The Hepatitis B pX Protein Promotes Dimerization and DNA Binding of Cellular Basic Region/Leucine Zipper Proteins by Targeting the Conserved Basic Region*

Giovanni Perini, Elke OetjenDagger , and Michael R. Green§

From the Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hepatitis B virus pX protein is a potent transcriptional activator of viral and cellular genes whose mechanism of action is poorly understood. Here we show that pX dramatically stimulates in vitro DNA binding of a variety of cellular proteins that contain basic region/leucine zipper (bZIP) DNA binding domains. The basis for increased DNA binding is a direct interaction between pX and the conserved bZIP basic region, which promotes bZIP dimerization and the increased concentration of the bZIP homodimer then drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one another and from that of the bZIP itself. Thus, through recognition of the conserved basic region, pX promotes dimerization, increases DNA binding, and alters DNA recognition. These properties of pX are remarkably similar to those of the human T-cell lymphotrophic virus type I Tax protein. Although Tax and pX are not homologous, we show that the regions of the two proteins that stimulate bZIP binding contain apparent metal binding sites. Finally, consistent with this in vitro activity, we provide evidence that both Tax and pX activate transcription in vivo, at least in part, by facilitating occupancy of bZIPs on target promoters.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatitis B virus (HBV)1 is the major cause of acute and chronic hepatitis and has been implicated in the initial stage of HBV-related hepatocarcinogenesis (1-5). The HBV genome is 3.2 kb in length and contains four open reading frames: pre-S/S, which encodes the surface antigens; core/e, which encodes the core protein; pol, which encodes the viral reverse transcriptase; and the X protein (pX) (for review, see Ref. 6). pX is required for viral infection (7, 8), can transform rodent cells (3, 4, 9, 10), and can cause hepatocellular carcinoma in transgenic mice (11).

HBV pX, a 154-aa protein, is a potent transcriptional activator required for efficient viral gene expression. Like several other viral activators, pX activity is promiscuous; in addition to the cognate HBV promoter, pX can activate transcription from a diverse set of cellular and viral promoters that appear to have little in common (12-24). The mechanism by which pX activates transcription is controversial; it has been proposed that pX acts through various protein kinase signal transduction pathways (25-28), is itself a protein kinase (29), has a ribo/deoxy ATPase activity (30), and is a protease inhibitor (31).

We have noted several similarities between HBV pX and the Tax protein of human T-cell lymphotrophic virus, type I (HTLV-I). First, like pX, Tax activates transcription from its own promoter as well as heterologous promoters (for review, see Ref. 32). Second, the HTLV-I long terminal repeat and the HBV enhancer contain binding sites for ATF proteins (ATFs) (33-35), an extensive family of cellular transcription factors that contain homologous basic region/leucine zipper (bZIP) DNA binding domains (36). These viral ATF binding sites have been directly implicated in the Tax (reviewed in Ref. 37) and pX (33) transcriptional responses. Third, like Tax, pX is present in the nucleus of expressing cells (10, 38). Finally, and of particular significance, Tax can substitute for pX in transcriptional activation of the HBV enhancer (2, 39, 40). Taken together, these observations suggest that pX and Tax may stimulate transcription by a common mechanism.

Recent studies have helped clarify the mechanism by which Tax functions. Tax can dramatically increase the in vitro DNA binding of proteins containing bZIP DNA binding domain (41-45). bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact (46-49). This DNA binding increase occurs by a mechanism in which Tax promotes dimerization of the bZIP domain in the absence of DNA and the elevated concentration of the bZIP homodimer then drives the DNA binding reaction (43, 44). Here we show that pX functions in a manner remarkably similar to Tax.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oligonucleotide Sequences for Gel-shift Assays-- Sequences were as follows: collagenase TRE, 5'-AGCTTTGACTCATCCGGA-3'; CRE, 5'-TCCTAAGTGACGTCAGTGGAA-3'; C/EBP binding site, 5'-TGCAGATTGCGCAAT CTGCA-3'; OR1 site of the lambda  phage PRM/PR promoter, 5'-TATCACCGCCAGAGGTA-3'.

Purification of pX Protein-- pX cDNA was cloned in the pRSET-C vector (Invitrogen) and expressed as a His-tagged derivative in the BL21(pLysE) Escherichia coli strain. BL21 cells were grown at 30 °C in the presence of 50 µM ZnCl2, and pX expression was induced with 0.5 mM IPTG for 2 h. Cells were harvested and resuspended in lysis buffer (1 M NaCl, 20% glycerol, 0.1% Tween 20, 20 mM Tris-HCl, pH 8, 20 µM ZnCl2, 1 mM PMSF, 10 mM beta -mercaptoethanol, 40 mM imidazole), frozen on dry ice, and then thawed on ice and sonicated. The bacterial lysate was centrifuged for 30 min at 12,000 rpm. The supernatant was incubated with NTA-Ni2+-agarose beads (Qiagen) for 4 h. Beads were washed four times with lysis buffer. pX protein was eluted with 0.5 M imidazole, pH 7.5, and dialyzed twice against buffer X (0.2 M NaCl, 20% glycerol, 20 mM Hepes, pH 8, 20 mM ZnCl2, 5 mM beta -mercaptoethanol, 0.1% Tween 20, 1 mM PMSF).

pX and Tax Deletion Mutants-- pX was first cloned in the pGEX-CS vector (50) and subsequently digested with the appropriate enzymes to generate CDelta 1, CDelta 2, and CDelta 3 mutants. To generate CDelta 1, pX vector was digested with HincII and HindIII, treated with Klenow, and self-ligated. CDelta 2 and CDelta 3 were obtained in the same way except that CSP1/HindIII and AatII/HindIII were used, respectively. Ligation products were transformed in the E. coli BL21 (pLysE) strain. CDelta 4 was instead obtained by PCR amplification of the region between aa 1 and 36. The primers were oligo X5' (CAT GCC ATG GCT GCT CGG GTG TGC TGC) and oligo XDelta 4 (CGG AAT TCT TAT TAG AGA GTC CCA ACC GGC CCG CA). The PCR product was then digested with NcoI and EcoRI and cloned in pGEX-CS vector. pX N-terminal deletions were also obtained by PCR amplification. NDelta 1 sequences were oligo X N1 (CAT GCC ATG CGT CCC GTC GGC GCT GAA TCC) and oligo X3' (CGG AAT TCT TAT TTA GGC AGA GGT GAA AAA CAA AC). NDelta 2 sequences were oligo X N2 (CAT GCC ATG CTC TCT TTA CGC GGT CTC CCC) and oligo X3. PCR products were digested with NcoI/EcoRI and cloned in the pGEX-CS vector. Protein expression was induced with 0.5 mM IPTG for 2 h at 30 °C. Protein mutants were purified on a glutathione affinity column and then dialyzed against buffer X. The GST moiety was removed by cleaving the GST fusion proteins with TEV protease (Life Technologies, Inc.) and by incubating the protein with fresh glutathione-agarose beads. Beads were spun down, and the supernatant containing the pX proteins was dialyzed against buffer X. Tax mutants were obtained by PCR amplification. Oligonucleotides used to generate Tax constructs were as follows: Tax wt: oligo T5' (TCC AAC AAC ATG GCC CAC TCC CCA GGG TTT GGA) and oligo T3' (AAA GGG GGA TCC TCA GAC TTC TGT TTC TCG GAA ATG); Tax CDelta 1: oligo T5' and oligo TD31 (AAA GGG GGA TCC TCA ATG AAA GGA AGA GTA CTG TAT GAG); Tax CDelta 2: oligo T5' and oligo TD32 (AAA GGG GGA TCC TCA GCC ATC GGT AAA TGT CCA AAT AAG); Tax CDelta 3: oligo T5' and oligo TD33 (AAA GGG GGA TCC TCA CCC TGT GGT GAG GGA AAT TTT ATA); Tax CDelta 4: oligo T5' and oligo TD34 (AAA GGG GGA TCC TCA GCA GAC AAC GGA GCC TCC CCA GAG); Tax CDelta 5: oligo T5' and oligo TD35 (AAA GGG GGA TCC TCA GGG TGG AAT GTT GGG GGT TGT ATG); Tax CDelta 6: oligo T5' and oligo TD36 (AAA GGG GGA TCC TCA CTG ATG CTC TGG ACA GGT GGC CAG). PCR products were digested with NcoI and BamHI and cloned in pGEX-CS vector. Proteins were expressed and purified as described previously (43).

DNA-binding Proteins-- cDNA sequences of CREB (aa 23-341), ATF-1 (aa 57-271), and ATF-2 (aa 144-505) were cloned in pGEX vectors and expressed as GST fusion proteins in E. coli. The C/EBP Delta 1-2 derivative (a gift from P. Rorth) was expressed by IPTG induction. After sonication, cell debris was removed by centrifugation and the supernatant used in gel-shift assays. Histidine-tagged JUN (aa 225-334) was obtained from T. Curran. Epstein-Barr virus ZTA peptide (aa 175-229) containing the DNA binding domain only was a gift from M. Carey. The FOS-ZTA chimera was obtained from D. S. Hayward. The construct containing the c-Fos basic region (aa 133-162) and the ZTA coiled-coil domain (aa 197-229) was PCR-amplified and cloned in the pGEX-2T vector. lambda -ZIP protein was obtained by IPTG induction of JH372(pJH370) E. coli (a gift from R. T. Sauer). Crude extracts were prepared and dialyzed against 1× buffer D. The synthetic peptide BR-CC (NH-ALKRARNTEAARRSRARKLQRMKQLEDVKELEEKLKALEEKLKALEEKLKALG-COOH; Ref. 51) was kindly provided by W. DeGrado, and the basic region peptide br1s (NH4-ALKRARNTEA-ARRSRARKLQRMKQGGC; Ref. 52) was a generous gift of P. Kim.

DNA Mobility Shift Assays-- Each reaction mixture contained 1 µg of BSA, 1 µg of poly(dI-dC), 0.5× buffer D, and 0.1 ng of a 32P-labeled CRE oligoduplex. The amount of DNA-binding protein and pX used is indicated in the figure legends. The reaction products were analyzed on a 5% polyacrylamide, 0.5× TBE gel except where otherwise noted.

Chemical Cross-linking-- The indicated amounts of GCN4 polypeptide were incubated with 200 ng of BSA or 100 ng of pX. Cross-linking was performed by adding glutaraldehyde (Fisher) to a final concentration of 0.02% and incubated for 30 min at room temperature. Reactions were terminated by addition of 0.5 µl of 0.5 M Tris, pH 8.0. Monomers and dimers were separated on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The filter was probed with an anti-GCN4 polyclonal serum (kindly provided by Peter Kim). 20 ng of H6-T7tag-pX were incubated for 10 min with glutaraldehyde to a final concentration of 0.01%. Multimers were separated on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with an anti-T7tag monoclonal antibody (Novagen).

Far-Western Analysis-- Purified proteins were separated on an SDS-polyacrylamide gel and transferred to an Immobilon membrane (Millipore). The filter was preincubated in far-Western buffer (150 mM KCl, 20 mM Hepes, pH 7.5, 5 mM MgCl2 0.5 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM PMSF, and 5% BSA). After 2 h of incubation, [35S]methionine-labeled pX was added and incubation continued for 4 h. The membrane was washed twice with far-Western buffer for 15 min, dried, and autoradiographed.

Cell Cultures and Transfections-- HepG2 cell were maintained in Dulbecco's modified Eagle's medium containing nonessential amino acids (Life Technologies, Inc.), 2 mM L-glutamine (Life Technologies, Inc.), antibiotics, and 10% FBS (HyClone). Cell transfections were carried out by using Opti-MEM reduced serum medium and LipofectAMINE reagent as described by the manufacturer (Life Technologies, Inc.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

pX Increases DNA Binding of Diverse bZIP Proteins-- To investigate the mechanism of pX action a series of recombinant bZIP proteins were tested for DNA binding in the presence and absence of pX. pX was expressed in E. coli as a histidine-tagged derivative and purified according to a nondenaturing procedure (see "Materials and Methods"). For reasons described below, DNA binding was performed at several protein concentrations, which, in the absence of pX, gave rise to a low level of DNA binding. Fig. 1 shows that, under these conditions, addition of purified pX greatly increased DNA binding of proteins of the ATF family (CREB, ATF-1, ATF-2), c-Jun family (c-Jun, GCN4), and C/EBP family (C/EBP). Significantly, however, pX did not stimulate DNA binding of all bZIPs (e.g. ATF-4; Fig. 5B). pX cannot enhance the DNA binding activity of the ATF-4 bZIP transcription factor (36). Neither an E. coli crude extract nor unrelated recombinant proteins stimulated DNA binding (data not shown; and see Ref. 43). pX comparably stimulated DNA binding of bZIP derivatives containing or lacking a GST moiety. Consistent with previous studies (33, 39), we could not detect an interaction between purified pX and DNA (data not shown).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   pX stimulates DNA binding of diverse bZIP proteins. Purified GST-ATF proteins, GST-JUN, C/EBP, and a GCN4 peptide were assayed for sequence-specific DNA binding. Proteins were titrated to minimize DNA binding in the absence of pX. Two different concentrations (1× = 5 ng, and 2×) of each affinity-purified GST fusion protein were tested in the presence of 1 µg of BSA (-) or 100 ng of affinity-purified His-tagged pX (+).

The pX-mediated DNA Binding Increase Is Dependent on bZIP Concentration-- To investigate the possible role of bZIP concentration in the pX-mediated DNA binding increase, we measured the effect of pX at GCN4 concentrations ranging from 0.05 to 20 nM. Fig. 2 shows that maximal stimulation of DNA binding was observed at peptide concentrations below 4 nM and, at higher protein concentrations, pX did not significantly increase DNA binding. The failure to increase DNA binding at higher bZIP concentrations was not due to a limiting amount of pX; DNA binding was again not affected when the concentration of pX was increased (data not shown). These results indicate that pX overcomes a concentration-dependent step that normally limits the extent of DNA binding.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2.   pX overcomes a concentration-dependent step that limits bZIP DNA binding. x axis, concentration of GCN4. y axis, pX-mediated DNA binding increase of GCN4. GCN4 at the indicated concentration was assayed for DNA binding to a TRE collagen oligonucleotide probe in the presence or absence of pX. Reaction products were fractionated on a 5% native polyacrylamide gel. The data were quantitated on a PhosphorImager.

pX Increases Formation of bZIP Homodimers in the Absence of DNA-- Dimerization of bZIP proteins occurs in the absence of DNA and is a prerequisite for DNA binding (51-54). pX could therefore stimulate DNA binding by increasing either dimerization or the subsequent interaction between the bZIP homodimer and DNA.

We measured the effect of pX on bZIP dimerization in the absence of DNA using a chemical cross-linking assay. Previous studies have shown that the two subunits of bZIP dimers can be cross-linked to one another with glutaraldehyde, a bifunctional cross-linking reagent (53). In Fig. 3A, GCN4 was incubated in the presence or absence of pX, and following addition of glutaraldehyde the products were fractionated on an SDS-polyacrylamide gel and analyzed by immunoblotting. At a concentration of 0.1 µM, GCN4 is predominantly a monomer (lane 2) and as the concentration of GCN4 was raised, homodimer formation increased (lane 5). Significantly, addition of pX dramatically increased the amount of GCN4 homodimer (compare lanes 2 to 3 and 5 to 6). An additional band with an approximate size of 46 kDa (arrow) appeared only when pX and GCN4 were both present. We suspect that this product results from a pX dimer cross-linked to a GCN4 dimer, but further experimentation would be required to confirm this supposition.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3.   pX promotes bZIP dimerization and requires an appropriate bZIP dimerization domain. A, chemical cross-linking of a GCN4 peptide was performed in the presence of 500 ng of BSA (lanes 2 and 5) or 100 ng of pX (lanes 3 and 6) at a final concentration of 0.02% glutaraldehyde. Reaction products were fractionated on a 15% SDS-polyacrylamide gel and detected by immunoblotting with a GCN4-specific polyclonal antiserum. The ~45-kDa product (arrow) is consistent with cross-linking of a GCN4 dimer to a pX dimer. B, increasing amounts (1× = 0.1 nM, 2×, 4×, 8×, or 16×) of a mixture containing equal molar concentrations of the GCN4 peptide and a cysteine-linked basic region peptide (br1s), were incubated together and assayed for DNA binding in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of 100 ng of pX. Reaction mixtures were incubated on ice and fractionated on an 8% polyacrylamide gel, run at 4 °C. Lower panel, amino acid sequence and schematic diagram of both peptides.

To confirm that bZIP dimerization was required for the pX-mediated DNA binding increase, we analyzed the ability of pX to stimulate DNA binding of a "pre-dimerized" protein. The synthetic peptide br1s (52) contains two GCN4 basic regions joined by a disulfide linkage. Fig. 3B shows that pX did not stimulate DNA binding of br1s, under the same conditions in which DNA binding of GCN4 was markedly enhanced. Thus, pX function requires a protein with an appropriate dimerization domain, consistent with the ability of pX to promote dimerization.

pX Does Not Recognize the Leucine Zipper-- The fact that pX can stimulate DNA binding of proteins containing only a bZIP (GST-JUN bZIP, GST-ATF2 bZIP, GCN4) implies that this domain is the target of pX action. To delineate the portion of the bZIP required for pX recognition, we analyzed a series of bZIP derivatives. Because pX increases dimerization, we began by examining the role of the leucine zipper. The synthetic peptide BR-CC contains the basic region of the yeast GCN4 bZIP protein attached to an idealized coiled-coil dimerization motif (51). BR-CC contains the canonical leucine repeat, but otherwise the dimerization domains of GCN4 and BR-CC bear no similarity (Fig. 4, bottom). BR-CC dimerizes and binds an AP-1 site with an affinity similar to GCN4 and Fig. 4 shows that pX stimulated DNA binding of BR-CC and GCN4 comparably.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   pX functions on bZIP proteins that contain a degenerate leucine zipper sequence. Two concentrations (1× = 0.1 nM, and 2×) of a GCN4 peptide, the idealized peptide BR-CC (51), a GST fusion of the Epstein-Barr virus ZTA protein, or a GST fusion of a FOS-ZTA chimera (55) were analyzed for DNA binding in the presence (+) or absence (-) of pX. Upper panel, schematic diagrams of the synthetic peptides and chimeric proteins. Lower panel, sequence alignment of the bZIP regions. Identical amino acids are marked by asterisks. The positions of the conserved leucines in the leucine zipper domain are highlighted and bold.

We next asked whether the leucines were important for pX recognition. The DNA binding domain of Zta, an Epstein-Barr virus protein, is a highly divergent member of the bZIP family (55, 56). Although the Zta basic region is similar to that of other bZIP proteins, the dimerization domain is not a typical leucine zipper. Nevertheless, this domain assumes a coiled-coil structure that supports dimerization and DNA binding. Fig. 4 (right) shows that pX efficiently stimulated binding of ZTA to the collagenase promoter AP-1 site. Similarly, pX increased DNA binding of a chimeric protein containing the human c-Fos basic region fused to the ZTA dimerization domain (Fig. 4, right). The combined data of Fig. 4 indicate that no specific sequence of the leucine zipper is required for pX function.

pX Recognizes the bZIP Basic Region-- To test whether the conserved basic region is the target for pX, we first analyzed a hybrid protein, lambda -ZIP, in which the DNA binding domain of bacteriophage lambda  repressor cI was fused in-frame to the GCN4 leucine zipper (57). lambda -ZIP efficiently dimerizes through the leucine zipper and binds to the OR1 site of bacteriophage phage PRM/PR promoter. Fig. 5A shows that pX failed to stimulate DNA binding of lambda -ZIP, indicating that the leucine zipper is not sufficient for pX responsiveness and suggesting an essential role for the basic region. Likewise, ATF4 (36), a bZIP protein with an atypical basic region that diverges at several residues from the consensus (Fig. 5B, bottom), was also not pX-responsive (Fig. 5B, top).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   pX requires the bZIP basic region to stimulate DNA binding. A, lambda -ZIP, a chimera containing the lambda  repressor DNA binding domain (aa 1-101) fused to the GCN4 leucine zipper (aa 250-281) was tested for binding to the OR1 site of the lambda  phage PRM/PR promoter in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of pX. A schematic diagram of the lambda -ZIP protein is shown below. B, several concentrations of GST-ATF4 protein were tested for binding to the ATF-C site (see "Materials and Methods") in the absence or presence of pX. Comparison of the ATF4 and GCN4 basic region is shown below. Non-conserved amino acids are shaded. C, upper panel, equal amounts of proteins were fractionated on a SDS-polyacrylamide gel and stained with Coomassie Blue for protein quantitation. Lower panel, far-Western analysis; purified GST-bZIP derivatives were fractionated on an SDS-polyacrylamide gel and transferred to an Immobilon membrane. The filter was probed with 35S-labeled pX. Excess probe was removed and bound pX visualized by autoradiography.

To confirm that pX interacted directly with the basic region, we performed a far-Western blotting experiment. bZIP derivatives were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with 35S-labeled pX. The results show that pX efficiently bound to proteins that contain a complete bZIP (Fig. 5C). In addition, pX interacted with a GST-ATF2 derivative containing the ATF2 basic region alone but not with ATF2 or c-Jun derivatives containing only the leucine zipper. Moreover, pX did not interact with ATF-4, consistent with its inability to stimulate ATF-4 DNA binding. These results provide strong evidence that pX binds to the bZIP basic region.

pX Alters DNA Recognition-- The fact that pX interacts with the basic region, which mediates DNA contact, prompted us to investigate the DNA binding specificity of pX-bZIP complexes. We analyzed the ability of pX to increase DNA binding of several bZIP proteins to four different DNA binding sites. These DNA probes contained an AP1 or an ATF consensus flanked by sequences derived from either the collagenase promoter (AP1-C, ATF-C) or a synthetic polylinker (AP1-S, ATF-S) (Fig. 6, bottom). Remarkably, the requisite combination of consensus binding site and flanking sequence differed for each bZIP protein tested. For example, pX stimulated DNA binding of CREB only to the AP1-C, ATF-C, and ATF-S sites. For GCN4, the pX-mediated DNA binding increase occurred with AP1-C, AP1-S, and ATF-C but was very modest with ATF-S. The effect of pX on FOS-ZTA DNA binding was maximal only with collagenase flanking sequences and independent of the core binding sites. In contrast, pX-mediated stimulation of ATF2 DNA binding depended only on the ATF core site.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 6.   A, pX alters bZIP DNA recognition. Two concentrations (1×, 2×) of GST-CREB (1× = 5 ng), GCN4 peptide (1× = 0.5 ng), GST-ATF2 (1× = 5 ng) and GST-Fos-ZTA (1× = 5 ng) were tested for DNA binding to a set of four DNA oligo-duplexes in the absence (-) or presence (+) of pX. DNA-bZIP complexes were separated on a 5% polyacrylamide gel at room temperature. The sequence of each DNA probe used is shown below the figure. The oligo-duplexes contain AP-1 (TGACTCA) or ATF (TGACGTCA) sites flanked either by the collagenase promoter sequence (italics) or the Bluescript polylinker sequence (shaded). B, intrinsic affinities of bZIPs to the AP-1 and ATF sites. Increasing concentrations of GST-CREB (5, 10, 20, and 40 ng), GST-ATF2 (5, 10, 20, and 40 ng), GST-Fos-ZTA (2.5, 5, 10, and 20 ng), and GCN4 peptide (0.5, 1, 2, and 4 ng) were tested for binding to the AP1-C, AP1-S, ATF-C, and ATF-S sites in a gel mobility assay.

To exclude that the diverse bZIP response to pX was not due to differences in their intrinsic binding activity, we analyzed the DNA binding of the four bZIPs alone to the AP-1 and ATF sites. The results of this experiment, summarized in Fig. 6B, show that CREB, ATF2, GCN4, and F-ZTA bind the four sites with comparable affinities. Taken together these results suggest that pX- mediated stimulation of bZIP binding requires an appropriate combination of core site and flanking sequences, and that DNA recognition of the pX-bZIP complex differs from that of the bZIP alone.

Delineation of the Regions of pX and Tax That Facilitate bZIP Binding-- To identify the region of pX required to enhance bZIP DNA binding, we constructed and analyzed several pX deletion mutants (Fig. 7A). The results show that the region between amino acids 13 and 105 is necessary and probably sufficient to stimulate bZIP DNA binding. Interestingly, the region between amino acids 49 and 85 is extremely conserved among human HBV isolates (58) and contains several histidines and cysteines suggestive of a metal binding domain.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Identification of the pX domain required for bZIP DNA binding stimulation. A, pX deletion mutants were expressed as GST fusion proteins. The GST moiety was then removed, and equal molar concentrations of each protein derivative were tested for stimulation of GCN4 DNA binding. The symbol - indicates that the pX mutant activity is at least 80% lower than that of wild-type pX. Gray box indicates the pX region that is highly conserved among several human HBV isolates and harbors a putative metal binding domain. Below, the conserved pX sequence between aa 49 and 85 is shown. Cysteine and histidine residues are shaded. B, Tax deletion mutants were tested for their ability to stimulate DNA binding of an H6-ATF-bZIP derivative. The symbol - indicates activity less than 5% of wild-type Tax. The N-terminal zinc binding domain is indicated (gray box).

HTLV-I Tax possesses an N-terminal zinc binding domain previously shown to be required for interaction with CREB (59) and stimulation of transcription in vivo (60-62). We investigated whether this region of Tax was also sufficient to enhance bZIP DNA binding. The results of Fig. 7B show that the Tax activity was abolished upon deletion of this N-terminal metal binding site.

Evidence That Tax and pX Activate Transcription in Vivo by Promoting bZIP DNA Binding-- We next designed an experiment to examine whether Tax and pX activated transcription in vivo by stimulating binding of bZIPs to the promoter. We reasoned that if Tax and pX function by promoting bZIP binding in vivo, their ability to stimulate transcription should decrease under conditions of increased promoter occupancy (i.e. high bZIP concentration).

To test this prediction, we took advantage of the human HepG2 cell line, which contains relatively low levels of certain bZIP proteins,2 enabling the intracellular bZIP concentration to be controlled by cotransfection. Fig. 8 shows that transcriptional stimulation of the somatostatin promoter by Tax decreased upon cotransfection of increasing amounts of CREB expression vector. Similarly, transcriptional stimulation by pX decreased upon co-transfection of increasing c-Jun expression vector. Thus, in both cases a significant reduction of Tax- and pX-mediated stimulation occurred at increased intracellular bZIP concentrations. These in vivo results recapitulate the in vitro DNA binding data in which the effects of Tax and pX were found to be inversely proportional to bZIP concentration (see Fig. 2 and Ref. 43).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8.   In vivo transcriptional activation by Tax and pX at various intracellular bZIP concentrations. -Fold activation was calculated as the ratio between the transcriptional activity in the presence or absence of the viral activator (y axis) and plotted as a function of intracellular bZIP concentration (x axis). Panel A, hepatitis B pX. In addition to the Rous sarcoma virus-JUN vector, whose concentration ranged from 0.1 to 3 µg, each transfection mixture contained 1 µg of -70/coll-luciferase plasmid and 0.5 µg of a CMV-X vector (a gift of Robert Schneider). Results from the luciferase assay were normalized based upon the protein concentration of the cell extract. Panel B, HTLV-I Tax. Tax-mediated stimulation was tested on the somatostatin promoter, which contains a CREB-responsive element (CRE). Except for the CREB vector, whose concentration varied from 0.01 to 0.5 µg, each transfection mixture contained 1 µg of somatostatin-CRE-CAT reporter (a gift from R. H. Goodman), 1 µg of Rous sarcoma virus-protein kinase A, 1 µg of CMV-Tax, and 1 µg of CMV-beta -galactosidase. CAT activity was normalized based upon the beta -galactosidase assay.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we have demonstrated that pX increases the DNA binding of diverse bZIP proteins. Our results confirm and extend several previous studies (33, 38, 63-65) and define novel aspects of the pX mechanism of action, which are discussed in greater detail below. Specifically, we show for the first time that: (i) pX increases bZIP dimerization through specific recognition of the basic region; (ii) pX itself can dimerize and/or multimerize, suggesting that the pX-bZIP complex contains at least two pX molecules; (iii) pX appears to increase promoter occupancy in vivo; (iv) pX alters bZIP DNA binding selectivity; (v) the region of pX required to stimulate bZIP DNA binding is highly conserved among HBV serotypes and resembles that of metal-binding proteins; (vi) pX resembles HTLV-I Tax, both structurally and functionally, despite lacking sequence similarity, supporting the idea that different viruses may have independently evolved promiscuous activators to target the same class of cellular transcription factors.

Stimulation of DNA binding requires only a minimal bZIP domain, and the two subdomains contribute different but essential functions; pX recognizes the basic region but requires a leucine zipper dimerization domain to increase DNA binding. The strong conservation of the basic region explains how pX can act upon a variety of bZIP proteins. Whereas previous studies have suggested that ATF-2 (33, 65), CREB, and other bZIPs (63, 64) might be the target of pX action, we have found that pX increases DNA binding of multiple bZIPs by promoting their homodimerization. The diverse bZIP proteins upon which pX can act are likely relevant to its ability to activate transcription promiscuously.

Previous studies have shown that bZIP proteins bind to DNA in a two-step reaction (54). In the first step, the bZIP dimerizes, followed by a second step in which the homodimer binds to DNA. We have shown that, through interaction with the bZIP basic region, pX increases dimerization in the absence of DNA. Why does binding of pX to the basic region promote bZIP dimerization? We have shown that pX can form dimers or multimers. Thus, we propose that a pX dimer (or multimer) interacts with the basic region of a relatively unstable bZIP dimer, thereby stabilizing the dimer form. The resulting increased concentration of the bZIP dimer then shifts the equilibrium toward formation of the bZIP-DNA complex.

Unexpectedly, we found that pX alters DNA binding site selectivity by a mechanism that remains to be determined. One possibility is that pX re-orients the bZIP so that it contacts DNA differently. For example, amino acids within the basic region, which make backbone contacts or do not normally interact with DNA (47), might be re-oriented by pX to contact a base. According to this model, minor differences among bZIP basic regions could account for the distinct selection of DNA sites by each bZIP. Alternatively, the pX portion of a pX-bZIP could contact DNA sequences flanking the consensus binding site.

The portion of pX necessary to stimulate bZIP DNA binding in vitro includes a 35-amino acid region that is highly conserved (>95%) among human HBV isolates. This region, which is also required for pX-mediated transcriptional activation in vivo (66-68), contains several histidine and cysteine residues and may comprise a metal binding domain. Significantly, Tax contains an N-terminal zinc-binding domain that is essential for stimulation of bZIP DNA binding in vitro (59) and transcription activation in vivo (60, 61). Thus, despite lacking conserved primary sequence, our results indicate that Tax and pX might, in fact, possess structurally and functionally similar domains. Structural and functional similarities between Tax and pX are also suggested by the ability of these viral proteins to facilitate bZIP binding in vivo, and transcriptionally activate the HTLV-I long terminal repeat and HBV enhancer (2, 39, 40). It is intriguing that several other transcriptionally related protein-protein interactions involve bZIP domain-metal binding site contacts (69, 70).

Our in vivo results support the view that Tax and pX function, at least in part, by increasing promoter occupancy. Specifically, activation by Tax and pX was diminished at high bZIP concentrations; if Tax and pX acted solely at a step subsequent to DNA binding (e.g. by contributing an activation domain), their effect should be largely independent of bZIP concentration. However, our results do not exclude the possibility that Tax and pX also contain an activation domain, a possibility supported by several previous studies (18, 66). In this regard, pX would be similar to the adenovirus E1a protein, which has a transcriptional activation domain and promoter-targeting region both required to activate their viral target genes (71-73). Furthermore, like pX, E1a's promoter-targeting region appears to function through interaction with the bZIP DNA binding domain of factors such as ATF-2 (74).

It has been proposed that pX has a cytoplasmic function that acts in a signal transduction cascade (26, 27, 38), and our results are not inconsistent with this possibility. In fact, a series of detailed experiments have shown that pX is present both in the nucleus and cytoplasm, and that different portions of pX are involved in nuclear and cytoplasmic functions (38). The cytoplasmic pX function appears to involve activation of the Ras, Raf, and mitogen-activated protein kinase, which ultimately acts through NF-kappa B and AP-1 factors (23, 24, 38, 75). The nuclear pX function is required to activate the HBV enhancer, most likely through the ATF/CREB bZIP protein family. It is for these reasons that pX has been referred to as "dual-specificity" activator (38).

Finally, our results have obvious implications with regard to the role of pX in viral infection and hepatocarcinogenesis. We have shown that pX increases DNA binding affinity and alters DNA binding selectivity. Both of these effects are likely important for the ability of pX to recruit the appropriate cellular bZIP protein to the HBV enhancer during viral infection. By altering the activity of cellular bZIP proteins, pX will also affect cellular gene expression. In particular, increasing the activity of known oncoproteins, such as Jun and Fos, may be the basis by which pX transforms cells.

    ACKNOWLEDGEMENTS

We thank M. Carey, P. Chirillo, T. Curran, W. DeGrado, R. Goodman, S. D. Hayward, J.-C. Hu, M. Karin, P. Kim, R. Rorth, R. Sauer, and A. Siddiqui for sharing reagents. We also thank M. Melegari, S. Roberts, J. Reese, P. P. Scaglioni, and R. J. Schneider for helpful discussion and assistance.

    FOOTNOTES

* This work was supported by a grant from the National Institutes of Health (to M. R. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Abteilung Biochemische Pharmakologie, 37070 Gottingen, Germany.

§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation St., Suite 309, Worcester, MA 01605. Tel.: 508-856-5331; Fax: 508-856-5473; E-mail: michael.green{at}ummed.edu.

2 G. Perini, E. Oetjen, and M. R. Green, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HBV, hepatitis B virus; aa, amino acid(s); kb, kilobase pair(s); CMV, cytomegalovirus; CREB, cyclic AMP response element-binding protein; CRE, CREB-responsive element; HTLV-I, human T-cell lymphotrophic virus, type I; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; GST, glutathione S-transferase; BSA, bovine serum albumin; bZIP, basic region/leucine zipper.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Koike, K., Shirakata, Y., Yaginuma, K., Arii, M., Takada, S., Nakamura, I., Hayashi, Y., Kawad, M., and Kobayashi, M. (1989) Mol. Biol. Med. 6, 151-160[Medline] [Order article via Infotrieve]
  2. Zahm, P., Hofschneider, P. H., and Koshy, R. (1988) Oncogene 3, 169-177[Medline] [Order article via Infotrieve]
  3. Sherker, A. H., and Marion, P. L. (1991) Annu. Rev. Microbiol. 45, 475-508[CrossRef][Medline] [Order article via Infotrieve]
  4. Koike, K., Moriya, K., Iino, S., Yotsuyanagi, H., Endo, Y., Miyamura, T., and Kurokawa, K. (1994) Hepatology 19, 810-819[Medline] [Order article via Infotrieve]
  5. Robinson, W. S. (1994) Annu. Rev. Med. 45, 297-323[CrossRef][Medline] [Order article via Infotrieve]
  6. Ganem, D., and Varmus, H. E. (1987) Annu. Rev. Biochem. 56, 651-693[CrossRef][Medline] [Order article via Infotrieve]
  7. Chen, H.-S., Kaneko, S., Girones, R., Anderson, R. W., Hornbuckle, W. E., Tennan, B. C., Cote, P. J., Gerin, J. L., Purcell, R. H., and Miller, R. H. (1993) J. Virol. 67, 1218-1226[Abstract]
  8. Zoulim, F., Saputelli, J., and Seeger, C. (1994) J. Virol. 68, 2026-2030[Abstract]
  9. Shirakata, Y., Kawada, M., Fujiki, Y., Sano, H., Oda, L., et al.. (1989) Jpn. J. Cancer Res. 80, 617-621[Medline] [Order article via Infotrieve]
  10. Hohne, M., Schaefer, S., Seifer, M., Feitelston, M. A., Paul, D., and Gerlich, W. H. (1990) EMBO J. 9, 1137-1145[Abstract]
  11. Kim, C.-M., Koike, K., Saito, I., et al.. (1991) Nature 351, 317-320[CrossRef][Medline] [Order article via Infotrieve]
  12. Twu, J.-S., and Schloemer, R. H. (1987) J. Virol. 61, 3448-3453[Medline] [Order article via Infotrieve]
  13. Spandau, D. F., and Lee, C.-H. (1988) J. Virol. 62, 427-434[Medline] [Order article via Infotrieve]
  14. Twu, J.-S., CHu, K., and Robinson, W. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5168-5172[Abstract]
  15. Aufiero, B., and Schneider, R. J. (1990) EMBO J. 9, 497-504[Abstract]
  16. Hu, K.-Q., Vierling, J. M., and Siddiqui, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7140-7144[Abstract]
  17. Seto, E., Mitchell, P. J., and Benedict Yen, T. S. (1990) Nature 344, 72-74[CrossRef][Medline] [Order article via Infotrieve]
  18. Unger, T., and Shaul, Y. (1990) EMBO J. 9, 1889-1895[Abstract]
  19. Zhou, D.-X., Taraboulos, A., Ou, J.-H., and Yen, T. S. B. (1990) J. Virol. 64, 4025-4028[Medline] [Order article via Infotrieve]
  20. Lopez-Cabrera, M., Letovsky, J., Hu, K.-Q, and Siddiqui, A. (1991) Virology 183, 825-829[Medline] [Order article via Infotrieve]
  21. Hu, K.-Q., Yu, C.-H., and Vierling, J. M. (1992) Proc. Natl. Acad. Sci U. S. A. 89, 11441-11445[Abstract]
  22. Kekule, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H. (1993) Nature 361, 742-745[CrossRef][Medline] [Order article via Infotrieve]
  23. Natoli, G., Avantaggiati, M. L., Chirillo, P., Costanzo, A., Artini, M., Balsano, C., and Levrero, M. (1994) Mol. Cell. Biol. 14, 989-998[Abstract]
  24. Henkler, F. F., and Koshy, R. (1996) J. Viral Hepat. 3, 109-121[Medline] [Order article via Infotrieve]
  25. Lucito, R., and Schneider, R. J. (1992) J. Virol. 66, 983-991[Abstract]
  26. Cross, J. C., Weng, P., and Rutter, W. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8078-8082[Abstract/Free Full Text]
  27. Benn, J., and Schneider, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10350-10354[Abstract/Free Full Text]
  28. Natoli, G., Avantaggiati, M. L., Chirillo, P., Puri, P., Ianni, A., Balsano, C., and Levrero, M. (1994) Oncogene 9, 2837-2843[Medline] [Order article via Infotrieve]
  29. Wu, J. Y., Zhou, Z. Y., Judd, A., Cartwright, C. A., and Robinson, W. S. (1990) Cell 16, 687-695
  30. De-Medina, T. I., Haviv, N. S., and Shaul, Y. (1994) Virology 202, 401-407[CrossRef][Medline] [Order article via Infotrieve]
  31. Takada, S., Kido, H., Fukutomi, A., Mori, T., and Koike, K. (1994) Oncogene 9, 341-348[Medline] [Order article via Infotrieve]
  32. Yoshida, M. (1993) Trends Microbiol. 1, 131-135[Medline] [Order article via Infotrieve]
  33. Maguire, H. F., Hoeffler, J. P., and Siddiqui, A. (1991) Science 252, 842-844[Medline] [Order article via Infotrieve]
  34. Beimling, P., and Moelling, K. (1992) Oncogene 7, 257-262[Medline] [Order article via Infotrieve]
  35. Xu, X., Kang, S. H., Heidenreich, O., Brown, D. A., and Neremberg, M. I. (1996) Virology 218, 362-371[CrossRef][Medline] [Order article via Infotrieve]
  36. Hai, T., Liu, F., Coukos, W. J., and Green, M. R. (1989) Genes Dev. 3, 2083-2090[Abstract]
  37. Feuer, G., and Chen, I. S. (1992) Biochim. Biophys. Acta 1114, 223-233[CrossRef][Medline] [Order article via Infotrieve]
  38. Doria, M., Klein, N., Lucito, R., and Schneider, R. J. (1995) EMBO J. 14, 4747-4757[Abstract]
  39. Faktor, O., and Shaul, Y. (1990) Oncogene 5, 867-872[Medline] [Order article via Infotrieve]
  40. Marriot, S. J., Lee, T. H., Slagle, B. L., and Butel, J. S. (1996) Virology 224, 206-213[CrossRef][Medline] [Order article via Infotrieve]
  41. Armstrong, A. P., Franklin, A. A., Uittenbogaard, M. N., Giebler, H. A., and Nyborg, J. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7303-7307[Abstract]
  42. Franklin, A. A., Kubik, M. F., Uittenbogaard, M. N., Brauweiler, A., Utaisincharoen, P., Matthews, M.-A. H., Dynan, W. S., Hoeffler, J. P., and Nyborg, J. K. (1993) J. Biol. Chem. 268, 21225-21231[Abstract/Free Full Text]
  43. Wagner, S., and Green, M. R. (1993) Science 262, 395-399[Medline] [Order article via Infotrieve]
  44. Baranger, A. M., Palmer, C. R., Hamm, M. K., Gleber, H. A., Brauweiler, A., Nyborg, J. K., and Schepartz, A. (1995) Nature 376, 606-608[CrossRef][Medline] [Order article via Infotrieve]
  45. Perini, G., Wagner, S., and Green, M. R. (1995) Nature 376, 602-605[CrossRef][Medline] [Order article via Infotrieve]
  46. Blatter, E. E., Ebright, Y. W., and Ebright, R. H. (1992) Nature 359, 650-652[CrossRef][Medline] [Order article via Infotrieve]
  47. Ellenberger, T. E., Brandl, C. J., Struhl, K., and Harrison, S. C. (1992) Cell 71, 1223-1237[Medline] [Order article via Infotrieve]
  48. Kim, J., Tzamarias, D., Ellenberger, T., Harrison, S. C., and Struhl, K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4513-4517[Abstract]
  49. Vinson, C. R., Hai, T., and Boyd, S. M. (1993) Genes Dev. 7, 1047-1058[Abstract]
  50. Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A., and Dougherty, W. G. (1994) Anal. Biochem. 216, 413-417[CrossRef][Medline] [Order article via Infotrieve]
  51. O'Neil, K. T., Hoess, R. H., and DeGrado, W. F. (1990) Science 249, 774-778[Medline] [Order article via Infotrieve]
  52. Talanian, R. V., McKnight, C. J., and Kim, P. S. (1990) Science 249, 769-771[Medline] [Order article via Infotrieve]
  53. O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1989) Science 243, 538-542[Medline] [Order article via Infotrieve]
  54. Weiss, M. A., Ellenberger, T., Wobbe, C. R., Lee, J. P., Harrison, S. C., and Struhl, K. (1990) Nature 347, 575-578[CrossRef][Medline] [Order article via Infotrieve]
  55. Chang, Y.-N., Dong, D. L.-Y., Hayward, G. S., and Hayward, S. D. (1990) J. Virol. 64, 3358-3369[Medline] [Order article via Infotrieve]
  56. Lieberman, P. M., Hardwick, J. M., Sample, J., Hayward, G. S., and Hayward, S. D. (1990) J. Virol. 64, 1143-1155[Medline] [Order article via Infotrieve]
  57. Hu, J. C., O'Shea, E. K., Kim, P. S., and Sauer, R. T. (1990) Science 250, 1400-1403[Medline] [Order article via Infotrieve]
  58. Kidd-Ljunggren, K., Oberg, M., and Kidd, A. H. (1995) J. Gen. Virol. 76, 2119-2130[Abstract]
  59. Goren, I., Semmens, O. J., Jeang, K.-T., and Moelling, K. (1995) Virol. 69, 5806-5811
  60. Smith, M. R., and Greene, W. C. (1991) Genes Dev. 4, 1875-1885[Abstract]
  61. Semmes, O. J., and Jeang, K. T. (1992) J. Virol. 66, 7183-7192[Abstract]
  62. Semmes, O. J., and Jeang, K. T. (1992) Virology 188, 754-764[Medline] [Order article via Infotrieve]
  63. Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819-3823[Abstract/Free Full Text]
  64. Barnabas, S., Hai, T., and Andrisani, O. M. (1997) J. Biol. Chem. 272, 20684-20690[Abstract/Free Full Text]
  65. Palmer, C. R., Gegnas, L. D., and Schepartz, A. (1997) Biochemistry 9, 15349-15355[CrossRef]
  66. Arii, M., Takada, S., and Koike, K. (1992) Oncogene 7, 397-403[Medline] [Order article via Infotrieve]
  67. Murakami, S., Cheong, J. H., and Kaneko, S. (1994) J. Biol. Chem. 269, 15118-15123[Abstract/Free Full Text]
  68. Renner, M., Haniel, A., Burgelt, E., Hofschneider, P. H., and Koch, W. (1995) J. Hepatol. 23, 53-65[CrossRef][Medline] [Order article via Infotrieve]
  69. Diamond, M. I., Miner, J. N., Yoshinaga, S. K., and Yamamoto, K. R. (1990) Science 249, 1266-1272[Medline] [Order article via Infotrieve]
  70. Jonat, C., Rahmsdorf, H. J., Park, K. K., Cato, A. C., Gebel, S., Ponta, H., and Herrlich, P. (1990) Cell 62, 1189-1204[Medline] [Order article via Infotrieve]
  71. Lillie, J. W., Loewenstein, P. M., Green, M. R., and Green, M. (1987) Cell 50, 1091-1100[Medline] [Order article via Infotrieve]
  72. Lillie, J. W., and Green, M. R. (1989) Nature 338, 39-44[CrossRef][Medline] [Order article via Infotrieve]
  73. Abdel-Hafiz, H. A., Chen, C. Y., Marcell, T., Kroll, D. J., and Hoeffler, J. P. (1993) Oncogene 8, 1161-1174[Medline] [Order article via Infotrieve]
  74. Liu, F., and Green, M. R. (1990) Cell 61, 1217-1224[Medline] [Order article via Infotrieve]
  75. Klein, N. P., and Schneider, R. J. (1997) Mol. Cell. Biol. 17, 6427-6436[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.