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Role of p50/CDC37 in hepadnavirus assembly and replication.

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pull-down assays showed that p50 or p50deltaC, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.

the specific interaction between the RT and ⑀ triggers two essential early steps in hepadnavirus assembly and replication, i.e. the protein-primed initiation of reverse transcription and the assembly of replication-competent nucleocapsids.
Using the duck hepatitis B virus (DHBV) as a model system, we have recently found that the RT requires the assistance of host cell factors to carry out specific ⑀ binding and protein priming functions (16,17). One such cellular factor is the 90-kDa heat shock protein (Hsp90). Hsp90 associates with the DHBV RT and is required for RT-⑀ interaction and protein priming in vitro and for pgRNA packaging and DNA synthesis in vivo. Hsp90 is thought to facilitate the functions of a specific subset of cellular proteins (the target or substrate proteins) by helping to establish and maintain certain poised but labile conformations of these target proteins through a dynamic multistage process (18 -22). In so doing, Hsp90 invariably collaborates with other factors (the so-called cochaperones or cofactors) and forms various multicomponent chaperone complexes. The precise composition of the Hsp90 chaperone complexes seems to vary depending upon the nature of the target proteins and the stage of the chaperoning process.
Hsp90 target proteins range from steroid receptors and protein kinases to reverse transcriptase (19,20,22). These proteins do not share any obvious structural or functional similarities. Hence, one of the most enigmatic, unresolved questions about Hsp90 is how the chaperone can recognize specifically such a diverse group of substrates. In our efforts to understand the molecular basis for the interaction between the Hsp90 complex and the viral RT, we searched the data base for Hsp90 target proteins that might share sequence and/or structural properties with the RT. For this purpose, we used a recently described sequence analysis program called protein property search or PropSearch (23), which was designed to detect weak structural and/or functional protein homologs that cannot be detected using conventional sequence alignment tools. PropSearch defines protein similarities not through sequence alignment, but as a weighted sum of compositional properties such as singlet and doublet amino acid compositions. By these criteria, we found that the RT is highly related to the serine/ threonine protein kinase c-Raf.
The protein kinase c-Raf and a selected group of several other serine/threonine and tyrosine-protein kinases (e.g. CDK4 and v-Src) are known to be Hsp90 target proteins, requiring the chaperone for their intracellular trafficking, assembly, and maturation (24,25). An Hsp90 cofactor called p50 (or CDC37) binds specifically to these kinases and to Hsp90 and is thought to be a "kinase-specific subunit" of a particular subset of Hsp90 complexes that targets Hsp90 to the kinase substrates by acting as a bridge between the kinases and Hsp90 (26 -28). In light of these results, we decided to examine whether p50/ CDC37 plays a role in the functions of the hepadnavirus RT, as suggested by the similarity between the RT and the protein kinases. Herein, we report that p50/CDC37 could indeed spe-cifically interact with the RT in vitro and in vivo. The functional significance of this interaction was underscored by the fact that p50/CDC37 was able to modulate RT functions, as determined by measuring its protein priming activity in vitro and RNA packaging and DNA synthesis activities in transfected cells. These results thus indicate that p50/CDC37 is a host cell cofactor for hepadnavirus replication and may play a more general role in Hsp90 chaperone function than originally proposed.

EXPERIMENTAL PROCEDURES
Plasmids-Plasmid pHTP was used for in vitro expression of the full-length DHBV RT (6). Plasmids expressing the DHBV mini-RT proteins, pcDNA-MiniRT1 and pcDNA-MiniRT2 (for in vitro and mammalian cell expression) and pGST-MiniRT1 and pGST-MiniRT2 (for bacterial expression of glutathione S-transferase (GST) fusion proteins) have been described before (29). pSP-c-Raf directs the in vitro expression of the human c-raf gene under the control of the SP6 promoter in the pSP64polyA vector (Promega) and was kindly provided by Zhijun Luo (Boston Medical Center). pGST-p50 and pGST-p50␦C were constructed in a modified pEBG vector and direct the expression of p50/ CDC37 and C-terminally truncated p50, respectively, in mammalian cells (27). pEBG-MiniRT1 and pEBG-MiniRT2 were constructed by substituting the GST-MiniRT1 and GST-MiniRT2 cassettes from pGST-MiniRT1 and pGST-MiniRT2, respectively, for the GST-p50 sequences in pGST-p50 and were used to express the GST-MiniRT fusion proteins in mammalian cells. All RT and mini-RT proteins were tagged with a synthetic hemagglutinin (HA) epitope inserted into the spacer domain. p50-FLAG, which directs the expression of FLAG epitopetagged p50 under the control of the cytomegalovirus immediate-early promoter/enhancer, was kindly provided by Gary Perdew (Pennsylvania State University) (30). pCMV-DHBV, which directs the expression of the DHBV pgRNA under the control of the cytomegalovirus promoter, and its derivative pCMV-YMHA, which harbors two amino acid substitutions in the RT active site abolishing polymerase activity, have been described (31)(32)(33). pCMV-␦XM(core) was derived from pCMV-DHBV by removing the DHBV sequences from the unique XhoI site (nucleotide 1217) to the MscI site (nucleotide 2375), abolishing the expression of the RT and viral envelope proteins, but retaining core protein expression.
In Vitro Transcription and Translation-RNAs used for in vitro translation were transcribed from linearized plasmids using an in vitro transcription kit (MEGAscript, Ambion) and were purified as described previously (16,29). Purified RNAs were then translated using the rabbit reticulocyte lysate in vitro translation system (Promega). Protein expression in the coupled transcription and translation reaction using the TnT rabbit reticulocyte lysate system (Promega) was carried out according to the manufacturer's instructions.
In Vitro Protein Priming-Approximately 10 ng of purified GST-MiniRT proteins were used in an in vitro protein priming reaction in a total volume of 10 l as described previously (29). Rabbit reticulocyte lysate (nuclease-treated; Promega) supplemented with an ATP regenerating system (5 mM ATP, 10 mM creatine phosphate, and 50 g/ml creatine phosphokinase) was used to reconstitute the RT protein priming activity as described (29). In all reactions, [␣-32 P]dATP was used as the labeled nucleotide.
Protein Expression and Purification-Two truncated minimal DHBV RT fusion proteins (GST-MiniRT1 and GST-MiniRT2) were expressed in Escherichia coli and purified as described (29). FLAG epitope-tagged p50 and p50␦C were purified from recombinant baculovirus-infected insect cells by immunoaffinity chromatography using agarose-crosslinked mAb M2 (Eastman Kodak Co.) as described (27).
Protein-Protein Interactions-Full-length DHBV RT and truncation mutants (labeled by in vitro translation using the reticulocyte lysate in the presence of [ 35 S]methionine) were diluted with lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) and incubated with p50 or p50␦C pre-bound to the mAb M2-agarose beads.
The unbound material was removed by extensive washing with lysis buffer, and the immunoprecipitates were then resolved by SDS-PAGE. Total proteins were detected by Coomassie Blue staining, and the labeled proteins by autoradiography. To detect direct binding between the RT and p50, purified GST-MiniRT proteins from bacteria were incubated with the FLAG-tagged p50-or p50␦C-bound mAb M2 immunoaffinity beads in lysis buffer, and unbound proteins were removed after extensive washing with lysis buffer. The RT proteins bound to the beads were detected by Western blot analysis using the anti-HA mAb following resolution of the immunoprecipitates by SDS-PAGE.
To assess p50-RT interaction in cultured cells, pcDNA-MiniRT1 and pcDNA-MiniRT2 were cotransfected with pGST-p50 or pGST-p50␦C into 293T or COS-1 cells. The transfected cells were lysed in lysis buffer 3 days after transfection, and the p50 proteins were purified by GSHagarose beads. The mini-RT proteins bound to the p50 proteins were eluted by GSH and detected by resolving the purified proteins by SDS-PAGE and Western blot analysis using either the anti-HA mAb or mAbs against the TP domains. In addition, the GST-MiniRT proteins were coexpressed using pEBG-MiniRT1 and pEBG-MiniRT2 in 293T or COS-1 cells together with FLAG-tagged p50. The GST-MiniRT fusion proteins were then purified by GSH beads and eluted by GSH. Proteins associated with the RT were detected by SDS-PAGE and Western blot analysis.
Analysis of Viral DNA, RNA, and Proteins-The human embryonic kidney cell line 293T and the monkey kidney cell line COS-1 were transfected with pCMV-DHBV, pCMV-␦XM(core), or pCMV-YMHA together with a p50 expression construct (pGST-p50 or pGST-p50␦C) or the vector alone (pEBG or GST) using the calcium phosphate (5 Prime 3 3 Prime, Inc.) procedure (16). Transfected cells were harvested 3 days after transfection. Replicative viral DNA intermediates were purified from cytoplasmic core particles and analyzed by Southern blot analysis as described (16,37).
Encapsidated pgRNA in cytoplasmic core particles obtained by polyethylene glycol precipitation (38) was detected by resolving the capsid particles on agarose gels followed by Southern blot analysis using a 32 P-labeled antisense riboprobe (spanning nucleotides 143-391 on the DHBV genome) (16) as described (39). The amount of assembled capsid particles was determined by subsequent reprobing of the same membrane using the anti-DHBV core antibody. To measure the steady-state levels of total DHBV core protein in the transfected cells, a portion of the cytoplasmic extract used for core DNA isolation was analyzed by SDS-PAGE and Western blotting using the anti-DHBV core antibody.

Similarity between the DHBV RT and c-Raf in Compositional
Properties as Detected by PropSearch-Because conventional sequence alignment methods have not revealed any similarity between the hepadnavirus RT and other Hsp90 target proteins, we attempted to determine whether the RT is related to any other known Hsp90 target proteins using an alternative sequence comparison method called PropSearch (23). PropSearch defines protein similarities not through sequence alignment, but as a weighted sum of compositional properties (a total of 144 parameters), including singlet and doublet amino acid composition and isoelectric point, and has been shown to detect weak structural and/or functional protein homologs that escaped detection by conventional sequence alignment tools.
A strong similarity in amino acid composition between the RT and c-Raf (kraf), a protein kinase target of Hsp90, was detected by PropSearch (Table I and additional search results not shown). In fact, the similarity between the RT and c-Raf proteins (Table I, ranks 7, 8, and 11) was judged to be stronger than that between the RT and other reverse transcriptases from other retroelements (rank 14) and just below that between the DHBV RT and other hepadnavirus RTs (ranks 1-5). It is not yet known whether the other proteins listed in Table I are Hsp90 targets or not. However, the strong similarity between the RT and c-Raf raised the intriguing possibility that the RT, like c-Raf, may be targeted to the Hsp90 chaperone via p50/ CDC37, an Hsp90 cofactor known to be responsible for targeting the kinase substrates to the chaperone.
Association between the RT and p50 in Vitro-To determine whether the RT could associate with p50 specifically, we ex-p50/CDC37 and Hepadnavirus Replication pressed the 35 S-labeled DHBV RT by in vitro translation in the rabbit reticulocyte lysate, in which an active DHBV RT can be expressed (4) and is associated with Hsp90 and one of the cochaperones, p23 (16,17). The labeled RT proteins were then co-immunoprecipitated with purified FLAG-tagged p50 prebound to antibody affinity beads. Bound proteins were detected by SDS-PAGE and autoradiography. As shown in Fig. 1A, the RT could be co-immunoprecipitated by the p50 beads (lane 1), similar to c-Raf (lane 4), which was used as a positive control. The negative control (in vitro translated luciferase) was not precipitated by the p50 beads (Fig. 1A, lane 7). As p50 is known to bind to Hsp90 (Fig. 1B, lanes 1, 4, and 7) (27,40), as does the RT (16), it was possible that the RT was associated with p50 via Hsp90. To test whether p50 could associate with the RT independently of Hsp90, a p50 mutant (p50␦C) with its C-terminal Hsp90-binding domain deleted, and thus unable to bind Hsp90 (Fig. 1B, lanes 2, 5, and 8) (27), was used to precipitate the RT. We found that p50␦C could also associate with the RT (Fig. 1A, lane 2) as well as c-Raf (lane 5), indicating that the interaction between p50 and the RT (like c-Raf) (27) is independent of its association with Hsp90. Interestingly, p50␦C seemed to bind even more in vitro translated RT proteins than wild-type p50, which was also the case when they were coexpressed in cultured cells (see Fig. 3

below).
To determine whether the RT could bind directly to p50, as c-Raf does (27,41), purified GST-MiniRT proteins expressed in bacteria were incubated with purified p50 proteins pre-bound to mAb M2 beads. We found that both GST-MiniRT1 and GST-MiniRT2 (but not GST alone) could bind directly to purified p50 and p50␦C ( Fig. 2) (data not shown). Therefore, these results showed that the DHBV RT could directly associate with p50 in vitro, independently of Hsp90 or any other cellular factors.
Association between the RT and p50 in Vivo-To determine whether the RT could associate with p50 in the cell, we coexpressed functional mini-RT proteins (29) together with GSTtagged p50 in 293T cells, which can be transfected efficiently. As shown in Fig. 3 (lanes 13-18), GST-tagged p50␦C proteins specifically pulled down the mini-RT proteins expressed in 293T cells. (Similar results were also obtained using COS cells (data not shown).) In addition to the intact mini-RT proteins, we noticed that a major degradation product derived from both mini-RT proteins was also pulled down by p50␦C. This degradation product reacted with both the anti-HA mAb (Fig. 3A) and several antibodies (both monoclonal and polyclonal) against the TP domain (data not shown), indicating that it represented the TP fragment with some residual sequences from the spacer region where the HA epitope was inserted. On the other hand, the binding of the RT to wild-type p50 seemed to be weaker and more variable (Fig. 3) (data not shown). In addition, we noticed that p50␦C (but not p50) was predominantly localized to the detergent-insoluble fraction and shifted a significant amount of the mini-RT proteins and the TP fragment into this fraction (Fig. 3, lanes 7-12). As p50␦C (unlike p50) could not associate with Hsp90 (Fig. 3B, lanes 13-18), these results indicated that the mini-RT proteins and the TP domain could associate with p50 in vivo independently of Hsp90.
To further assess the association of the RT with p50 (and other cellular proteins) in the cell, we expressed the GST-MiniRT proteins in 293T cells. The tagged mini-RT proteins were purified by GSH beads, and RT-associated proteins in the GSH eluate were detected by Western blot analysis using specific antibodies against various cellular proteins. As GST-MiniRT1 was rapidly degraded in the transfected cells, it was difficult to purify significant amounts of MiniRT1 for this purpose. However, we found that GST-MiniRT2 was significantly more stable, and substantial amounts of GST-MiniRT2 could be purified (Fig. 4, fourth panel). When a FLAG-tagged p50 protein was coexpressed with GST-MiniRT2 in 293T cells, the overexpressed exogenous p50-FLAG protein was associated with the MiniRT2 protein (Fig. 4, second panel). The association between the mini-RT protein and endogenous p50 was also detectable in some experiments (data not shown). In addition, we could consistently detect the association between GST-Mi-niRT1 and endogenous Hsp90 and another cochaperone, p23 (Fig. 4, first and third panels). We noticed that in some experiments, the amount of p23 in association with the RT seemed to be decreased when p50-FLAG was overexpressed (Fig. 4, third  panel, lane 1).
In summary, we demonstrated that the RT could associate with several components of the Hsp90 complex, including

p50/CDC37 and Hepadnavirus Replication
Hsp90, p50, and p23 in vivo. Furthermore, we found that the association between the RT and p50 in vivo, as in vitro, was independent of Hsp90.
Inhibition of in Vitro RT Activation by p50␦C-To determine whether p50 plays a role in the functions of the RT, we measured the protein priming activity of purified GST-MiniRT1 from bacteria using our recently established in vitro reconstitution assay (29). When the purified mini-RT protein was incubated with the reticulocyte lysate, it was activated for in vitro priming (Fig. 5, lanes 1 and 2) (29). The addition of increasing amounts of p50␦C led to a progressive inhibition of protein priming (Fig. 5, lanes 3 and 4). On the other hand, the addition of wild-type p50 to the priming reaction had little or no effect (Fig. 5, lanes 5 and 6) (data not shown). As p50␦C is known to be a dominant-negative inhibitor of p50 function (27), these results suggested that p50 function was important for protein priming, but was not limiting under these in vitro conditions. On the other hand, they could not exclude the possibility that the inhibitory effect of p50␦C on RT activation was simply due to the sequestration of the RT away from the Hsp90 chaperone by p50␦C.
Increase in Viral Replication by p50 and Decrease by p50␦C in Vivo-To obtain evidence for a role of p50 in RT function and in viral replication in vivo, we transfected replication-competent DHBV DNA into 293T cells together with p50 expression plasmids. As shown in Fig. 6, the overexpression of p50 led to a significant increase in viral DNA synthesis, whereas the dominant-negative mutant (p50␦C) showed just the opposite effect and decreased viral replication. The stimulatory and inhibitory effects of p50 and p50␦C, respectively, were particularly evident on the replicative viral single-stranded DNA intermediates (Fig. 6A). Although the effects of p50 and p50␦C on viral DNA synthesis were modest (ϳ3-fold increase by p50 and 3-fold decrease by p50␦C), the opposing effects of p50 and its dominant-negative inhibitor (p50␦C) clearly demonstrated that p50 plays a role in viral DNA replication.
To understand the mechanism of p50 effect on viral DNA synthesis in the cell, we determined the levels of viral pgRNA  35 S-labeled RT protein was then incubated with purified FLAG-tagged p50 (p50FLAG) or p50␦C (p50␦CFLAG) pre-bound to mAb M2-agarose beads. Control beads were protein G-agarose beads with pre-bound nonimmune mouse IgG. 35 S-Labeled c-Raf (Raf) and luciferase (Lucif) were also translated and used as positive and negative controls, respectively, for the binding reaction. Bound proteins were eluted by boiling in SDS sample buffer and resolved by SDS-PAGE. 35 S-Labeled proteins were then detected by autoradiography (A, lanes 1-9), and total proteins by Coomassie blue staining (B). Note the presence of Hsp90 in the p50 (but not p50␦C) immunoprecipitates (IP) in the Coomassie blue-stained gel (B). Lanes 10 -12 in A show the translation reactions used for the binding reaction (Input). IgH and IgL, immunoglobulin heavy and light chains, respectively. Note that the control antibody Ig heavy chain migrated faster than the mAb M2 Ig heavy chain and comigrated with p50-FLAG.

FIG. 2. Association of purified mini-RT proteins with purified p50 and p50␦C.
GST-MiniRT1 and GST-MiniRT2 purified from bacteria were incubated with p50-FLAG-bound (A) or p50␦C-FLAG-bound (B) mAb M2-agarose beads or control beads. Bound proteins were eluted in SDS sample buffer and resolved by SDS-PAGE. Total proteins were then detected by Coomassie blue staining (upper panels), and the mini-RT proteins by Western blot analysis (lower panels) using the anti-HA mAb against the HA epitope inserted into the mini-RT proteins. IP, immunoprecipitates; IgH and IgL, immunoglobulin heavy and light chains, respectively. Note that the control antibody Ig heavy chain migrated faster than the mAb M2 Ig heavy chain and comigrated with p50-FLAG.

p50/CDC37 and Hepadnavirus Replication
packaged into the nucleocapsids. Because pgRNA packaging in hepadnaviruses depends on RT function, we predicted that p50, through its effect on the RT, may be required for efficient pgRNA packaging. The preferential effect of p50 and p50␦C on the single-stranded DNA intermediate (rather than the mature relaxed circular DNA) (Fig. 6A) also suggested that they might have affected pgRNA packaging. Using a native agarose gel assay of nucleocapsid assembly and RNA packaging (39), we could indeed show that the overexpression of p50 led to a stimulation of pgRNA packaging, whereas the expression of the dominant-negative mutant (p50␦C) had just the opposite effect and inhibited pgRNA packaging (Fig. 7). DISCUSSION In our attempt to understand the molecular basis for the interaction between the hepadnavirus RT and the host cell Hsp90 chaperone complex, we have now obtained in vitro and in vivo evidence for a specific interaction between the RT and one of the known Hsp90 cofactors, p50/CDC37. This study was initially inspired by the intriguing data base search result suggesting a close relationship between the RT and the protein kinase c-Raf, as judged by their amino acid compositional properties (PropSearch) (23). As p50 is thought to bind specifically to both Hsp90 and c-Raf (and several other Hsp90 kinase substrates) and thus target the chaperone to the kinase substrates, this result prompted us to test whether p50 might also help to target the RT to Hsp90. We have shown here that p50 could bind to the RT in vitro and in vivo independently of Hsp90. Indeed, the RT and p50 could interact directly, at least in vitro. In addition, the in vitro protein priming activity of the FIG. 3. Association of mini-RT proteins with p50 and p50␦C in cells. pcDNA-MiniRT1 and pcDNA-MiniRT2 were transfected into 293T cells together with pGST-p50, pGST-p50␦C, or pGST (as a vector control). Transfected cells were then lysed in lysis buffer. Insoluble materials were removed by pelleting in a microcentrifuge. The supernatant (soluble fraction) was incubated with GSH beads to pull down GST-p50, GST-p50␦C, or GST alone. Bound proteins were eluted with GSH (eluate). The soluble (lanes 1-6), insoluble (lanes 7-12), and eluate (lanes 13-18) materials were resolved by SDS-PAGE and detected by Western blot analysis using the anti-HA mAb against the HA epitope inserted into the mini-RT proteins (A) or by Coomassie blue staining (B). The intact MiniRT1 and MiniRT2 proteins and the degradation product containing the TP domain are indicated in A. In B, the arrowheads denote GST-p50 (lanes 3, 6, 9, 12, 15, and 18), GST-p50␦C (lanes 2, 5, 8, 11, 14, and 17), and GST (lanes 1, 4, 13, and 16). p50-associated Hsp90 evident in lanes 15 and 18 is also indicated.  3 and 4), or buffer alone (lanes 1 and 2). All reactions were supplemented with reticulocyte lysate (1 l), which is required to activate the RT. The 32 P-labeled mini-RT protein was then resolved by SDS-PAGE and detected by autoradiography.

p50/CDC37 and Hepadnavirus Replication
RT was inhibited by a dominant-negative inhibitor of p50, p50␦C. Furthermore, both viral DNA synthesis and RNA packaging in vivo were stimulated by p50 and inhibited by p50␦C.
It is not yet known how p50 recognizes the different protein kinases or the viral RT. As the PropSearch program indicated that the RT and c-Raf are highly related in their amino acid composition (but apparently not by sequence alignment), it is possible that this similarity in composition may in turn dictate certain common folding characteristics (pathways) or folding intermediates that are recognized by p50. Interestingly, the same program also identified other classes of Hsp90 substrates, including certain steroid hormone receptors, as related to the RT and c-Raf. Although p50 was originally thought to be a kinase-specific subunit of the Hsp90 complex (26), more recent results suggest that this may not be entirely the case, as other classes of Hsp90 target proteins, including some steroid receptors, may also interact with p50 (42)(43)(44). Furthermore, p50 has been found to be present in Hsp90 complexes together with other cochaperones which had been previously thought to be irrelevant for kinase functions (45,46) but have since been found to associate with at least some kinase substrates and to be required for their functions (47,48). Our results are therefore consistent with the emerging notion that p50 may not be a dedicated Hsp90 cofactor specific for the kinase substrates, as originally envisioned, but rather functions as a more general cochaperone interacting with a variety of different Hsp90 target proteins. This concept is also supported by the failure, so far, to identify any "dedicated" cochaperones that would target the Hsp90 complex to its increasingly wide array of substrates (19,20), except for the kinases. Furthermore, genetic experiments in yeast have suggested that Hsp90 folds structurally very different target proteins (e.g. glucocorticoid receptor and v-Src) by a similar mechanism and uses a comparable set of cochaperones (49,50). If, indeed, no specific targeting factors are responsible for the interaction between the Hsp90 chaper- The replication-incompetent DHBV construct YMHA, which harbors two amino acid substitutions in the RT active site abolishing viral DNA synthesis, but still allowing pgRNA packaging, was transfected into 293T cells together with pGST-p50 (lane 1), pGST-p50␦C (lane 2), or pGST (lane 3). A negative control plasmid expressing only the DHBV core protein (thus, no pgRNA packing was possible) was also transfected into 293T cells together with pGST-p50 (lane 4). Viral nucleocapsids harvested from transfected cells were resolved on a native agarose gel and transferred to nylon membrane. pgRNA packaged in the nucleocapsids was then detected by Southern blot analysis using an antisense DHBV riboprobe (see "Experimental Procedures" for details) (A, upper panel), and the viral capsid was detected by subsequent reprobing of the same membrane by Western blot analysis using a rabbit antiserum against the DHBV core protein (lower panel). pgRNA packaged in nucleocapsids was quantified by phosphorimaging and normalized to viral core protein levels. The normalized viral RNA packaging efficiencies are shown in B. p50/CDC37 and Hepadnavirus Replication one and its diverse substrates, the challenge now will be to identify the common properties that have to be shared among these different substrates and that are recognized by the chaperone. Whatever these properties turn out to be, it appears that both the TP and RT domains of the hepadnavirus RT may share some of these properties, as each of them could independently associate with p50 ( Fig. 3) (data not shown) and Hsp90 (29).
The functional importance of p50-RT association in RT activity and viral replication was supported by both in vitro and in vivo experiments. In vitro, a dominant-negative inhibitor of p50 (p50␦C) decreased reconstitution of protein priming activity by the reticulocyte lysate, suggesting that endogenous p50 present in the reticulocyte lysate played a positive role in RT reconstitution. Furthermore, the overexpression of p50 stimulated viral RNA packaging and DNA synthesis in transfected cells, whereas p50␦C, a dominant-negative inhibitor of p50 function, inhibited viral RNA packaging and DNA replication. These results thus strongly support a role for p50 in viral replication in vivo. Recently, we have developed a defined reconstitution system whereby five purified components of the Hsp90 complex, i.e. Hsp90, Hsp70, Hsp40, p60/Hop, and p23, are sufficient to partially reconstitute a functional mini-RT protein (51). Preliminary results so far suggest that p50 does not appear to further enhance the activity of the RT in this defined in vitro reconstitution system. 2 These results may suggest that p50 is required only for RT functions under the more physiological conditions either in the complex mixture of the cell lysate or in the living cell, e.g. by helping to target the RT to the Hsp90 complex. In the defined reconstitution system using purified factors, the requirement for p50 may be bypassed when the RT and the Hsp90 chaperone system are brought together under the selected in vitro conditions. However, other interpretations are possible, and we are currently investigating conditions under which p50 may play a role in RT functions also in this defined system.
Because p50␦C could still bind the RT, but not Hsp90, it probably inhibited RT activation by sequestering the RT away from the Hsp90 complex. In fact, we found that p50␦C could bind to the RT more strongly compared with p50 and shifted most of the RT into a detergent-insoluble fraction in the cell. Although the nature of the detergent-insoluble compartment in the cell to which the RT was shifted remains to be characterized, other Hsp90 target proteins have also been shown to partition to detergent-insoluble fractions when the chaperone function is impaired (52). The finding that p50␦C associated with the RT more strongly compared with p50 also suggests that p50 may only transiently associate with the RT: it helps to bring the RT to Hsp90 and then dissociates from the RT. In this scenario, p50␦C would remain bound to the RT because it cannot "transfer" the RT to Hsp90. The dynamics of chaperone-RT association in the cell are still unknown. Steroid receptors have been shown to associate with several different Hsp90 subcomplexes (each with a different cochaperone complement) along the maturation pathway (53,54). It is possible that the RT may also undergo a similar pathway of conformational maturation/activation. Preliminary results presented here showing that the overexpression of p50 appears to decrease the association of another cochaperone (p23) with the RT are consistent with this suggestion. Additional work will be necessary to elucidate the intricate and dynamic pathway of RT activation by the Hsp90 chaperone complex.


extfieldsofstudy: Biology Medicine

text:
The SUMO Ubiquitin-Protein Isopeptide Ligase Family Member Miz1/PIASx (cid:1) /Siz2 Is a Transcriptional Cofactor for TFII-I*

We have shown previously that a TFII-I-related protein, hMusTRD1/BEN, represses transcriptional activity of TFII-I. The repression by hMusTRD1/BEN was hy-pothesized to occur via a two-step competition mechanism involving a cytoplasmic shuttling factor and a nuclear cofactor required for transcriptional activation of TFII-I. Employing a two-hybrid approach with both yeast genomic and mouse cDNA libraries in parallel, we have identified the RING-like zinc finger containing Miz1/PIASx (cid:1) /Siz2, which is a ubiquitin-protein isopeptide ligase in the SUMO pathway, as the potential nuclear cofactor that interacts with both TFII-I and hMus-TRD1/BEN. Our conclusion is based on the following observations. First, the interactions are biochemically confirmed in mammalian cells where Miz1/mPIASx (cid:1) interacts with both TFII-I and hMusTRD1/BEN when these proteins are ectopically co-expressed. Second, co-expression of a nuclear localization signal-deficient mutant of Miz1/mPIASx (cid:1) with wild type TFII-I fails to alter the subcellular localization of the former. Finally, ectopically expressed Miz1/mPIASx

TFII-I belongs to a family of proteins characterized by the presence of I-repeats first identified in the founding member TFII-I (1)(2)(3)(4)(5)(6)(7)(8). Although TFII-I functions as a signal-induced transcriptional activator, the role of the related family member hMusTRD1/BEN is less clear. Both TFII-I and hMusTRD1/ BEN are mapped to the breakpoint regions of the 7q11. 23 Williams-Beuren syndrome deletion (reviewed in Ref. 2). Furthermore, genetic and biochemical analyses show that each of these proteins have multiple isoforms both in mice and in humans (7, 9 -11). The transcription functions of hMusTRD1/ BEN have not yet been well characterized biochemically. It was first reported (3) as a muscle specific activator of the troponin I gene. It also seems to function as an activator in yeast onehybrid assays (6). However, clear demonstration of its activator function is not yet obtained. Results from our laboratory suggest that hMusTRD1/BEN behaves as a specific repressor of TFII-I (10). Although each protein when individually expressed exhibits predominant nuclear localization in eukaryotic cells, TFII-I is excluded from the nucleus when it is co-expressed with hMusTRD1/BEN. Nuclear exclusion of TFII-I results in the repression of the TFII-I-responsive c-fos gene. A key to this novel nuclear exclusion function appears to be the serine stretch (ss) 1 in hMusTRD1/BEN because deletion of this stretch results in co-occupancy of both proteins in the nucleus (10). However, although the ⌬ss hMusTRD1/BEN failed to prevent nuclear localization of TFII-I, the transcriptional repression of TFII-I was still observed. The latter data led us to postulate that the repression by hMusTRD1/BEN involves the following two-step mechanism: a competition for a common cytoplasmic factor required for nuclear translocation and a competition for a nuclear cofactor required for transcriptional activation.
In an attempt to identify these putative factors that are shared by both the activator (TFII-I) and the repressor (hMus-TRD1/BEN), we performed a two-hybrid screen using TFII-I and hMusTRD1/BEN as baits. We screened both yeast genomic and mouse cDNA libraries with the rationale that these screens would ideally yield homologous proteins. We further set the criteria such that (a) the interacting proteins should interact with both hMusTRD1/BEN and TFII-I and (b) the nuclear cofactor must interact with both the wild type and the ⌬ss hMusTRD1/BEN, whereas the cytoplasmic factor should not interact with the latter. By employing such a double screen, we report that we have identified protein Siz2/Miz1/PIASx␤ that appears to be the nuclear cofactor that interacts with TFII-I as well as the wild type and the ⌬ss hMusTRD1/BEN. The yeast library screen yielded NFI1 (neck filament interacting protein) also known as Siz2 (after SAP and Miz) (12-14, 19 -21), whereas the mouse library screen yielded the corresponding homologue, Miz1/mPIASx␤. NFI1/Siz2 and Miz1/ mPIASx␤ interact more avidly with hMusTRD1/BEN than TFII-I both in yeast and in mammalian interaction assays. This RING-like zinc finger-containing factor is localized exclusively in the nucleus and failed to alter the nuclear residency of TFII-I or hMusTRD1/BEN in mammalian localization assays. Consistent with these properties, ectopic Miz1/mPIASx␤ augments transcriptional activation by TFII-I and alleviates ⌬ss hMusTRD1/BEN-mediated transcriptional repression.

EXPERIMENTAL PROCEDURES
Cell Culture COS7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 50 units/ml penicillin, and 50 g/ml streptomycin (Invitrogen). * This work was supported by National Institutes of Health Grant AI45150 (to A. L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *

Yeast Two-hybrid Assay
Yeast was transformed using the lithium acetate method (16). To screen the yeast genomic library and the mouse cDNA library, the S. cerevisiae pJA69-4A strain was sequentially transformed with a Gal4-BD bait fusion construct followed by transformation with the library plasmid. We used three yeast genomic DNA libraries (Y2HL-C1, Y2HL-C2, and Y2HL-C3) into pGAD-C(x) vectors (2 m, ADH1 promoter driving GAL4-AD-library fragment fusions, ADH1 terminator, LEU, Amp r ) (15) and various pools of WEHI-3 cDNA libraries (WEHI-3 is a mouse myelomonocytic leukemia cell line) (ATCC TIB 68) into pGADNOT vector (2 m, ADH1 promoter driving a NLS-GAL4-AD-cDNA library fragment fusions, ADH1 terminator, LEU, Amp r ) (17). The initial screen was done by selecting positives in SD ϪUra, ϪLeu, ϪAde or SD ϪUra, ϪLeu, ϪHis ϩ 1 mM 3-aminotriazole (3-AT; Sigma) plates. From the colonies that grew under these conditions, a ␤-galactosidase colony-lift assay was performed. The ones with strong ␤-galactosidase activity were selected, and the library plasmid DNA was isolated and re-transformed with the bait construct in yeast. Final selection of true positive interacting proteins was done by growing co-transformed yeast in SD ϪUra, ϪLeu, ϪAde, ϪHis ϩ 50 mM 3-AT ϩ X-␣-gal (Clontech) selection plates.

Transient Transfection and Luciferase Assays
Transfections were performed with LipofectAMINE (Invitrogen) as described (10). Before harvesting, cells were serum-starved for 14 -16 h and stimulated with 25 ng/ml of recombinant human epidermal growth factor (rhEGF; Sigma) for 4 h. Total transfected DNA was kept constant by the empty vectors pEBB or pEBG. Luciferase activity was assessed using the Dual Luciferase kit (Promega). Experiments were done at least three times with triplicate sets included each time.

Cell Extracts, GST Pull-down, and Immunoblotting
At 42 h post-transfection COS7 cells were harvested, washed twice in phosphate-buffered saline buffer, and lysed in lysis buffer (25 mM Tris-HCl (pH 8.0), 25°C, 100 mM KCl, 10% glycerol, 5 mM NaF, 2 mM Na 3 VO 4 , 0.1% Triton X-100) containing 1ϫ antiprotease mixture EDTA-free (Roche Molecular Biochemicals) for 30 min at 4°C. The lysate was clarified by centrifugation for 10 min at 14,000 rpm at 4°C. Total protein concentration was measured by the Bradford method (Bio-Rad). 800 g of whole cell extracts were used for immunoprecipitation with anti-TFII-I antibody, and 300 g were used for GST pull down. Incubations with protein A-agarose or glutathione-agarose beads (50 l, 1:1 slurry; Invitrogen and Sigma, respectively) were done for 2 h, rocking at 4°C. At the end of the incubation, the beads were washed 2 times with 1 ml of lysis buffer without detergents. After the final wash, 4ϫ SDS sample buffer was added to the beads, boiled at 100°C for 3 min, and loaded onto a 10% SDS-PAGE. Semi-dry transfer to nitrocellulose was done as described (18). The mouse monoclonal antibodies, anti-GST (GST-2, Sigma) and anti-GFP (JL-18, Clontech), were used at 1:3500 and 1:2000, respectively. Detection was done by enhanced chemiluminescence (PerkinElmer Life Sciences) using standard methods.

Identification of Siz2/Miz1/mPIASx␤ as a hMusTRD1/BEN and TFII-I Interacting Protein by Two-hybrid Screen-
In an attempt to identify the putative factors that are shared by both TFII-I and hMusTRD1/BEN, we performed a two-hybrid screen using both of these proteins sequentially as baits. We screened both yeast genomic library and a mouse cDNA library with the rationale that these screens would ideally yield homologous proteins. Indeed, by using this approach, the yeast library screen yielded NFI1/Siz2 (12)(13)(14), whereas the mouse library screen yielded the corresponding homologue, Miz1/mPIASx␤ (19) (Fig. 1). Although in all our analyses we have used the cDNA corresponding to Miz1, sequence analysis revealed that Miz1 is actually a truncated form of mPIASx␤ missing the N-terminal 124 amino acids. The PIAS proteins belong to a large family of proteins that share two highly conserved motifs ( Fig. 2) as follows: a SAP domain (a putative DNAbinding motif) presumably involved in chromosomal organization (12,20,21) and a RING-like zinc finger motif implicated in protein-protein interactions (22)(23)(24). Importantly, both NFI1/SIZ2 and Miz1/mPIASx␤ interacted better with ⌬ss hMusTRD1/BEN. Our final selection of true positive clones was determined under extremely stringent conditions ( Fig. 1). Hence, the observation that TFII-I yeast cotransformants grow much slower than wt or ⌬ss hMusTRD1/ BEN yeast co-transformants reflects the strength of interaction. We conclude that Siz2/Miz1/mPIASx␤ interaction with hMusTRD1/BEN in yeast is stronger than the interaction of Siz2/Miz1/mPIASx␤ with TFII-I.
In Vivo Interactions of Miz1/mPIASx␤ with TFII-I or hMus-TRD1/BEN-After establishing interactions of Siz2/Miz1/mPI-ASx␤ with both TFII-I and hMusTRD1/BEN in yeast, we wanted to corroborate these interactions in mammalian cells. Furthermore, to determine which regions of Miz1/mPIASx␤ are important for potential physical and functional interactions with TFII-I and hMusTRD1/BEN, we also generated a series of C-terminal mutants (Fig. 3A). For interaction studies, we either expressed GST-tagged Miz1/mPIASx␤ (wild type or mutants) alone or with GFP-tagged TFII-I (Fig. 3B) or with GFPtagged hMusTRD1/BEN (Fig. 4) in COS7 cells. The potential interactions were analyzed either by immunoprecipitation (for TFII-I, Fig. 3B, upper panel) or by GST pull down (for hMus-TRD1/BEN, Fig. 4, upper panels). These results revealed that wild type GST-Miz1/mPIASx␤ was immunoprecipitated when co-expressed with GFP-TFII-I (  2 and 4). Whereas deletion of a portion of the RING/zinc finger (amino acids 231-236, ⌬ZF) or deletion of a putative nuclear localization signal (⌬NLS1) did not alter the interaction of Miz1/mPIASx␤ with TFII-I (lanes 5 and 6), deletion of the putative second NLS (⌬NLS2) significantly diminished the interaction with TFII-I (lane 7). The amount of immunoprecipitated TFII-I in all lanes was comparable except in lane 5 (bottom panel). However, when normalized to the precipitated protein in lane 1, it appears that the interaction of TFII-I with ⌬ZF is even better than the wild type Miz1/mPI-ASx␤. These results suggested that interaction of Miz1/mPI-ASx␤ with TFII-I is mediated largely through the N-terminal portion of Miz1/mPIASx␤ that does not include the RING/zinc finger.
Identical results were obtained with hMusTRD1/BEN (Fig.  4, top and bottom panels). ⌬ZF Miz1/mPIASx␤ interacted better than the wild type protein with hMusTRD1/BEN (compare lanes 3 and 8). Because hMusTRD1/BEN is expressed less than TFII-I in general, we observed fainter bands in GST pull-down assays with GST-Miz1/mPIASx␤. The GST pulldown assay was performed due to a lack of specific antibodies against either hMusTRD1/BEN or Miz1/mPIASx␤. Despite these differences in assay conditions, it is clear that both TFII-I and hMusTRD1/BEN interact with Miz1/mPIASx␤ through the N-terminal end of Miz1/mPIASx␤. It is also worth mentioning that ⌬ss hMusTRD1/BEN also interacted with Miz1/mPIASx␤ in GST pull-down assays (data not shown).
Identification of a Functional Nuclear Localization Signal (NLS2) in Miz1/mPIASx␤-Although members of PIAS family of proteins have been shown to exhibit nuclear residency, no definitive localization for Miz1/mPIASx␤ has been documented. To determine the functional nuclear localization signal in Miz1/mPIASx␤, either wild type or various mutants were expressed as GST fusion proteins in COS7 cells, and their localization was determined by indirect immunofluorescence (Fig. 5). Miz1/mPIASx␤ (wt) was localized exclusively to the nucleus when expressed individually. Although deletion of NLS1 (⌬NLS1) did not have any significant effect, deletion of NLS2 (⌬NLS2) profoundly altered the nuclear residency of Miz1/mPIASx␤, suggesting that NLS2 is the functional sequence under our conditions and in this cell type. Neither deletion of the zinc/RING finger motifs (⌬ZF) nor deletion of the C-terminal 141 amino acids (⌬C141) had significant effect on the nuclear localization of Miz1/mPIASx␤. Interestingly, ⌬ZF localizes in nuclear dots far more than the wild type protein. Also a slight cytoplasmic spreading out observed with overexpression of ⌬C141 may be due to the fact that the deletion is too close to NLS2. As anticipated, further deletion from the Cterminal end (⌬C237 and ⌬C382) had severe effect on the FIG. 1. NFI1/SIZ2 and Miz1/mPI-ASx␤ interact with TFII-I and hMus-TRD1/BEN wt or ⌬ss in yeast. GAL4-BD-TFII-I, GAL4-BD-hMusTRD1/BEN wt, and GAL4-BD-hMusTRD1/BEN ⌬ss were used as baits in the two-hybrid screening of a yeast genomic DNA library and a mouse cDNA library, both containing GAL4-AD fusion proteins. Using wild type GAL4-BD-hMusTRD1/BEN as the bait yielded NFI1/SIZ2 from the yeast library and the mammalian homologue Miz1/mPIASx␤ from the mouse library (denoted as mPIASx␤ in all figures). Both NFI1/SIZ2 and Miz1/mPIASx␤ also interact with TFII-I, wt hMusTRD1/BEN, or ⌬ss hMusTRD1/BEN in yeast, as shown by growth in SD ϪUra, ϪLeu, ϪAde, ϪHis ϩ 50 mM 3-AT ϩ X-␣-gal (Clontech) selective plates.
nuclear localization of Miz1/mPIASx␤ because both of these mutations remove the functional NLS2 (Fig. 5).
Localization of Miz1/mPIASx␤ NLS Mutant Is Unaltered Upon Co-expression With TFII-I or hMusTRD1/BEN-We ra-tionalized that if Miz1/mPIASx␤ is indeed a nuclear cofactor for TFII-I, then it should have a nuclear residency even in the presence of TFII-I. To test this idea, either wild type or various mutants of GST-Miz1/mPIASx␤ were co-expressed with GFP-TFII-I or GFP-hMusTRD1/BEN in COS7 cells, and their localization was determined by indirect immunofluorescence. As expected, there was no change in the nuclear localization of either Miz1/mPIASx␤ or TFII-I when these proteins were coexpressed and both co-localized in the nucleus (Fig. 6). Identical results were also obtained when Miz1/mPIASx␤ was coexpressed with hMusTRD1/BEN (Fig. 6). However, these results do not indicate whether the interactions between Miz1/ mPIASx␤ and TFII-I or hMusTRD1/BEN occur in the nucleus. In order to test whether the interactions are occurring in the cytoplasm or nucleus, we employed the ⌬NLS2 mutant of Miz1/ mPIASx␤. Our rationale was that if the two proteins interact stably in the cytoplasm, wild type TFII-I or wild type hMus-TRD1/BEN should be able to "carry" the NLS mutant Miz1/ mPIASx␤ to the nucleus. However, no significant change in cytoplasmic localization of ⌬NLS2 Miz1/mPIASx␤ was observed when co-expressed with either TFII-I or with hMus-TRD1/BEN. These data suggests that either interaction between these proteins is not strong enough or that it occurs inside the nucleus. Based on the fact that we observe a greatly diminished interaction of the ⌬NLS2 Miz1/mPIASx␤ with TFII-I or hMusTRD1/BEN, we prefer the latter explanation. In converse experiments both a nuclear signal deficient-TFII-I or a nuclear-deficient hMusTRD1/BEN remains in the cytoplasm when co-expressed with wild type Miz1/mPIASx␤ further corroborating our idea (data not shown). We have also observed that the nuclear localization of TFII-I does not change substantially when it is ectopically expressed in a yeast strain that lacks the SIZ2 gene and subsequently complemented with ectopic Miz1/mPIASx␤. 2 Because we observed an enhanced interaction between the ⌬C382 and TFII-I or hMusTRD1/BEN, we also wanted to test whether such interactions might lead to alteration in nuclear localization of the latter proteins. However, upon co-expression of TFII-I or hMusTRD1/BEN with ⌬C382 Miz1/mPIASx␤, we did not observe any significant  change in nuclear localization of TFII-I or hMusTRD1/BEN. Although 77% of ectopically TFII-I remained in the nucleus when co-expressed with wild type Miz1/mPIASx␤, 70% of nuclear TFII-I was found when it is co-expressed with ⌬C382 Miz1/mPIASx␤. Thus, the enhanced interaction of TFII-I or hMusTRD1/BEN with ⌬C382 Miz1/mPIASx␤ most likely occurred at a post-lysis stage.
Miz1/mPIASx␤ Is a Co-activator of TFII-I Transcriptional Activity-Because TFII-I and Miz1/mPIASx␤ co-localize in the nucleus, we were interested to test whether Miz1/mPIASx␤ would augment or inhibit transcriptional activity of TFII-I. However, if Miz1/mPIASx␤ is indeed the nuclear cofactor that we sought, then it should co-activate rather than repress. In transient co-transfection assays with c-fos as a reporter and in the presence of rhEGF, TFII-I activated the c-fos promoter in a dose-dependent fashion (Fig. 7, lanes 2 and 3) over the vectoronly lanes (lane 1). Miz1/mPIASx␤ alone also gave a 3-fold increase in promoter activity in the presence of rhEGF (lanes 4 and 5). Co-expression of low but constant levels of TFII-I with increasing levels of Miz1/mPIASx␤ resulted in nearly an additive activation of the c-fos promoter (lanes 6 and 7). We conclude that Miz1/mPIASx␤ augments transcriptional activity of TFII-I, although the co-activity is not very high. Nevertheless, the increase in transcriptional activity is not due to an increase in TFII-I expression as the levels of expression of ectopic TFII-I decreases when both proteins were co-expressed (compare lanes 2 and 3 with 6 and 7, bottom panels). The increase in transcriptional activity seen with Miz1/mPIASx␤ alone most likely reflects activation of endogenous TFII-I, although this remains to be formally proven. In the same assay we also tested the co-activation potentials of two mutants of Miz1/ mPIASx␤, ⌬ZF and ⌬C382. ⌬ZF showed modest activation potential on its own (compare lanes 1 with 8 and 9) and retained modest co-activation potentials with TFII-I (compare lanes 2 with 10 and 11) despite the fact that the activities were lower than observed with wild type Miz1/ mPIASx␤. However, ⌬C382 showed neither activation (lanes 12 and 13) nor any significant co-activation (lanes 14 and 15) potentials. We conclude that the zinc finger may not be essential for co-activation function of Miz1/mPIASx␤ and that nuclear residency of Miz1/mPIASx␤ is required for its TFII-I-dependent transcription function.

Miz1/mPIASx␤ Can Alleviate hMusTRD1/BEN ⌬ss-mediated Repression of TFII-I Transcriptional Activity-
We showed before (10) that both wild type and serine stretch mutant (⌬ss) hMusTRD1/BEN repress TFII-I-dependent transcriptional activity. We also proposed a two-step mechanism for such inhibitory effects. In the first step, hMusTRD1/BEN excluded TFII-I from the nucleus either by impeding nuclear import or by accelerating nuclear export. This was primarily attributed to a functional region with a stretch of 12 serines because the ⌬ss mutant failed to impede nuclear occupancy of TFII-I. However, although ⌬ss mutant co-localized to the nucleus with TFII-I, it still inhibited transcriptional activity of TFII-I (10). This led us to postulate that there must be an additional step involving a nuclear component responsible for such inhibition. This nuclear component could be a co-activator of TFII-I that is titrated by either wild type or ⌬ss mutant hMusTRD1/BEN (10). Therefore, this putative nuclear cofactor must interact with TFII-I, wild type, and ⌬ss mutant hMusTRD1/BEN in the nucleus. Given that Miz1/mPIASx␤ interacted with all three proteins and appeared to be exclusively in the nucleus, we tested whether Miz1/mPIASx␤ could be the potential nuclear cofactor. If it is, then upon overexpression Miz1/mPIASx␤ would alleviate ⌬ss-mediated repression of TFII-I transcriptional activity. For this experiment we chose suboptimal levels of TFII-I (Fig.  8, lane 2). As seen in the previous experiment (Fig. 7), although high levels of Miz1/mPIASx␤ alone showed 3-fold activation over the vector-only lanes (compare lanes 1 and 3), ⌬ZF and ⌬C382 failed to do so (compare lanes 1 with 4 and 5). The ⌬ss mutant hMusTRD1/BEN slightly repressed even the basal transcriptional activity (compare lanes 1 and 6). This repression was significantly relieved by wild type Miz1/mPIASx␤ but not by ⌬ZF or ⌬C382 mutants (compare lanes 7-9). Moreover, as observed before, ⌬ss also repressed TFII-I-dependent activation (compare lane 2 with 10). Most importantly however, ectopic co-expression of wild type Miz1/mPIASx␤ with TFII-I and hMusTRD1/BEN ⌬ss almost fully relieved ⌬ss-mediated transcriptional repression in a dose-dependent fashion ( lanes  11 and 12). In contrast, ⌬ZF had a moderate effect on relief of repression (lanes 13 and 14) and ⌬C382 totally lacked such ability (lanes 15 and 16). Western blot analyses show that the levels of expression of ectopic proteins were comparable between different lanes. Taken together, these data suggest that  6 and 7), GST-Miz1/mPIASx␤ ⌬C141 (lanes 9 and 10), GST-Miz1/mPIASx␤ ⌬C237 (lanes 11 and 12), or GST-Miz1/mPIASx␤ ⌬C382 (lanes 13 and 14) alone or together with 10 g of plasmid encoding GFP-hMusTRD1/BEN (lanes 1, 3, 5, 7, 8, 10, 12, and 14). Blots were probed first with anti-GFP antibody (␣-GFP) (upper panels), stripped, and reprobed with anti-GST antibody (␣-GST) (lower panel).
Miz1/mPIASx␤ is indeed a nuclear cofactor for TFII-I that can be titrated out by hMusTRD1/BEN leading to transcriptional repression of TFII-I-dependent promoters. Moreover, such function of Miz1/mPIASx␤ is dependent upon its nuclear localization.

DISCUSSION
The TFII-I family of proteins currently has two members, each with multiple isoforms (25). One distinguishing feature about these proteins is that they are genetically linked in human chromosome 7 (7q11.23) and are deleted in Williams-Beuren syndrome (25). Perhaps a more interesting feature is the fact that although TFII-I is a transcriptional activator, under the same conditions its family member hMusTRD1/BEN behaves as a repressor (10). In an attempt to elucidate the molecular mechanisms governing this family of proteins, we undertook a two-hybrid screen with the notion that interacting partners may provide useful hints to the biochemical modes of action of these proteins. We performed a parallel screen with both yeast and mammalian libraries. Although this was not the most important criterion for screening, we rationalized that if homologous proteins are pulled out from these two screens, it will provide a compelling evidence for bona fide interactions. Out of the three clones that survived this criterion, only Siz2/ Miz1/PIASx␤ appeared to be a potential nuclear factor with transcriptional properties (26 -30). Although we used hMus-TRD1/BEN as our primary bait for screening these libraries, we also tested both TFII-I and ⌬ss hMusTRD1/BEN as interacting partners of the isolated putative clones. In yeast interaction studies, NFI1/Siz2 and Miz1/mPIASx␤ interacted with TFII-I, hMusTRD1/BEN, as well as ⌬ss mutant of hMusTRD1/ BEN (Fig. 1). Hence, we proceeded to characterize the physical and functional interactions of Miz1/mPIASx␤ with TFII-I and hMusTRD1/BEN in mammalian cells and to test whether Miz1/mPIASx␤ is the nuclear cofactor of TFII-I that is competed by hMusTRD1/BEN leading to transcriptional repression.
The interactions observed in yeast were recapitulated in mammalian cells upon co-expression of Miz1/mPIASx␤ with either TFII-I or hMusTRD1/BEN (Figs. 3 and 4). Although it appears that a fraction of input TFII-I or hMusTRD1/BEN interacts with Miz1/mPIASx␤ under the assay conditions, whether this is also true under more physiologic conditions could not be determined because an antibody against endogenous Miz1/mPIASx␤ is not available, and thus co-immunoprecipitation experiments could not be performed. Moreover, whether under normal conditions these interactions take place in the nucleus also could not be determined. Although Miz1/ mPIASx␤ belongs to a large family of proteins (Fig. 2), the majority of which have nuclear localization, precise subcellular localization of Miz1/mPIASx␤ has not been demonstrated. Hence, to follow further characterization of Miz1/mPIASx␤ and its interactions with TFII-I family proteins, it was important for us to determine unequivocally the subcellular localization of Miz1/mPIASx␤ at least when ectopically expressed. Under our assay conditions, Miz1/mPIASx␤ localized exclusively to the nucleus. Interestingly, the staining revealed a speckled pattern with nuclear pod-or dot-like structures that are reminiscent of promyelocytic leukemia (PML) bodies. Our preliminary data suggest that the nuclear dots observed with Miz1/mPIASx␤ indeed co-localize with PML bodies. However, the functional consequences of this co-localization are unclear at present. The localization of TFII-I and hMusTRD1/BEN in PML bodies is under study at present.
Data base scanning (PSORT) (31) revealed two consensus nuclear localization signals in Miz1/mPIASx␤, termed NLS1 and NLS2. Of these, NLS1 appears to be non-functional at least under our assay conditions because deletion of NLS1 did not alter the nuclear localization of Miz1/mPIASx␤. The NLS2 is functional because the deletion of NLS2 causes a major change in the subcellular localization of Miz1/mPIASx␤. It is also clear that co-expression of TFII-I or hMusTRD1/BEN does not increase the nuclear localization of NLS mutant Miz1/mPIASx␤. Conversely, wild type Miz1/mPIASx␤ also failed to alter the localization of a nuclear localization deficient mutant of TFII-I (9) or a nuclear localization deficient mutant of hMusTRD1/ BEN when both proteins were co-expressed (data not shown). Despite the fact that the wild type proteins interact, the failure of a wild type protein to alter the localization of the partner mutant could be either due to the fact that these proteins interact weakly or transiently or that they interact exclusively in the nucleus. Although these scenarios are not yet clear, the fact that protein substrates must be targeted to the nucleus in order to undergo SUMO-1 conjugation (32), a process dependent on PIAS family proteins, favors the latter idea. TFII-I, on its own, is a weak transcriptional activator. Miz1/mPIASx␤ also has a weak transcriptional activity in the absence of ectopically expressed TFII-I. Together they appear to have an additive effect. It is not clear at present whether Miz1/mPI-ASx␤ binds to c-fos promoter directly in a sequence-specific fashion or whether it is a transcriptional cofactor for TFII-I. Although Miz1/mPIASx␤ can bind sequence specifically to dou- ble strand DNA in gel shift assays, the precise consensus sequence or the target genes are not well characterized yet (19). It is worthwhile to point out that various members belonging to the PIAS family (Fig. 2), viz. androgen receptor interacting protein (ARIP3) (26,27), protein inhibitor of activated STATs (PIAS1, PIAS3, and PIASy) (28 -30), and Miz1 (19), have all been shown to possess varying degrees of transcriptional activity. However, it is not clear at present whether Miz1/mPIASx␤ can indeed behave as a gene-specific transcriptional activator or it behaves more as a transcriptional co-regulator together with TFII-I. Although it is very likely that the full range of transcriptional activity by Miz1/PIASx␤ requires its interaction with TFII-I and its N-terminal regions, the activation of c-fos by Miz1/PIASx␤ in the absence of ectopic TFII-I makes it more difficult to pinpoint its mechanism of action. Accordingly, changes in transcriptional activity of the mutants of Miz1/ PIASx␤ could be independent of TFII-I. This issue has to be addressed by determining whether Miz1/mPIASx␤ has a consensus binding site in the c-fos promoter and, if so, whether Miz1/mPIASx␤ binds to this site and activates it. Given the fact that STAT1 and STAT3 activate the c-fos promoter, we also tested the effect of PIAS1 and PIAS3 on TFII-I-dependent activation. However, in our hands, we failed to see any significant effects of either PIAS1 or PIAS3 on TFII-I-dependent activation of the c-fos promoter (data not shown). Most importantly, Miz1/mPIASx␤ relieved the ⌬ss hMusTRD1/BEN-mediated transcriptional repression. Thus, under conditions in which TFII-I-dependent activation was completely abrogated by ⌬ss hMusTRD1/BEN, co-expression of Miz1/mPIASx␤ rescued such repression. Miz1/mPIASx␤ alone, in the absence of TFII-I, also prevented ⌬ss hMusTRD1/BEN-mediated transcriptional repression, although the relief of repression was less than achieved in the presence of TFII-I. This result suggests that Miz1/mPIASx␤ requires TFII-I for its full range of activity under our assay conditions, leading us to believe that Miz1/mPIASx␤ in this context might be acting more as a transcriptional co-regulator of TFII-I rather than as an independent transcriptional activator. Regardless, it is clear that Miz1/ mPIASx␤ is a nuclear co-regulator or cofactor for TFII-I that is titrated away by hMusTRD1/BEN leading to transcriptional repression. The precise mechanism of this pathway remains to be elucidated.
The PIAS family of proteins is characterized by the presence of an N-terminal SAP domain believed to be involved in chromosome organization (12,21,22). However, because a truncated form of mPIASx␤ (Miz1, lacking the SAP domain) interacts with TFII-I, the SAP domain is probably dispensable for such interactions. This family is also characterized by the presence of a RING-like zinc finger motif, presumably involved in protein-protein interactions (22)(23)(24). Compelling evidence supports the involvement of a number of different and functionally distinct RING finger proteins in ubiquitin-and SUMO-dependent pathways (22,23). Recent studies (13,14,(33)(34)(35)(36)(37) have demonstrated that SIZ2, PIASx␣, PIASx␤, PIASy, PIAS1, and PIAS3 behave as ubiquitin-protein isopeptide-SUMO-ligases that sumoylates septins and several transcription factors (13,14,(33)(34)(35)(36). In this regard, it has been shown that the enzymatic activity of PIAS family proteins is dependent on its zinc/RING finger domains (33,34). However, our data reveal that deletion of amino acids 231-236 coding for central CTH-LQC zinc/RING finger of Miz1/mPIASx␤ (⌬ZF) does not significantly affect its interaction with TFII-I or hMusTRD1/BEN and that the functional effects of these mutations are rather moderate. However, lack of enzymatic activity of this mutant needs to be formally proven.
Despite the fact that Miz1/mPIASx␤ is not yet well characterized, many of its family members have been shown to have exciting properties that control multiple aspects of chromosome structure and function by establishing or maintaining chromosome organization in interphase nuclei (12,13,21,33,38). Taken together these data suggest that besides its role in transcriptional activation or repression, the PIAS family of proteins will have a definite role in other chromosomal functions as well. One of the other clones obtained from the twohybrid screens that interacted with both TFII-I and hMus-TRD1/BEN was Ubc9, a ubiquitin-conjugating enzyme involved in SUMO modification. 2 These data raise the possibility that TFII-I family of proteins may undergo SUMO modification and are also likely to be involved in SUMO-dependent pathways that control transcription. However, given that ⌬ZF interacts with TFII-I and partially functions as a co-activator, it appears that the interactions of TFII-I family proteins with Miz1/mPIASx␤ may be independent of enzymatic activity of Miz1/mPIASx␤. It has been observed in other cases that PIAS proteins can tether other sumoylated proteins in a noncovalent fashion (37). Whether TFII-I family proteins serve a regulatory role in this latter process remains to be determined.