Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein

Proteins of the PIAS family enhance the sumoylation of thepapillomavirus E1 protein*

Germán Rosas-Acosta1 and Van G. Wilson1,2

1Department of Medical Microbiology and Immunology, College of Medicine Texas A&MUniversity System Health Science Center, College Station, TX 77843-1114

SUMMARYThe E1 protein, a papillomavirus (PV)-encoded origin-binding helicase essential for PV DNAreplication, is post-translationally modified by sumoylation. As this modification is essential forthe nuclear accumulation of the bovine PV E1 (BPV E1), factors modulating the sumoylation ofE1 could ultimately alter the outcome of a papillomavirus infection. Therefore, we systematicallytested the known sumoylation enhancing factors (E3 SUMO ligases), namely RanBP2 and PIASfamily proteins, to determine their ability to bind to E1 and stimulate its sumoylation, using invitro assays. We found that RanBP2 bound to BPV E1 but failed to bind to the E1 from a humanPV (HPV11 E1), and lacked any sumoylation enhancing activity for both BPV E1 and HPV11 E1.In contrast, proteins of the PIAS family (except for PIASy) bound to both BPV E1 and HPV11 E1and stimulated their sumoylation, with PIASxβ (Miz1) exerting the largest stimulatory effect. Thestructural integrity of the RING finger domain of the PIAS proteins was required for their E3SUMO ligase activity on PV E1 sumoylation, but was dispensable for their PV E1 bindingactivity. Furthermore, the sumoylation enhancing activity exerted by the PIAS proteins on BPVE1 was more pronounced than on HPV11 E1, and appeared to favor SUMO1 versus SUMO2 asthe SUMO modifier. Altogether, this study identifies PIAS proteins as possible modulators of PVE1 sumoylation during papillomavirus infections.

KeywordsPapillomavirus; E1; BPV E1; HPV11 E1; sumoylation; E3 ligase; RanBP2; PIAS; Miz1

INTRODUCTIONPapillomaviruses (PVs1) are the etiological agents of warts in humans and animals, are theprimary risk factor associated with cervical cancer, and are associated with anogenitalcancers and other pathologies (1). PVs replicate their small genome (approximately 8000bp) episomally in the nucleus of the infected cell. This ability is conferred by the E1 protein,a viral-encoded protein that recognizes the viral origin of replication in conjunction with theE2 viral protein (2), recruits host cell replication proteins to the origin (3–5), and initiatesDNA replication via its helicase activity (6). Thus, the E1 and the E2 proteins are the only

*This work was supported by grant CA89298 from the NIH.2To whom correspondence should be addressed: Dr. Van G. Wilson, Professor, Department of Medical Microbiology andImmunology, College of Medicine – Texas A&M University System Health Science Center, College Station, TX 77843-1114, Tel:(979) 845-5207, Fax: (979)845-3479, [email protected] abbreviations used are: PVs, Papillomaviruses; BPV E1, bovine papillomavirus type 1 E1; HPV11 E1, human papillomavirustype 11 E1; PIAS, protein inhibitor of activated STAT1; STAT, signal transducers and activators of transcription; RanBP2, Ranbinding protein; Ran, ras-related nuclear protein; Sae2/1, sumo activating enzyme 2 and 1; GST, glutathione S-transferase; PAGE,polyacrylamide gel electrophoresis

NIH Public AccessAuthor ManuscriptVirology. Author manuscript; available in PMC 2012 October 26.

Published in final edited form as:Virology. 2005 January 5; 331(1): 190–203. doi:10.1016/j.virol.2004.10.025.

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viral-encoded proteins required for viral DNA replication, with the rest of the replicationmachinery being provided by the host (7).

At present, there are at least 80 distinct genotypes of human PVs (HPVs), and the E1 ORF isthe second most conserved ORF among the different types (1). A comparison between thepredicted HPV E1s and the well-characterized bovine papillomavirus type 1 (BPV) E1reveals several shared physical features, such as an acidic isoelectric point (6.48 for BPV E1and an average of 5.44 for the HPV E1s), a similar molecular weight (68 kD for BPV E1and an average of 72 for HPV E1s), and a similar tripartite organization (an N-terminalregion of undefined activity, a variable-length spacer, and a larger C-terminal region withrelatedness to ATPases and helicases) (8). BPV E1 and HPV E1s also share post-translational modification by phosphorylation at multiple serine and threonine residues,mediated by several kinases including cyclin E-cdc2 kinase (9–11), protein kinases A and C(12), and casein kinase II (13,14). In spite of these similarities, BPV E1 and HPV E1s alsoexhibit subtle differences in oligomerization properties, DNA binding affinity, and hostprotein interactions (8).

Besides phosphorylation, E1 proteins are also post-translationally modified by a processknown as sumoylation (15,16). Sumoylation consists of the covalent attachment of a smallubiquitin-related protein moiety (SUMO) to a substrate protein via an iso-peptide bondformed between the carboxyl group of the C terminal glycine in SUMO and the epsilonamino group of a lysine residue in the substrate protein (17). Sumoylation involves fourenzymatic steps: 1) proteolytic cleavage of the last 4 amino acid residues in SUMO toexpose the internal di-glycine motif required for conjugation, 2) activation of SUMO via athioester bond formed between the terminal carboxyl group of SUMO and Cys 173 in theSae2 subunit of the heterodimeric SUMO activating enzyme Sae2/Sae1, 3) transfer ofSUMO to the conjugating enzyme Ubc9 (also via a thioester linkage involving Cys 93 inUbc9), and 4) final conjugation of SUMO to the target substrate protein (17). The secondstep (activation) requires the presence of ATP. Thus, sumoylation can be recapitulated invitro by incubating the substrate with a mixture of purified Sae2/Sae1, Ubc9, and a truncatedSUMO protein (lacking the last 4 amino acid residues), in the presence of ATP (18,19).Recently, several groups have presented evidence that, although no other enzymatic activityis required for sumoylation in vitro, there are proteins that enhance the sumoylation ofspecific substrates both in vitro and in vivo (20–26). This enhancing effect resembles theeffect mediated by the E3 ligases in the ubiquitination pathway. Thus, these sumoylation-enhancers have been dubbed as E3 SUMO ligases (23). At present, two major types ofSUMO E3 ligases have been described: RanBP2, and proteins belonging to the PIAS family.

RanBP2 (also known as Nup358) is the largest structural component of the nuclear porecomplex, where it is localized to the cytosolic extensions or fibrils of the nuclear pore (27).Depletion of this protein in Xenopus egg extracts produces no detectable import defects intothe nucleus, but leads to a nuclear pore devoid of cytosolic extensions, thus showing thatRanBP2 is the major component of these structures (28). RanBP2 is sumoylated, containstwo Zinc fingers, four RanGTP binding domains, a domain containing two internal repeatmotifs, and a series of loosely spaced FG repeats thought to play a relevant role in nucleartraffic by providing docking sites for nuclear traffic factors (transportins)(for a schematicrepresentation, see reference 23). Besides binding RanGTP and transportins in conjunctionwith transport complexes, RanBP2 also binds RanGDP and RanGAP, the GTPase activatingprotein for RanGTP. This last interaction depends on the sumoylation of RanGAP (29,30).The region in RanBP2 responsible for its interaction with sumoylated RanGAP has beenmapped to the domain containing the two internal repeat motifs. This region also containsthe requirements needed for SUMO1 modification of RanBP2 and binding to Ubc9 (29).Recently, a smaller version of this region, lacking the FG repeat (referred to as

Rosas-Acosta and Wilson

Virology. Author manuscript; available in PMC 2012 October 26.

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RanBP2ΔFG), was shown to enhance its own sumoylation and stimulate the sumoylation ofSp100 (23). Subsequent studies have shown that RanBP2ΔFG also acts as a SUMO E3ligase for other substrates, including HDAC4 (25) and Mdm2 (31).

PIAS proteins were initially characterized by their ability to inhibit STAT-mediatedsignaling pathways (hence their name, Protein Inhibitors of Activated STAT) (32), but yeasttwo-hybrid analyses also demonstrated that PIAS1 was an interacting partner for p53 (33)and SUMO1 (21), thus leading to the prediction that PIAS1 could act as a SUMO E3 ligasefor p53. This was tested and confirmed by Kahyo et. al.(21). Simultaneously, Johnson andGupta described that the yeast PIAS homologues, Siz1 and Siz2, promote sumoylation of theseptins (the major sumoylation target in yeast), thus further supporting PIAS proteins asSUMO E3 ligases (20). PIAS proteins are characterized by the presence of an N-terminalSAP (SAF-A/B, Acinus, PIAS) domain, a central RING finger domain where two consensuscysteine residues for a Zinc finger domain seem to have been replaced by serine and asparticacid, and a C-terminal acidic domain. The SAP domain in PIASy mediates the binding ofthis protein to LEF1 (22), and may play a role in the interaction with other sumoylationsubstrates (34). The RING finger domain has been determined to be structurally required forthe SUMO E3 ligase of the PIAS proteins, as mutations disrupting its integrity preclude theenhancing activity mediated by PIAS proteins (21,24,26,35,36). However, such mutationsexert little or no effect on the ability of PIAS proteins to interact with the sumoylationsubstrates for which this has been tested (21). The current list of substrates for the SUMOE3 ligase activity of the members of the PIAS family includes p53 (21,24), GRIP1 (26), AR(26,36), LEF1 (22), c-Jun (24,26), c-Myb (37), Mdm2 (31), IRF-1 (35), C/EBPalpha (38),Sp3 (39), and the yeast Septins (20).

BPV E1 contains a Leu-Lys motif at positions 420 and 421 that is critical for Ubc9 binding(15), and mutations affecting this motif drastically impair the ability of BPV E1 to bind toUbc9 and become sumoylated (15,16). BPV E1 is sumoylated at a single residue, lysine 514,and replacing this residue by arginine abolishes BPV E1 sumoylation (16). Importantly, allthe mutations affecting BPV E1 sumoylation impair the intranuclear accumulation of BPVE1, leading to a mostly cytoplasmic distribution of this otherwise nuclear protein (16).Therefore, sumoylation of BPV E1 is essential for its nuclear accumulation, which in turn isrequired for the replicative functions of E1 (16). Thus, factors regulating E1 sumoylationmay ultimately regulate and alter the outcome of a BPV infection, and more generally, ofany PV infection as other papillomavirus E1 proteins are also sumoylated (16).Consequently, we sought to determine if the currently known SUMO E3 ligases exerted anyeffect on PV E1 sumoylation using an in vitro sumoylation system. We found that all of thePIAS family proteins tested, except PIASy, were able to specifically interact with both BPVE1 and HPV11 E1, and enhance their sumoylation, with PIASxβ (Miz1) producing thelargest sumoylation enhancing effect. Such enhancing activity was dependant upon theintegrity of the RING finger domain, although the disruption of this domain had no effect onthe PV E1-binding activity of the PIAS proteins. RanBP2 exhibited significant bindingactivity only toward BPV E1, but failed to enhance its sumoylation. The enhancing effectmediated by the PIAS proteins on BPV E1 was more pronounced than on HPV11 E1, thussuggesting slight differences in the way the sumoylation of these proteins is regulated.Furthermore, the enhancing effect of PIASxβ on PV E1 sumoylation was greatly reducedwhen SUMO2 was used as the modifier, thus indicating that the E3 SUMO ligase activityexerted by PIAS proteins on PV E1 favors their SUMO1 modification. Altogether, this studysupports PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirusinfections.



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EXPERIMENTAL PROCEDURESExpression plasmids, Protein expression and purification

The PIAS1 expression plasmid (21) was provided by Dr. Hideyo Yasuda. The pGEX4T3-ARIP3, pGEX4T3-ARIP3(W383A), and pGEX5X1-Miz1 plasmids for the expression ofPIASxα (ARIP3), the RING finger domain mutant of PIASxα (ARIP3[W383A]), andPIASxβ (Miz1), respectively (26,40) were provided by Dr. Jorma J. Palvimo. The pGEX2T-mPIASy plasmid for the expression of PIASy (22) was provided by Dr. Rudolf Grosschedl.The pGEX3X plasmid for the expression of RanBP2ΔFG (23) was provided by Dr. FraukeMelchior. All of the above SUMO E3 ligases were expressed as GST-fusion proteins andpurified by affinity chromatography on glutathione-Sepharose 4B beads (AmershamPharmacia Biotech, Piscataway, NJ) using a modified version of a previously describedmethod (41). Briefly, Escherichia coli BL21 bacteria containing the appropriate plasmidwere grown in 250 ml of liquid media at 37°C up to an optical density of 1.0 (wave length:600 nm), incubated on ice for 4 min, induced with 0.2 mM isopropyl-thio-β-D-galactoside,and incubated at room temperature for 4 h. The cells were collected by centrifugation,resuspended in 1× PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4,pH 7.3) containing 5 mM DTT and 1/100 volume of Protease Inhibitor cocktail (SIGMA-Aldrich Corp., St. Louis, MO), treated with lysozyme at a final concentration of 0.1 mg/ml,lysed by two passages through a prechilled French press cell at 16,000 lb/in2, and sonicatedbriefly. The resulting extract was clarified at 12,000 g for 15 min, and the supernatant wasincubated with the glutathione-Sepharose 4B beads for 2 h at 4°C. The beads were washedwith 20 bead volumes of 1× PBS, 10 bead volumes of 1× PBS containing 750 mM NaCl,and another 10 bead volumes of 1× PBS, and bound proteins were eluted with 20 mMreduced glutathione, 100 mM Tris pH 8.0, 120 mM NaCl. The eluted proteins were dialyzedovernight against 1× TBS (50 mM Tris pH 7.4, 150 mM NaCl) supplemented with 10%Glycerol, aliquoted into 20–50 μl aliquots, and stored at −70°C. The purified proteins werechecked by SDS-PAGE followed by Coomasie blue staining or western blotting performedusing goat anti-GST antibodies (Amersham Pharmacia Biotech) and peroxidase-conjugatedrabbit anti-goat IgG secondary antibodies (SIGMA-Aldrich Corp.), developed bychemoluminescence using the Western Lightning chemoluminescence reagent (PerkinElmerLife Sciences, Inc., Boston, MA). Protein concentrations were determined by a Bradfordassay. Sae2/Sae1 were co-expressed and co-purified as above from the GST-Sae2/Sae1expression plasmid (42), provided by Dr. Ronald T. Hay, but the proteins were eluted off thebeads by Thrombin digestion. Ubc9 was expressed using a previously describedpGEX-5X-1-based expression plasmid (15), and purified as above but the elution step wasexecuted by Factor Xa digestion. GST-SUMO2 was expressed from a pGEX expressionplasmid provided by Dr. Hisato Saitoh and purified as above. Human SUMO1 was clonedinto plasmid pRSET (Invitrogen Corp., Carlsbad, CA) from a plasmid provided by Dr. JoanaDesterro, and expressed and purified in E. coli BL21(DE3) bacteria as above, with themodifications indicated below. First, the cells were resuspended in a 1× PBS containing 500mM NaCl and 5 mM Imidazole; second, a Ni2+-NTA-Agarose resin (QIAGEN Inc.,Valencia, CA) was used for the purification; and third, the washes and the elution wereperformed by using a 1× PBS buffer containing increasing concentrations of Imidazole (50mM for the final wash, 500 mM for the elution).

Plasmids and procedures for in vitro protein expressionpRSET constructs containing either full length or truncated forms of BPV E1 were describedin a previous study (15). Plasmid pCR3-HPV11-E1, containing the full length HPV11 E1(43), was provided by Dr. Jacques Archambault. Plasmid HA-Sp100A/pSG5, coding for aHA-tagged Sp100 (44), was provided by Dr. Jacob S. Seeler and was used as a control toverify the E3 SUMO ligase activity of the purified GST-RanBP2ΔFG. All the above



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constructs were used in coupled in vitro transcription and translation reactions toproduce 35S-labeled proteins. Briefly, approximately 1–2 μg of plasmid DNA were mixedwith 32 μci of Redivue L-(35S)-Methionine (Amersham Pharmacia Biotech) and 25 μl ofthe TNT® T7 Quick Coupled transcription/translation system (Promega Corp., Madison,WI), and incubated at 30°C for 90 min. Upon incubation, the samples were flash-frozen at−70°C or used directly in pulldown experiments or in vitro sumoylation assays.

Pulldown experiments2, 6, or 12 μg of the appropriate purified GST-fusion protein were incubated with 20 μl ofglutathione-Sepharose 4B beads and 3 μl of the 35S-labeled in vitro translated protein, in a1× TBS buffer supplemented with 1% Tween 20 and 5 mM MgCl2 (1× TTBS-MgCl2), in afinal volume of 500 μl. The incubations were performed at 4°C in a circular rotator for 3 h.Then, the samples were centrifuged at 5,000 g for 30 sec, the supernatant was discarded, andthe beads were resuspended in 1 ml of ice cold 1× TTBS-MgCl2. This procedure wasrepeated three additional times to wash any unbound proteins away from the beads. After thefinal wash, the supernatant was discarded and the beads were resuspended in 25 μl of 4×SDS-PAGE sample buffer, incubated at 95°C for 3 min, and the resulting mixture wasloaded on an SDS-PAGE gel. The proteins in the gel were blotted onto Immobilon™

(Millipore Corp., Bedford, MA), and the membrane was developed by autoradiography.Quantitative analysis of the resulting profile of 35S-labeled proteins on the membrane wasperformed by phosphordensitometry analysis using a Storm™ 860 laser scanning system andImageQuant® analysis software (both from Molecular Dynamics Inc., AmershamPharmacia Biotech).

In vitro sumoylation assaysIn vitro sumoylation assays were carried out mostly as described (16), except that thepartially purified SUMO-activating enzyme Sae2/Sae1 derived from NIH 3T3 cellspreviously used was replaced by a bacterially-expressed affinity purified Sae2/Sae1. Briefly,2 μl of 35S-labeled in vitro translated BPV E1 or HPV11 E1 were incubated with or without1 μg of Sae2/Sae1, 280 ng of Ubc9, 1.5 μg of SUMO1 or GST-SUMO2, and the indicatedamounts of the purified E3 SUMO ligases. All the in vitro sumoylation reactions wereperformed in a buffer containing 50 mM Tris pH 8.0, 5 mM MgCl2, 5 mM ATP, and 0.5mM DTT, in a final volume of 25 μl, at 30°C for 90 min. The reactions were stopped byadding 9 μl of 4× SDS-PAGE sample buffer. Then, the sample were incubated at 95°C for 3min, loaded on an SDS-PAGE gel, and processed and quantified as described for thepulldown experiments.

RESULTSSumoylation of the bovine papillomavirus E1 protein regulates E1 function by controllingits access to the nuclear regions where viral DNA replication takes place (16); therefore,factors that regulate the sumoylation of this viral protein may ultimately affect viralreplication. Recently, two major types of proteins exhibiting features of an E3 ligase forsumoylation have been characterized: the PIAS family and the nucleoporin RanBP2. Eachone of these E3 SUMO-ligases appear to exhibit a specific range of protein targets. Wesought to investigate if either of these E3 SUMO ligases enhanced the sumoylation of thebovine and human papillomavirus E1 proteins.

PIAS proteins interact with both BPV E1 and HPV11 E1First, we examined the ability of bovine papillomavirus E1 (BPV E1) to interact with eitherof the known E3 SUMO ligases. To this end, we performed in vitro translations of BPV E1in the presence of 35S-methionine and tested the ability of the in vitro translated BPV E1 to



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bind in pulldown experiments to a purified member of the PIAS family, PIAS1, expressed asa glutathione-S-transferase (GST) fusion protein. Since the SUMO conjugase, Ubc9, couldpotentially stabilize the interaction between BPV E1 and PIAS1, we also performedpulldown experiments supplemented with purified recombinant Ubc9. BPV E1 did not bindto GST alone (fig. 1, lanes 2–5), but bound to GST-PIAS1 in a protein-concentrationdependent manner (fig. 1, lanes 6–8); such binding did not seem to be enhanced orcompromised by Ubc9, as judged by the intensity of the BPV E1 signals obtained (fig. 1,compare lanes 8 and 9). Similar pulldown experiments were performed to determine ifRanBP2 interacted with BPV E1, using in vitro translated BPV E1 and a GST fusion proteincorresponding to the region in RanBP2 reported to contain the necessary determinants forSUMO modification and E3 ligase activity (GST-RanBP2ΔFG)(23). As for PIAS1, BPV E1bound to GST-RanBP2ΔFG in a protein-concentration dependent manner (fig. 1, lanes 10–12), and Ubc9 did not seem to alter the binding of BPV E1 to GST-RanBP2ΔFG (fig. 1,compare lanes 12 and 13). An unrelated in vitro translated protein control (luciferase) didnot bind to GST-PIAS1 or to GST-RanBP2ΔFG (fig. 1, lanes 15–25), thus indicating thatthe binding was not due to an intrinsic unspecific stickiness of the GST fusion proteinstested.

In mammals, the PIAS family comprises five different PIAS proteins, namely PIAS1, thetwo alternative splicing forms of PIASx (PIASxα/ARIP3 and PIASxβ/Miz1), PIASy, andPIAS 3. The binding of BPV E1 to one of the members of the PIAS family suggested thepossibility that BPV E1 could interact with other members of the PIAS family. To test thispossibility, we affinity purified GST-fusions of other members of the PIAS family,performed pulldown experiments with in vitro translated BPV E1 and each of the purifiedPIAS proteins, and quantitated the data by phosphordensitometry. The purified GST fusionproteins used for this and all subsequent experiments were evaluated by SDS-PAGEfollowed by Coomasie blue or immunoblotting using α-GST antibodies. Notably, all thepurified GST-PIAS proteins displayed complex patterns of protein bands when resolved bySDS-PAGE (fig. 2, a and b), hence indicating that they were obtained largely as mixtures oftruncated forms which contained very little of their respective full size protein. In contrast,GST-RanBP2ΔFG appeared predominantly as a single band of the expected molecularweight, comparatively exhibiting a very limited abundance of truncated forms (fig. 2a lane6, and fig. 2b lane 7). All of the tested members of the PIAS family bound to BPV E1,except for PIASy which exhibited binding levels similar to those observed for GST (fig. 3, aand b). Likewise, a GST-ARIP3 fusion protein containing a single amino acid replacementinvolving a Trp residue located in the middle of the RING finger domain (GST-ARIP3[W383A]) exhibited BPV E1 binding activity similar to that of the wild type GST-ARIP3 (fig. 3, a and b). Since the above mutation is known to disrupt the structure of theRING finger domain (26), this indicates that the RING finger domain is not essential for thebinding of BPV E1 to GST-ARIP3. In these experiments the amount of BPV E1 retained byGST-RanBP2ΔFG was slightly lower than that retained by any of the PIAS proteins (exceptfor PIASy)(fig. 3, a and b).

A previous study indicated that, in addition to BPV E1, the E1 proteins from one cutaneousand one mucosal human PV were also sumoylated, consistent with a general role forsumoylation in PV E1 function (16). To determine if the known E3 SUMO ligases couldalso bind to the E1 protein from a human papillomavirus, we performed similar pulldownexperiments using in vitro translated E1 from human papillomavirus 11 (HPV11 E1). Asobserved for BPV E1, all of the members of the PIAS family tested bound to HPV11 E1,with the exception of PIASy; however, unlike BPV E1, HPV11 E1 did not bind to GST-RanBP2ΔFG (fig. 3, c and d).



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A large central portion of the BPV E1 protein is responsible for its interaction with PIASproteins

Next, we aimed to map the region in BPV E1 responsible for its interaction with themembers of the PIAS family and with RanBP2ΔFG, as several specific protein activitieshave been associated with well-defined subregions within E1 (8). To this end, constructsspanning subregions of E1 (represented in figure 4e) were in vitro translated in the presenceof 35S-methionine and used in pulldown experiments performed with GST, GST-PIAS1,GST-Miz1, or GST-RanBP2 ΔFG as baits. As shown in figure 4 (a and b), constructsspanning exclusively the C-terminal (amino acid residues 312–605) or the N-terminal(residues 1–312) halves of BPV E1 exhibited limited binding to GST-PIAS1. However, aconstruct spanning the N-terminal half of BPV E1 but lacking the first 120 amino acidresidues (121–311, which corresponds to the DNA binding domain), and a constructspanning the N-terminal half and part of the C terminus of BPV E1 (1–458), exhibitedsimilar or more abundant binding to GST-PIAS1 than the full-length BPV E1 protein (fig. 4,a and b). Similarly to the binding observed to GST-PIAS1, constructs spanning exclusivelythe N-terminal region (constructs 1–100 and 1–312) or the C-terminal half (312–605) ofBPV E1, exhibited very little binding to GST-Miz1, while a construct spanning the N-terminal half and part of the C-terminus (1–458) exhibited abundant binding to GST-Miz1(fig 4, c and d). Notably, the construct spanning the DNA binding domain did not appear tobind efficiently to GST-Miz1, unlike as observed for GST-PIAS1 (compare in fig. 4 panelsb and d). Thus, the construct spanning residues 1–458 of BPV E1 exhibited the mostconsistent binding to the PIAS proteins tested. Altogether, these data indicate that aminoacid sequences contained within a large central region of BPV E1 spanning residues 121–458 are involved in binding to PIAS proteins. The slight differences observed in BPV E1binding between GST-PIAS1 and GST-Miz1 may indicate minor differences in the waysmall subregions within this large central core of BPV E1 contribute to the binding of thefull-length protein. Alternatively, such differences could be partially due to the quality of thepurified proteins used as baits, as all the purified PIAS proteins were obtained as partiallytruncated products, as previously indicated (fig. 2).

The results with GST-RanBP2ΔFG were not definitive as all of the tested subregions withinBPV E1, except for the construct spanning residues 1–458, bound GST-RanBP2ΔFG eitheras efficiently or even more efficiently than the full-length protein, although the constructspanning amino acid residues 121–311 consistently exhibited the most abundant binding(fig. 4, a, c, and e). Thus, the data obtained suggest the possible presence of multiple regionswithin BPV E1 that may mediate its binding to RanBP2ΔFG, though some regions may notactually be accessible for binding to RanBP2ΔFG in the full-length E1 protein.

The in vitro sumoylation of PV E1 is enhanced by PIAS1The ability of BPV E1 to establish protein interactions with the two previously characterizedE3-SUMO ligases, RanBP2ΔFG and PIAS proteins, suggested that these proteins couldpotentially act as E3 ligases for BPV E1 sumoylation. To test this hypothesis, we developedan in vitro sumoylation assay using purified E. coli-expressed sumoylation enzymes aspreviously reported (42), and performed sumoylation assays of in vitro translated BPV E1with and without each E3-ligase. The in vitro sumoylation reactions were resolved in SDS-PAGE gels, transferred to immobilon membranes, and analyzed by autoradiography andphosphordensitometry. Since RanBP2ΔFG stimulates its own sumoylation (23), weperformed preliminary GST-RanBP2ΔFG sumoylation assays to test the activity of ourpurified sumoylation enzymes; such assays indicated that our purified sumoylationcomponents were highly active (data not shown). The in vitro sumoylation assays performedwith BPV E1 showed the appearance of high molecular forms of E1 in the presence of Ubc9plus Sae2/Sae1 (fig. 5a). The molecular weights of the novel forms of E1 were consistent



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with the addition of one, two, or several SUMO molecules to E1. The apparentconcentration of such high molecular weight forms increased in the presence of GST-PIAS1,and continued to increase as increasing amounts of GST-PIAS1 were added to the reaction(fig. 5a). Phosphordensitometric quantification of the predominant sumoylated form of BPVE1 indicated that GST-PIAS1 reached a maximum stimulatory effect of 2–3 fold on BPV E1sumoylation at 5 μg per reaction, and that such stimulatory effect decreased slightly athigher concentrations (fig. 5c). In sharp contrast, the addition of GST-RanBP2ΔFG atconcentrations slightly lower than those used for GST-PIAS1 seemed to exert an inhibitoryeffect on BPV E1 sumoylation, leading to a decrease in the amount of BPV E1 sumoylatedproducts; such decrease became more prominent as the amount of GST-RanBP2ΔFGincreased (fig. 5, a and c). This apparent inhibitory activity of GST-RanBP2 ΔFG on BPVE1 sumoylation prevailed over the stimulatory activity mediated by GST-PIAS1 when thetwo E3 ligases were combined in the same in vitro sumoylation reaction (fig. 5a, lane 11).

To determine if GST-PIAS1 and GST-RanBP2ΔFG exerted similar effects on thesumoylation of other PV E1 proteins, we performed similar in vitro sumoylation assaysusing HPV11 E1. Similarly as observed for BPV E1, the addition of GST-PIAS1 appearedto have a slight stimulatory effect on HPV11 E1 sumoylation, as increasing amounts ofGST-PIAS1 led to larger amounts of sumoylated HPV11 E1 (fig. 5b). However, while suchstimulatory effect appeared to continue to increase up to a concentration of 25μg of GST-PIAS1 per reaction, it did not appear to be as dramatic as the one observed for BPV E1sumoylation (compare figs. 5c and d). GST-RanBP2ΔFG appeared to exert an inhibitoryeffect on HPV11 E1 sumoylation (fig. 5, b and d), which prevailed over the stimulatoryactivity of GST-PIAS1 (fig. 5b, lane 11), as previously seen for BPV E1.

Immunoblotting analyses of the in vitro sumoylation reactions performed using anti-SUMO1 antibodies indicated the presence of large amounts of sumoylated GST-RanBP2ΔFG in the in vitro sumoylation reactions performed with GST-RanBP2ΔFG, thussuggesting that the apparent inhibitory effect of GST-RanBP2ΔFG on BPV E1 sumoylationwas due to competition for free SUMO1 (data not shown). As very small amounts ofRanBP2ΔFG are sufficient to produce the E3 ligase activity mediated by RanBP2ΔFG (inthe range of 1–5 ng per reaction), and larger amounts of RanBP2ΔFG are inhibitory for thesumoylation of Sp100 (a known substrate for the E3 ligase activity of RanBP2ΔFG)(23), weperformed additional in vitro sumoylation assays of BPV E1 using smaller amounts of GST-RanBP2ΔFG (1 to 20 ng per reaction). Under these conditions GST-RanBP2ΔFG stilllacked a stimulatory effect on BPV E1 sumoylation, although it was able to stimulate thesumoylation of Sp100 (data not shown). However, the inhibitory effect on E1 sumoylationobserved at higher concentrations GST-RanBP2ΔFG was now absent, consistent with acompetitive inhibition mechanism.

Miz1 (PIASxβ) is the PIAS family member protein that exerts the highest stimulatoryactivity on PV E1 sumoylation

To evaluate if the different members of the PIAS family differed in their ability to stimulatethe sumoylation of BPV E1, we performed additional sets of in vitro sumoylation reactionssupplemented with the different purified GST-PIAS proteins at the concentration estimatedto be optimal for GST-PIAS1. GST-RanBP2ΔFG was also evaluated in these assays, at aconcentration of 20 ng per reaction. Large differences among the members of the PIASfamily were observed in their ability to stimulate BPV E1 sumoylation. GST-PIASy did notexert any stimulatory activity on BPV E1 sumoylation, in agreement with the inability ofthis PIAS protein to interact with BPV E1 (fig. 6a). In contrast, GST-PIAS1, GST-ARIP3,and GST-Miz1 exerted a noticeable stimulatory activity on BPV E1 sumoylation, increasingthe relative intensity of some of the sumoylation products observed, thus producing bothqualitative and quantitative changes in the sumoylation patterns (fig. 6a). Out of the 3



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members of the PIAS family that stimulated BPV E1 sumoylation, GST-Miz1 exerted thehighest stimulatory activity on BPV E1 sumoylation, leading to a 2.3 fold increase in theapparent concentration of the most predominant form of sumoylated BPV E1, while GST-PIAS1 and GST-ARIP3 appeared to exert similar stimulatory activities, leading to a 1.5 foldincrease in sumoylated BPV E1, as determined by phosphordensitometry (fig. 6c). Studiesby Kotaja, et. al. (26), had indicated that the integrity of the RING finger domain wasessential for the SUMO-E3 ligase activity exerted by the members of the PIAS family, as asingle amino acid substitution known to disrupt this domain (i.e., a W to A substitution inposition 383 of ARIP3) leads to inactivation of the sumoylation stimulatory activity ofARIP3. Importantly, no significant stimulatory activity was observed for the RING fingerdomain mutant GST-ARIP3(W383A), thus indicating that the RING finger domain is alsorequired for the stimulatory activity mediated by GST-ARIP3 (fig. 6 a and c). As expected,no stimulatory activity on BPV E1 sumoylation was observed for GST-RanBP2ΔFG (fig. 6,a and c).

Similar experiments performed with HPV11 E1 showed that the effects mediated by thedifferent PIAS proteins on HPV11 E1 sumoylation had the same profile as those observedon BPV E1 sumoylation, although the stimulatory effects were less pronounced. Forinstance, GST-Miz1 exerted the highest stimulatory activity on HPV11 E1 sumoylation, butled only to a 1.5 fold increase in HPV11 E1 sumoylation as compared to the un-stimulatedreactions. Similarly, no stimulatory activity on HPV11 E1 sumoylation was observed forPIASy, the RING finger domain mutant GST-ARIP3(W383A), and GST-RanBP2ΔFG (fig.6, b and d).

Miz1 (PIASxβ) stimulates the SUMO1 and SUMO2 sumoylation of BPV E1E3 ligases are thought to enhance the transfer of SUMO from the E2 conjugating enzyme(Ubc9) to the substrate while providing specificity to the reaction. To determine if thestimulatory activity on PV-E1 sumoylation observed for the PIAS proteins was exclusive forSUMO1 sumoylation, we performed in vitro sumoylation assays using either BPV E1 orHPV11 E1 (in vitro translated), SUMO1 or SUMO2, and different concentrations of GST-Miz1 (the PIAS family member that exhibited the highest stimulatory activity on PV-E1SUMO1 sumoylation). As shown in figure 7 (a and b), GST-Miz1 appeared to be able tostimulate BPV E1 sumoylation with both SUMO1 and SUMO2, reaching a maximumstimulatory activity at approximately the same concentration for both SUMO modifiers.However, the apparent yield of sumoylated BPV E1 was noticeably higher when SUMO1was used as the modifier, thus suggesting that SUMO2 was a poor modifier for BPV E1sumoylation. A more dramatic bias against SUMO2 sumoylation was observed for HPV11E1. While GST-Miz1 clearly stimulated the SUMO1 sumoylation of HPV11 E1 (fig. 7c), nosignificant SUMO2 sumoylation was observed for HPV11 E1 even in the presence of GST-Miz1 (fig. 7d).

DISCUSSIONPV E1 plays a crucial role during the natural history of papillomavirus infections byallowing the episomal replication of the viral DNA in the host cell via its origin-binding andhelicase activity. Previous studies by our group showed that BPV E1 is modified bysumoylation and that this post-translational processing is essential for the nuclearaccumulation of BVP E1 (15,16). As nuclear accumulation of BPV E1 is a prerequisite forreplication function, the biological activity of BPV E1 is coupled to its ability to besumoylated. Thus, sumoylation can regulate BPV E1 function, and factors that stimulate orrestrict the sumoylation of BPV E1 may alter the outcome of a papillomavirus infection.



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In this study we sought to investigate the ability of the known SUMO E3 ligases to stimulateBPV E1 and HPV11 E1 sumoylation, using in vitro approaches. Although the precisemechanism of action of the currently identified SUMO E3 ligases is still unknown, it iscertain that binding between the sumoylation target and the E3 ligase is required for thesumoylation enhancing activity to occur. Thus, our first goal was to determine if E1 couldinteract with any of the known SUMO E3 ligases. We found that BPV E1 and HPV11 E1are able to interact with all of the members of the PIAS family tested, except for PIASy, andthat such interaction was not dependant on the structural integrity of the RING fingerdomain; furthermore, we found that BPV E1 (but not HPV11 E1) was able to interact withthe other known SUMO E3 ligase, RanBP2. We also determined that a large N-terminalregion within BPV E1 is responsible for its interaction with the PIAS proteins. This regionof E1 defined for PIAS binding at least partially overlaps the Ubc9 interaction region (15),though the actual interaction sites for PIAS and Ubc9 must be distinct since addition ofUbc9 had no effect on E1-PIAS binding (Fig. 1). Nonetheless, the apparent proximity of thePIAS and Ubc9 binding regions on E1 is consistent with the possible formation of an E1-Ubc9-PIAS complex during the sumoylation process. In addition, although we did not mapthe BPV E1 binding region within the PIAS proteins, a direct comparison of the tertiarystructure of PIASy with that of the other members of the PIAS family used in this studysuggests that the C-terminal end of the PIAS proteins might be critical for their binding toPV E1. The variation in PIAS family members is most extensive at the C-terminus withPIASy being the smallest at 507 amino acids (fig. 8). ARIP3 with an additional 65 aminoacids at its C-terminus binds E1 well, implicating this small region as critical for theinteraction, and suggesting that the longer C-terminal regions of PIAS1 and Miz1 containinga serine-rich motif are not essential for E1 interaction.

Next, we tested the ability of the PIAS proteins and RanBP2 to stimulate PV E1sumoylation, as previous reports show that binding of a given substrate to an E3 ligase doesnot imply per se that the ligase will enhance the sumoylation of the substrate (for instance,PIAS3 and PIASxβ interact with the human Androgen Receptor but do not enhance itssumoylation)(36). Even though the complex profile of sumoylated forms observed for BPVE1 and HPV11 E1 complicated the quantification of the stimulatory activity exerted by thedifferent E3 ligases tested, we found that RanBP2 did not exhibit SUMO ligase activity oneither BPV or HPV E1, although it bound to BPV E1; in contrast, all the PIAS proteins thatexhibited PV E1 binding activity were able to enhance the in vitro sumoylation of PV E1.These results were based on the intensity of the major sumoylated form observed on each setof reactions, to allow the cross-examination of all the samples in the same experiment andthe direct comparison of different experiments. However, this method does not take intoaccount the additive effect that would be obtained if several of the bands corresponding tosumoylated forms of E1 were used for the quantification. Thus, the data obtained probablyrepresents an underestimation of the sumoylation enhancing effect mediated by the ligasestested. Consequently, even though the apparent sumoylation enhancements mediated by thedifferent SUMO E3 ligases tested in this study were not always quantitatively striking, weconsider that the differences observed in the protein profiles obtained upon sumoylation inthe presence of some of the PIAS proteins were qualitatively significant and reflected thecontribution of the ligase activity. The meaning of these qualitative changes remains to bedetermined.

Even with the above limitations, members of the PIAS family enhanced the in vitrosumoylation of both BPV and HPV E1 proteins, consistent with SUMO ligase activity. Miz1was the most active on both HPV and BPV E1, with a 2–3 fold stimulation of BPV E1sumoylation. Additionally, we also observed that the integrity of the RING finger domain,while not required for E1 binding, was essential for the PV E1 sumoylation enhancingactivity displayed by the PIAS proteins. This requirement for an intact RING finger domain



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for the SUMO E3 ligase activity displayed by the PIAS proteins, observed here and inseveral other reports (21,24,26,35,36), is attributable to the fact that the RING finger domainmediates the interaction between PIAS proteins and Ubc9 (36). Furthermore, the differencesin the PV E1 sumoylation enhancing abilities displayed by the different members of thePIAS family did not correlate with the E1 binding activity as PIAS1, ARIP3, and Miz1bound E1 equally well, yet Miz1 was consistently more active in stimulating sumoylation.Such differences further support a role for the C-terminal region of the PIAS proteins intheir PV E1 sumoylation enhancing activity, as PIASxα (ARIP3) and PIASxβ (Miz1) onlydiffer from each other at the C-terminal end (fig. 8) yet exhibit substantial differences intheir ability to enhance the sumoylation of BPV E1. Interestingly, even though the SUMOE3 ligase activity exerted by the PIAS proteins on BPV E1 sumoylation was not restricted toSUMO1 sumoylation, as it also enhanced the SUMO2 sumoylation of BPV E1, theenhancing effect observed on SUMO2 sumoylation was notably lower. Furthermore, PIASproteins failed to enhance the SUMO2 sumoylation of HPV11 E1. Altogether, thedifferences observed among the PIAS proteins in their ability to stimulate PV E1sumoylation, and the differences observed in the type of SUMO that is preferentially used asa modifier upon PIAS stimulation, indicate a certain degree of specificity in the regulation ofPV E1 sumoylation by PIAS proteins. However, as there may still be additional unknownSUMO ligases, it is possible that these other E3 proteins may exert a much more profoundenhancing effect on PV E1 sumoylation. Alternatively, as several post-translationalmodifications affecting PV E1 have been identified, including phosphorylation (10,12–14)and ubiquitination (45), it is possible that one of these (or both) may regulate PV E1sumoylation, further enhancing or decreasing PV E1 sumoylation. This possibility issupported by recent reports showing that phosphorylation of Heat Shock Factor 1 enhancesits sumoylation (46,47). Future studies will try to establish the role of PIAS proteins in E1sumoylation in vivo, identify additional SUMO ligases, and evaluate how the different post-translation modifications of PV E1 interact together in the cellular environment to regulatethe function of this protein.

Sumoylation appears to be predominantly a nuclear event as most SUMO substrates arenuclear proteins, and sumoylation of several nuclear proteins has been shown to requireintact nuclear import capacity since mutants lacking a functional nuclear localizationsequence (NLS) remain unmodified (25,48). Recently, evidence was presented (31) thatsumoylation can be a biphasic process with addition of SUMO both during nuclear import(utilizing the nuclear pore-associated RanBP2 E3 ligase activity) and subsequently withinthe nucleus via PIAS proteins which are exclusively confined to the cell nucleus(22,26,49,50). Even though the details of nuclear trafficking by PV E1 proteins remainsmostly uncharacterized, BPV E1 protein resides primarily in the nucleus and possess atypical nuclear localization sequence (NLS) (9,51), suggesting that sumoylation of E1proteins will be a nuclear or perinuclear event. The lack of PV E1 sumoylation enhancingactivity displayed by RanBP2 in vitro suggests that the binding observed between BPV E1and RanBP2 may represent an interaction established by BPV E1 along its traffic to thenucleus rather than an interaction established to enhance E1 sumoylation. In contrast, asPIAS proteins exert a sumoylation enhancing effect on BPV E1, it is tempting to proposethat BPV E1 traffics to the nucleus in an un-sumoylated form, and once in the nucleus it issumoylated in conjunction with nuclear PIAS E3 ligase activity. Intranuclear sumoylation ofE1 may be necessary for its retention within the nucleus by SUMO-dependent proteininteractions, and/or modulation of E1 biochemical activities necessary for replicativefunction. Should the PIAS proteins be the primary SUMO E3 ligases for PV E1 sumoylationin vivo, it will be necessary to determine if the expression of these cellular proteins isregulated during keratinocyte differentiation, as papillomavirus viral replication is intimatelyassociated with the cellular differentiation of their natural host.



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Lastly, the profile of sumoylated forms observed for BPV E1 in this study differssubstantially from the one previously reported (16). In the former studies, a single,predominant sumoylated form of BPV E1 was observed, while in the current studies a morecomplex array of sumoylated products formed in vitro, even in the absence of PIASproteins. The profile obtained in the present study suggests two possible scenarios: 1) BPVE1 may contain other potential sumoylation sites that are being used under the sumoylationassay conditions used in this study, leading to its sumoylation at multiple sites, or 2) theSUMO1 added at the single sumoylation site in BPV E1 may be undergoingpolysumoylation (SUMO1 chain formation). SUMO1 was initially considered unable toundergo chain formation due to the lack of a lysine displaying the consensus sumoylationsequence and no experimental data indicative of SUMO1 polysumoylation (42). However,later reports have given strong support to the idea that SUMO1 can form chains in vitro (23),and we have also observed this for sumoylation of RanBP2 in vitro (unpublishedobservations). Although at present we can not discard either of these scenarios, we considerthat an important contributing factor to the differences observed is the use of E. coli-expressed, purified Sae2/Sae1 in this study versus the use of partially enriched Sae2/Sae1from mammalian cell extracts in our previous reports. Hence, a higher enzymatic activitycombined with a decrease in the total pool of possible sumoylation substrates could result ineither of the scenarios described above. Alternatively, an unknown E3 ligase or other factorthat limits the spectrum of SUMO-modifiable lysine residues in BPV E1 (thus increasing thespecificity of sumoylation) may have been present in the cell extracts previously used.

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Fig. 1. BPV E1 interacts with GST-PIAS1 and GST-RanBP2ΔFG2, 6, or 12 μg of either GST-PIAS1 or GST-RanBP2ΔFG were incubated with 20 μl ofglutathione-Sepharose 4B beads and 3 μl of 35S-labeled in vitro translated BPV E1 orLuciferase. Bound proteins were resolved on a 8% SDS-PAGE gel and analyzed byautoradiography. i represents 50% of the in vitro translation protein mixture used as initialmaterial (input). Underlined samples were supplemented with 10 μg of Ubc9. Numbers tothe left indicate molecular weight markers (in kDa).



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Fig. 2. The GST-PIAS proteins were purified mostly as truncated forms of the fusion proteinsA. 10 μg of each purified GST-fusion protein were resolved on a 10% SDS-PAGE gel andstained by Coomasie blue. B. 2 μg of each purified GST-fusion protein were resolved on a7.5% SDS-PAGE gel, transferred to an Immobilon membrane, and immunoblotted withanti-GST antibodies. Numbers to the left indicate molecular weight markers (in kDa).



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Fig. 3. Interactions between PV E1 proteins and the known SUMO-E3 ligases12 μg of each purified GST-fusion protein were incubated with 20 μl of glutathione-Sepharose 4B beads and 3 μl of 35S-labeled in vitro translated BPV E1 or HPV11 E1.Bound proteins were resolved on a 10% SDS-PAGE gel and analyzed byphosphordensitometry. A and C. Autoradiographs showing the binding obtained inrepresentative experiments using BPV E1 and HPV11 E1, respectively. B and D. Graphicrepresentation of the data obtained in two or three experiments performed with BPV E1 andHPV11 E1, respectively. The binding of the PV E1 proteins to the different GST-fusionproteins is represented as percentage of the initial material (% input). Error bars presented inthis figure and throughout the paper represent standard error.



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Fig. 4. Mapping of the region in BPV E1 responsible for its binding to SUMO-E3 ligases12 μg of either GST-PIAS1, GST-Miz1, or GST-RanBP2ΔFG were incubated with 20 μl ofglutathione-Sepharose 4B beads and 3 μl of 35S-labeled in vitro translated proteinsrepresenting subregions within BPV E1. Bound proteins were resolved on a 10% SDS-PAGE gel and analyzed by phosphordensitometry. i: 50% of the input material; 1: GST; 2:GST-PIAS family protein; 3: GST-RanBP2ΔFG. The numbers above the line indicate theamino acid residues in the primary structure of BPV E1 covered by the in vitro translatedprotein. A and C. Autoradiographs showing the binding obtained in representativeexperiments performed with GST-PIAS1 and GST-RanBP2ΔFG, and GST-Miz1 and GST-RanBP2ΔFG, respectively. B, D, and F. Graphic representation of the data obtained in 2different experiments performed with GST-PIAS1, GST-Miz1, and GST-RanBP2ΔFG,respectively. E. Diagram of the regions in BPV E1 covered by the different constructs usedin this series of experiments. Numbers indicate amino acid residues within BPV E1. Krepresents Lys 514, the lysine residue previously identified as the major sumoylation site inBPV E1 (16). U represents the LK motif at positions 420 and 421, a motif previouslyidentified as critical for interaction with Ubc9 (15).



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Fig. 5. The in vitro sumoylation of PV E1 is enhanced by GST-PIAS1 but not by GST-RanBP2ΔFG2 μl of 35S-labeled in vitro translated BPV E1 or HPV11 E1 were incubated with (+) orwithout (-) 1 μg of Sae2/Sae1, 1.5 μg of SUMO1, 280 ng of Ubc9, and the indicatedamounts of purified GST-PIAS1 or GST-RanBP2ΔFG. Upon incubation, the samples wereresolved on an 8% SDS-PAGE gel and analyzed by phosphordensitometry. A and B.Autoradiographs showing the sumoylation profile obtained for BPV E1 and HPV11 E1,respectively. Numbers to the left indicate molecular weight markers (in kDa). C and D.Graphic representation (in arbitrary units) of the data obtained by phosphordensitometrymeasurement of the major sumoylated product obtained for BPV E1 and HPV11 E1,respectively.



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Fig. 6. Miz1 (PIASxβ) is the PIAS family member protein that exerts the highest stimulatoryactivity on PV E1 sumoylation2 μl of 35S-labeled in vitro translated BPV E1 or HPV11 E1 were incubated with bufferonly (None), SUMO1 and Ubc9 (No Sae2/1), SUMO1+Sae2/1+Ubc9 (ALL), orSUMO1+Sae2/1+Ubc9 supplemented with 2 μg (PIAS proteins) or 20 ng (RanBP2ΔFG) ofthe indicated purified GST-fusion proteins. The amounts used per reaction of the differentsumoylation components were 1 μg of Sae2/Sae1, 280 ng of Ubc9, and 1.5 μg of SUMO1.Upon incubation, the samples were resolved on 8 or 10% SDS-PAGE gels and analyzed byphosphordensitometry. A and B. Autoradiographs showing the sumoylation profile obtainedfor BPV E1 and HPV11 E1, respectively, in the presence of the different GST-fusion



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proteins in representative experiments. The unmodified E1 (arrow) and the sumoylatedproducts (S-E1, bracket) are indicated. C and D. Graphic representation of the data obtainedby phosphordensitometry quantification of the major sumoylated product obtained for BPVE1 and HPV11 E1, respectively, in 2 or 3 experiments. The data is expressed as percentagesumoylation activity as determined by assigning 100% sumoylation activity to the reactionperformed in the presence of all the sumoylation components (Sae2/Sae1+Ubc9+SUMO1).



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Fig. 7. Miz1 (PIASxβ) stimulates the SUMO2 sumoylation of BPV E1 but not of HPV11 E11.5 μl of 35S-labeled in vitro translated BPV E1 or HPV11 E1 were incubated with (+) orwithout (−) 1μg of Sae2/Sae1, 280 ng of Ubc9 and 2 μg of either SUMO1 or GST-SUMO2,and the indicated amounts of purified GST-Miz1. Upon incubation, the samples wereresolved on an 8% SDS-PAGE gel and analyzed by autoradiography. A and B. Sumoylationof BPV E1 with SUMO1 and GST-SUMO2, respectively. C and D. Sumoylation of HPV11E1 with SUMO1 and GST-SUMO2, respectively.



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Fig. 8. Schematic representation of the primary structure of the PIAS family protein membersused in this studyNumbers indicate the amino acid residues limiting the different domains identified withinthe primary structure of the PIAS proteins. SAP: SAF-A/B, Acinus, PIAS domain. Noticethat the differences in length observed among the different PIAS proteins are all due tovariations in the C-terminal end of the proteins.



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NIH


NIH


Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein

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Transcript of Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein