Saitoh & Hinchey, 2000
). To study the conjugation of the SUMO variants to IE1, we co-transfected 293T cells with IE1 and SUMO-1 (His-tagged) or HASUMO-2 or HASUMO-3 expression vectors and detected the conjugated forms of IE1 by IE1 IP and Western blotting using anti-SUMO-1 or anti-HA antibodies. The first notable observation was that in the absence of SUMO-1 overexpression, a single IE1 protein of 170 kDa was detected following IP and Western blotting (Fig. 2
A, lane 2, top panel), suggesting that SUMOylated IE1s are present at much lower levels than unconjugated IE1 or that SUMO peptidases are very active and only partly inhibited by NEM in our lysis buffer. When SUMO-1 was overexpressed, the overall levels of IE1 were increased (Fig. 2A
, lane 3, top panel), as also shown in Fig. 1(C)
and reported previously (Stanton et al., 2002
). In addition, slower-migrating IE1 species, corresponding to SUMO-1-conjugated IE1s were detected (Fig. 2A
, lane 3, middle panel). HASUMO-2 (Fig. 2A
, lane 4) and HA-SUMO-3 (Fig. 2A
, lane 5) overexpression also led to increased levels of IE1, in a manner similar to that of SUMO-1. When the immunoprecipitated IE1s were analysed for SUMO-2 or -3 conjugation by reprobing the blot with anti-HA antibodies, one major immunoreactive IE1 protein of approximately 210 kDa (arrowhead) was detected (Fig. 2A
, lanes 4 and 5, bottom panel). Higher and lower molecular mass IE1 species containing SUMO-2 and -3 were also detected following film overexposure (not shown). The results presented in Fig. 2(A)
indicated that IE1 was efficiently conjugated by SUMO-1, -2 and -3. Two differences of likely biological significance between SUMO-1 and SUMO-2/3 exist. First, compared with SUMO-1, SUMO-2 and -3 are much more abundantly expressed within cells. Secondly, and possibly directly related to the first difference, there are large quantities of free SUMO-2/3 available for conjugation in contrast to the majority of SUMO-1, which is attached to target proteins (Saitoh & Hinchey, 2000
). To determine whether IE1 was preferentially SUMOylated by SUMO-1, -2 or -3, we overexpressed SUMO-1 and SUMO-2 or SUMO-1 and SUMO-3 together with IE1 in 293T cells and analysed the SUMOylation patterns of IE1. Overexpression of SUMO-1 (Fig. 2B
, lanes 35) led to the detection of a major protein of approximately 195 kDa, identified as a SUMO-1-conjugated IE1 (indicated by an asterisk), as well as several minor larger IE1SUMO-1 proteins (Fig. 2B
, middle panel). When SUMO-2 and SUMO-1 were both overexpressed (Fig. 2B
, lane 4), three major IE1 proteins of 170, 195 and 210 kDa were detected (top panel). These respectively corresponded to unconjugated, SUMO-1-conjugated (Fig. 2B
, middle panel) and SUMO-2-conjugated IE1 (Fig. 2B
, bottom panel). The 195 kDa band observed in Fig. 2(B)
(lane 4, bottom panel) corresponds to IE1 linked to a single SUMO-2 residue (asterisk). The 195 kDa band is therefore a mixture of SUMO-1 and SUMO-2 conjugated IE1. Although we could not firmly conclude that both SUMO-1 and SUMO-2 were simultaneously conjugated to IE1, co-migration of a 210 kDa SUMO-1-conjugated (Fig. 2B
, middle panel) and SUMO-2-conjugated (Fig. 2B
, bottom panel) IE1 (arrowhead) suggested that this was the case. This was not unexpected considering that SUMO-2 contains an internal SUMOylation site allowing its conjugation to SUMO proteins (Tatham et al., 2001
). Simultaneous overexpression of both SUMO-1 and -3 (Fig. 2B
, lane 5) had little effect on the SUMOylation patterns of IE1 with detection of multiple SUMO-1-conjugated IE1s (Fig. 2B
, middle panel) and a major 210 kDa SUMO-conjugated IE1 (Fig. 2B
, bottom panel, arrowhead), most likely linked to a SUMO-3/SUMO-1 dimer. Whether polySUMOylated proteins behave differently from monoSUMOylated ones requires a clearer understanding of the biological differences between SUMO-1 and SUMO-2/3. To confirm and compare the SUMOylation of IE1 by SUMOs, we generated matured GSTSUMO-1, -2 and -3 fusion proteins, as described previously (Tatham et al., 2001
). A GSThUBC9 recombinant protein was used as a source of SUMO-conjugating enzyme. WT and K mutants (K281R, K802R and DM) of IE1B were in vitro transcribed/translated and incubated with 1 µg GSTSUMO-1, -2 or -3, 1 µg GSThUBC9 or 1 µg GST in a 20 µl reaction volume. In the presence of GST or GST plus GSThUBC9, only the unmodified form of IE1 was detected (Fig. 2C
, lanes 1 and 2). Transcription/translation of an empty pBK control vector under similar conditions yielded background noise only (data not shown). The K mutants of IE1 were as efficiently transcribed/translated as WT IE1 with a single major band of 170 kDa detected. When GSTSUMO-1 was added to the reaction mixture (Fig. 2C
, lane 3), SUMOylation of WT IE1 and the K281R mutant was observed, while the K802R and DM mutants failed to undergo SUMO-1 conjugation. These results were in accordance with those presented in Fig. 1(B)
indicating that K-802 represents the target residue for SUMO-1 conjugation in transfection experiments. Similar experiments were carried out using GSTSUMO-2 and -3 proteins (Fig. 2C
, lanes 4 and 5). The results obtained indicated that WT IE1 and the K281R mutant could be efficiently modified by both SUMO-2 and SUMO-3. The K802R and DM mutants failed to undergo SUMOylation in the presence of SUMO-2 or SUMO-3 suggesting that K-802 is the only residue capable of conjugating all forms of SUMO. Interestingly, both SUMO-2 and SUMO-3 have a
KXE motif within their primary amino acid sequence, raising the possibility that multimers of SUMO can form. In fact, a recent paper has indicated that this is indeed the case (Tatham et al., 2001
). Considering that only K-802 of IE1 is a target for SUMOylation, we presumed that the high molecular mass IE1 proteins observed (>195 kDa) in Fig. 1(A)
represented IE1 proteins carrying multimers of SUMO-1, -2 and/or -3 on K-802. Post-translational protein modifications by SUMO residues are often technically difficult to study. This is partly attributable to deSUMOylating peptidase activity in cellular extracts. One recently characterized deSUMOylating enzyme is SENP1 (Gong et al., 2000
). We cloned and purified a GSTSENP1 protein to near homogeneity and its deSUMOylating activity was tested using the RanGAP1 protein. RanGAP1 was the first SUMO-modified protein to be reported (Matunis et al., 1996
) and is one of the easiest to study. In vitro-transcribed/translated RanGAP1 undergoes spontaneous SUMOylation in the presence of rabbit reticulocyte lysate. In vitro-generated RanGAP1 protein was incubated with purified GST or GSTSENP1 for 1 h at 37 °C, then electrophoresed and exposed to imaging plates. The results obtained (Fig. 2D
, upper panel) confirmed that GSTSENP1
is capable of removing SUMO residues from RanGAP1 as revealed by the disappearance of the slower-migrating RanGAP1 species (Gong et al., 2000
). We next proceeded to determine whether SENP1 could remove SUMO-1, -2 and/or -3 from IE1. In vitro SUMO-conjugated IE1 was incubated with GST or GSTSENP1 and analysed by gel electrophoresis. Our results (Fig. 2D
, lower panel) indicated that SUMO-1-conjugated IE1 (Fig. 2D
, lane 3) could be efficiently deconjugated by SENP1 (Fig. 2D
, lane 4). These results also indicated that SENP1 could cleave IE1SUMO-2 (Fig. 2D
, lanes 5 and 6) and IE1SUMO-3 (Fig. 2D
, lanes 7 and 8) residues, highlighting a potential role for SENP1 in controlling the levels of SUMO-modified proteins within cells. So far, three mammalian (SMT3IP1, SENP1 and SUSP1) and one yeast (Ulp1) SUMO peptidases have been identified (Gong et al., 2000
; Kim et al., 2000
; Li & Hochstrasser, 1999
; Nishida et al., 2000
; Suzuki et al., 1999
). Although these enzymes share some homologies within the C-terminal region, their distribution within cells differs considerably. For example, SENP1 has a diffuse nuclear distribution (Gong et al., 2000
) and SUSP1 is exclusively cytoplasmic (Kim et al., 2000
), while SMT3IP1 associates with the nucleoli (Nishida et al., 2000
). Although sharing a common property in cleaving SUMO residues from conjugated proteins, the differing cellular localization of the SUMO peptidases suggests that they act on different SUMOylated target proteins. How SUMO peptidases can modulate the course of a virus infection remains unclear, but considering that the functionality of some viral proteins, such as the HCMV IE86 (Ahn et al., 2001
; Hofmann et al., 2000
), are influenced by their SUMOylation status, these enzymes are expected to play an integral part in the infectious process.
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IE1 and IE2 transcriptional modulating activities
The function of IE1 from HHV-6 variant B remains largely elusive, with the ability to weakly transactivate heterologous promoters the only known activity of IE1 (Gravel et al., 2002). We recently cloned a full-length transcript encoding IE2, the second major protein from the IE-A locus of HHV-6 (Gravel et al., 2003
). In contrast to IE1, IE2 is a strong promiscuous transcriptional activator. Given the fact that both genes are expressed with similar kinetics (Gravel et al., 2003
), we tested the effects of IE1 on the ability of IE2 to activate several promoters, including the HIV LTR, as well as more simple promoters containing NF-
B-binding consensus sequences, nuclear factor of activated T cells or cyclic-AMP-responsive elements. Similar results were obtained using all these promoters; Fig. 4
shows the results using the HIV LTR promoter. Molt-3 T cells transfected with an IE1B expression vector promoted marginal activation (<twofold) of the HIV LTR, as previously reported (Gravel et al., 2002
). In contrast, transfection with an expression vector for HHV-6 IE2 led to an 18-fold activation in promoter activity. By combining IE1 and IE2 expression vectors, the activity increased up to almost 30-fold, suggesting that both proteins may act additively in promoting transcription. We next tested the impact of IE1 SUMOylation-deficient mutants of IE1 on transactivation capability. First, the mutants were individually tested and shown to have poor transactivation potential, similar to that of WT IE1B. Next, in combination with IE2, all IE1 mutants demonstrated levels of activation almost identical to that of WT IE1. These results indicated that IE1, together with IE2, can efficiently transactivate heterologous promoters and that this effect is independent of the SUMOylation status of IE1.
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ACKNOWLEDGEMENTS |
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Received 28 August 2003;
accepted 7 January 2004.