Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK1
Author for correspondence: Peter OHare. Fax +44 1883 714375. e-mail P.OHare{at}mcri.ac.uk
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Abstract |
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Introduction |
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NBs are also enriched with proteins that are modified at specific lysine residues by a small ubiquitin-like molecule, known as SUMO-1 [also termed Sentrin (Okura et al., 1996 ), PIC1 (Boddy et al., 1996
), or GMP-1 (Mahajan et al., 1997
; Matunis et al., 1996
)]. It has been suggested that SUMO-1 modification of PML itself may be important for recruiting PML to the NB structure (Kamitani et al., 1998b
; Müller et al., 1998
), although for a number of other NB proteins the SUMO-1 modification does not appear to be essential for localization.
ICP0 promotes the loss of PML NBs in a proteasome-dependent manner (Everett & Maul, 1994 ). ICP0 also promotes the proteasome-mediated degradation of other cellular proteins, including CENP-C, CENP-A and the regulatory subunit of the cellular DNA-dependent protein kinase (Everett et al., 1998
, 1999a
; Lomonte et al., 2001
; Parkinson et al., 1999
). The ability of ICP0 to promote targeted NB disruption and protein degradation is dependent on the conserved RING finger domain. Earlier work had established that the ICP0 RING domain is essential for transactivation, providing robust evidence that NB disruption, protein degradation and promotion of gene expression are linked (Everett et al., 1995
; ORourke & OHare, 1993
).
Emerging evidence indicates that RING finger domains may act as ubiquitin E3 ligases (for reviews see Aravind & Koonin, 2000 ; Freemont, 2000
; Tyers & Jorgensen, 2000
). ICP0 has also now been shown to function as a ubiquitin E3 ligase, although the precise mechanism has been disputed (Boutell et al., 2002
; Van Sant et al., 2001
) and direct evidence of ICP0-mediated conjugation of ubiquitin to specific cellular targets has yet to be established. However, consistent with the role of an E3 ligase function, ICP0 has been reported to induce the accumulation of conjugated ubiquitinated protein species (Everett, 2000
) and to interact with a ubiquitin-specific protease (USP7/HAUSP) that is also present within the PML NB (Everett et al., 1997
, 1999b
).
In order for PML degradation to occur during virus infection, there may be a requirement for the removal of SUMO-1 from the SUMO-1-modified protein isoforms prior to degradation of PML or other target protein(s). Recent work has identified a number of SUMO-1-specific proteases that reverse the covalent modification of SUMO-1 from the target protein (Gong et al., 2000 ; Kim et al., 2000
; Li & Hochstrasser, 1999
; Suzuki et al., 1999
), although little is understood of their regulation and activity. Some evidence of substrate specificity for SUMO-1 deconjugation has been obtained from yeast. Yeast has two proteases (Ulp1 and Ulp2) and mutants which lack either protease show the accumulation of different Smt3 (yeast homologue of SUMO-1)-conjugated species, suggesting that the enzymes may exhibit selectivity in controlling the conjugation profile (Li & Hochstrasser, 1999
, 2000
). This has not been reported for the mammalian system, nor is it known if a spatial regulation of SUMO-1 conjugation/deconjugation occurs.
In this work we examine the effects of ICP0 on the SUMO-1 modification status of the cell, with particular emphasis on the association of ICP0 with a SUMO-specific protease (SENP1). To facilitate analyses of the SUMO-1 pathway, we constructed a cell line that constitutively expresses epitope-tagged SUMO-1. Our results demonstrate that ICP0 promotes the generalized loss of SUMO-1 from the nucleus. We also demonstrate that SENP1 localizes to a number of subnuclear domains and also promotes the loss of SUMO-1 from the nucleus and from PML. Interestingly, PML remains in NB structures suggesting that removal of the SUMO-1 moiety alone is not sufficient for NB dispersal. We observe that co-expression of ICP0 with SENP1 results in the recruitment of SENP1 into ICP0 subnuclear structures, and that this occurs during early viral infection. The relevance of these results to the loss of SUMO-1 modified PML during virus infection is discussed.
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Methods |
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Construction of the SENP1 expression vector.
The 1·9 kb SENP1 ORF was amplified from a human testis cDNA library (Clontech #7414-1) using the sense (5' GAGCTCGCTAGCATGGATGATATTGCTGATA 3') and antisense (5' CTCGAGTCTAGATCACAAGAGTTTTCGGTGG 3') primers, based on published sequence (Gong et al., 2000
). The PCR product was inserted into pCR-II-Topo, then excised as an NheIScaI fragment and inserted into a vector containing an in-frame Flag-HA-tag. The Flag-Ha-SENP1 EcoRIKpnI fragment was then excised and inserted into pcDNA3 for expression of Flag and HA epitope-tagged SENP1. Sequencing of the SENP1 clone demonstrated that the clone differed from the published sequence due to the presence of an additional glutamic acid residue at aa 593. Comparison with database sequences indicated that this sequence was identical to the orf predicted by the ENSEMBL project (ID: ENSG00000079387).
Construction of stable cell lines expressing His-Myc-tagged SUMO-1.
A His-Myc-tagged SUMO-1 clone (hmSUMO-1) was constructed by inserting a 6xHis tag linker (sense: 5' AGCTTATGCATCATCATCATCATCA 3'; antisense: 5' CATGTGATGATGATGATGATGCATA 3') and a 1·1 kb NcoIXbaI Myc-tagged SUMO-1 fragment from pMLV-Myc-PIC1 (Boddy et al., 1996
) in a three-way ligation with a HindIII/XbaI-digested pcDNA3 backbone, to generate pcDNA3-Myc-SUMO-1. The His-Myc SUMO-1 fragment was then subcloned into a vector, pIRES-P, which had been digested with XhoI, blunt-ended with Klenow DNA polymerase and then digested with XbaI. pcDNA3-Myc-SUMO-1 was digested with HindIII, filled-in with Klenow DNA polymerase and further digested with XbaI. This generated a 1·1 kb fragment that was then inserted into the cleaved pIRES-P backbone to produce vector pIRES-HIS-SUMO-1. The pIRES-HIS-SUMO-1 vector was transfected into HEp2 cells and stable line clones selected using 2 µmg/ml puromycin. Following selection, 12 independent HEp2 cell colonies were isolated and analysed for His-Myc-tagged SUMO-1 expression. Immunofluorescence analysis demonstrated that 100% of cells from all the independent colonies showed approximately similar levels of nuclear Myc/SUMO-1 expression. Cells derived from a colony (HEp2-SUMO) were used in these studies. Similar Myc/SUMO-1 modification profiles of cellular proteins were observed when analysed by Western blot, with some variation in expression levels between clones (data not shown). Similar results were also obtained when generating the tagged SUMO-1-expressing stable lines independently in HeLa cells (HeLa-SUMO).
Transfections.
Transfections were performed using the calcium phosphate precipitation procedure modified by the use of BES-buffered saline (pH 7·06) as previously described (Batchelor & OHare, 1992 ). The total amount of DNA was equalized to 2 µg with pUC19 DNA.
Immunofluorescence studies.
Approximately 40 h post-transfection cells, plated on glass coverslips, were washed in PBS and fixed with ice-cold methanol. Primary antibodies, diluted in PBS10% FCS, were anti-c-Myc 9E10 (1:400, Boehringer Mannheim) for the Myc-tag; anti-GMP-1 (1:1000, Zymed) or anti-PIC1 (1:200) for SUMO-1; anti-11060 (1:1000), R191 and Rb95 (1:200) for ICP0; anti-Flag M2 (1:200, Stratagene) and anti-OctA (1:10, Santa Cruz) for the Flag-tag. A rabbit polyclonal antibody against a purified GSTPML bacterial expression product was generated by standard methods. Specificity was verified by comparing against an existing anti-PML monoclonal antibody (5E10). Secondary antibodies conjugated to fluorochromes Alexa 488 or Alexa 543 dyes (Molecular Probes) were diluted 1:200 in PBS10% FCS. After washing in PBS, cells were visualized using a Zeiss LSM 410 confocal microscope. Images for each channel were captured sequentially with 8-fold averaging. Composite illustrations, representative of numerous images gathered for each test construct and condition, were prepared using Adobe software.
Western blot analysis.
After separation by SDSPAGE, proteins were transferred to nitrocellulose membranes which were blocked with PBS0·05% Tween 20 (PBST) containing 5% dried non-fat milk. The membranes were incubated (1 h) with primary antibody in PBST5% dried milk, washed three times in PBS1% Triton X-100 and incubated for a further 1 h in PBST5% dried milk containing the appropriate horseradish peroxidase-conjugated secondary antibody. After further washing in PBS1% Triton X-100, membranes were processed with chemiluminescence detection reagents (Pierce). Primary antibodies used for immunoblot were anti-c-Myc 9E10 (1:400, Boehringer Mannheim), anti-SV5 (1:5000, kindly supplied by R. Randall, University of St Andrews) and anti-LP1 (1:4000, kindly supplied by T. Minson, University of Cambridge) and anti-GMP-1 (1:1000, Zymed).
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Results |
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Construction of stable cell lines expressing epitope-tagged SUMO-1
Nevertheless, the technical difficulties in detecting the very low levels of endogenous SUMO-1 limited ready analysis of effects of SENP1 in both immunofluorescence and Western blotting applications. To facilitate the analysis of the activity of SENP1 upon SUMO-1, we established cell lines (HEp2 or HeLa cells) maintaining expression of an epitope-tagged SUMO-1 (hmSUMO-1) using a bicistronic vector system and puromycin selection (Fig. 3a). In these cells hmSUMO-1 was present in predominantly diffuse nuclear staining (Fig. 3a
), with a subset of the protein frequently accumulating in foci that co-localized with PML (data not shown). To confirm that the introduced hmSUMO-1 acted as a substrate for conjugation into high molecular mass conjugates, we performed Western blot analysis on the parental cells or the hmSUMO-1-expressing cell lines with antibodies to SUMO-1 or an anti-Myc monoclonal antibody to specifically detect hmSUMO-1. In the control HeLa cells a major band was detected with the anti-SUMO-1 antibody (Fig. 3b
, left-hand panel) which from previous reports most likely represents Ran-GAP (Matunis et al., 1996
; Saitoh & Hinchey, 2000
). The detection of additional SUMO-1-modified species in the normal HeLa cells requires long exposures of the blot (data not shown). In the hmSUMO-1-expressing cells, numerous high molecular mass conjugates were observed and these species were also detected with the anti-Myc antibody (Fig. 3b
, right-hand panel), confirming that the hmSUMO-1 was indeed a substrate for conjugation. The majority of hmSUMO-1 within these cell lines appeared to be conjugated in high molecular mass species with comparatively minor amounts of free SUMO-1 observed, indicating that the SUMO-1 conjugation machinery was not limiting in the established lines. We noted that the band around the size of Ran-GAP1 migrated as a doublet, most likely representing modification of Ran-GAP1 with the endogenous SUMO-1 or the hmSUMO-1, which would result in a slightly slower migration.
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Effect of SENP1 expression upon PML and high molecular mass SUMO-1 conjugates
The majority of SUMO-1 in the cell is reported to be present in high molecular mass conjugates, both from previous reports (Desterro et al., 1998 ; Matunis et al., 1996
) and from analysis of our hmSUMO-1 stable cell lines (Fig. 3b
). To obtain biochemical confirmation that the SENP1 clone was active in promoting the loss of SUMO-1, the epitope-tagged SENP1 clone was co-transfected into COS cells with an hmSUMO-1 plasmid. Cells were harvested as previously described and analysed by Western blot. In the presence of SENP1 the general profile of the SUMO-1 modified species was significantly reduced (Fig. 4a
), consistent with the generalized loss of SUMO-1 from the nucleus as observed by immunofluorescence. We noted also that levels of the unconjugated SUMO-1, migrating at approx. 18 kDa (Fig. 4a
, free SUMO-1), were increased in the presence of SENP1, consistent with deconjugation from target proteins. To specifically examine the status of the modified forms of PML in the presence of SENP1, an SV5 epitope-tagged PML560 construct was transfected into COS cells, either in the presence or absence of the SENP1 construct. Expression of PML alone resulted in a major species migrating at approx. 80 kDa, with the presumptive SUMO-1 modified forms of PML migrating within the range 90200 kDa (Fig. 4b
). The expression of SENP1 in conjunction with PML abolished the majority of the modified high molecular mass forms of PML (Fig. 4b
, long arrows, bands 13), despite similar levels of expression of the unmodified PML species. Interestingly, some high molecular mass isoforms of PML were unaffected by SENP1 (Fig. 4b
, short arrows, bands 4, 5), indicating the possibility that these represent other post-translational modifications of PML (or perhaps stable dimers). In control experiments, levels of expression of a control protein (VP16), expressed from a control plasmid, were unaffected by SENP1 expression (Fig. 4b
, right-hand panel). These results, taken together, suggest that SENP1 promotes the deconjugation of SUMO-1 from PML and other high molecular mass conjugates.
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Discussion |
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Certain previous reports have suggested that SUMO-1 modification of PML is an important targeting signal for PML recruitment to the NB and for NB formation (Ishov et al., 1999 ; Kamitani et al., 1998a
; Lallemand-Breitenbach et al., 2001
; Zhong et al., 2000
), and it could therefore have been that SENP1-mediated loss of SUMO-1 from PML was sufficient to disrupt the NBs. However, we show that in the absence of ICP0, the SENP1 deconjugation of SUMO-1 from PML did not disrupt PML localization from the NBs. Our results are consistent with a more recent report showing maintenance of NBs despite the SENP1-induced loss of SUMO-1 modification on PML (Gong et al., 2000
). Moreover in unpublished work, we have shown that a mutated version of PML, which could not be modified by SUMO-1, was nevertheless recruited to NBs with similar efficiency to the parental PML species. Although the activity of PML within the NBs could certainly be affected by its conjugation status, our data imply that removal of SUMO-1 is not sufficient to disperse PML and that other functions may be required to accelerate this process.
In cells constitutively expressing the epitope-tagged version of SUMO-1, hmSUMO-1 was distributed in a diffuse nuclear pattern with some accumulation in punctate foci. Western blot analysis demonstrated that the vast majority of hmSUMO-1 was also present in high molecular mass conjugates, indicating, perhaps not unexpectedly, that the diffuse nuclear SUMO-1 pattern represented numerous conjugated species. The restriction of SUMO-1 to the nucleus is consistent with previous results indicating that SUMO-1 conjugation is either a nuclear event or coupled to entry. While there may well be shuttling of SUMO-1-modified species from the nucleus, in the equilibrium position the majority of SUMO-1-modified species appears to be in the nucleus.
Several non-mutually exclusive proposals can be envisaged relating SENP1 recruitment by ICP0 to NBs, with the loss of SUMO-1-conjugated species and of NBs. It is possible that upon SENP1 recruitment the key target for deconjugation is PML itself. Having lost SUMO-1, PML would then be degraded, with this event requiring additional distinct events (see above), possibly ubiquitin conjugation catalysed by the ICP0 RING finger (Boutell et al., 2002 ; Everett, 2000
). After the disruption of the NBs, it could then be that generalized loss of SUMO-1-conjugated species is a downstream event, a consequence of the loss of the major site, rather than SENP1 actively deconjugating multiple numerous species. It could also be, however, that with the recruitment of SENP1 to the NBs, SENP now catalyses the loss of SUMO-1 from the resident PML and likewise from numerous species which are trafficking through the NBs. In either model though, the PML NBs act as a central processing centre, regulating SUMO-1 status of substrates which dynamically traffic through the sites by conjugation/deconjugation. Clearly, it is possible that ICP0, in addition to a generalized loss of SUMO-1-modified species, may promote the loss and degradation of specific protein conjugates. Indeed, during HSV-1 infection the loss of modified PML was accelerated compared to the generalized loss of SUMO-1-modified isoforms (Everett et al., 1998
). Furthermore, analysis of the activity of other herpesvirus homologues of ICP0 on the NB protein Sp100 suggested that loss of SUMO-1 occurred first, prior to a general increase in unconjugated proteins (Parkinson & Everett, 2000
).
Whilst significant evidence has accumulated indicating that HSV-1 promotes the loss of PML NBs by promoting the degradation of PML via the proteasome-dependent pathway (Everett, 2000 ; Everett et al., 1998
), it has proved difficult to demonstrate that PML is ubiquitinated (even in the presence of ICP0). Nevertheless, PML (and other SUMO-1-modified proteins) are likely to be modified by ubiquitin conjugation. It may be that PML is co-modified by ubiquitin and SUMO-1, and that in order for PML degradation to occur SUMO-1 has to be first removed. Alternatively, SUMO-1 may have to be removed prior to ubiquitin conjugation on the same (or different lysines). SENP1-deconjugation activity could participate in either of these models. Our preliminary analysis with certain ICP0 mutants which lack the RING finger, an important component in their ubiquitin-ligase activity (Boutell et al., 2002
), indicates that they are still able to recruit SENP1 (unpublished data). Previous data showed that these mutants were unable to promote degradation of PML or loss of NBs (Everett et al., 1998
; ORourke et al., 1998
), suggesting that recruitment of SENP1 by itself is not sufficient for degradation of the deconjugated species, and that additional factors linked to the ubiquitination pathway are involved.
There is presently little information on the activity of the mammalian SUMO-specific proteases. One recent paper (Hang & Dasso, 2002 ) examined SENP2 with results that, in contrast to SENP1 (this work, see also Gong et al., 2000
), indicated that SENP2 was specifically located to the inner side of the nuclear membrane. This localization required an N-terminal domain not conserved in SENP1 which was shown to confer binding to a nuclear pore component. Interestingly, while native SENP2 showed little activity in vivo, a deletion mutant which lacked the nuclear rim-targeting region showed significantly increased activity in deconjugating SUMO-1 from target proteins. Therefore, both SENP1 and SENP2 show activity on SUMO-1 conjugates in vivo, but it is currently unclear for either protein whether they exhibit target selectivity with regard to the protein or the particular SUMO-1 species. In conclusion, while further work remains to be done on this class of proteins, our results indicate that SENP1 recruitment may be an important (though not sufficient) role in the modulation of SUMO-1, PML and NBs by ICP0. The cell line constitutively expressing epitope-tagged SUMO-1 should also prove useful in further characterization of SUMO-1, its regulation by SENP1 and its interaction with ICP0. The identification of target species and an understanding of how modification is regulated by specific signals should aid in understanding events occurring during virus infection.
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Acknowledgments |
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Received 17 June 2002;
accepted 26 August 2002.