©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Group of Cellular Cofactors That Stimulate the Binding of RNA Polymerase II and TRP-185 to Human Immunodeficiency Virus 1 TAR RNA (*)

(Received for publication, August 31, 1995; and in revised form, December 5, 1995)

Foon Wu-Baer (1) William S. Lane (2) Richard B. Gaynor (1)(§)

From the  (1)Divisions of Molecular Virology and Hematology-Oncology, Departments of Internal Medicine and Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8594 and the (2)Protein Microchemistry Facility, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.


INTRODUCTION

The regulation of HIV-1 gene expression is dependent on a number of cis-acting regulatory elements in the long terminal repeat(1) . Cellular factors binding to each of these elements are important for the assembly of transcription complexes that are responsive to the transactivator protein Tat. Mutagenesis of the HIV-1 long terminal repeat has demonstrated that the SP1(2, 3) , TATA(4, 5, 6, 7) , and TAR (8, 9, 10, 11, 12) elements are each critical for Tat activation. The TAR element which extends between +1 and +60 appears to be the major regulatory element required for Tat transcriptional activation(4, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . Thus, to determine the mechanism of Tat activation will require a better understanding of the interplay of these different regulatory elements.

The TAR element when inserted downstream of a variety of heterologous viral and cellular promoters will confer on these promoters the ability to be activated by Tat(4, 9, 13, 18) . This suggests that TAR is necessary and, in many cases, sufficient for Tat activation although the TATA and SP1 sites are also critical in modulating the degree of Tat activation. TAR DNA binds a variety of proteins including UBP/LBP-1 (22, 23, 24) , YY-1(25) , and TDP-43 (26) and induces the synthesis of short or nonprocessive transcripts in both the HIV-1 long terminal repeat and heterologous promoters(18, 27) . Although TAR DNA likely plays a regulatory role in the control of HIV-1 gene expression, it appears that the ability to form a stable RNA stem-loop structure transcribed from TAR and extending between +1 and +60 is critical for Tat activation(10, 12, 15, 20, 21) . Two elements within TAR RNA, a three-nucleotide bulge structure (15, 28, 29, 30, 31) and a six-nucleotide loop sequence(10, 11, 15, 30, 32) , are both critical for Tat activation. This is likely due to the ability of the bulge to serve as the binding site for Tat(28, 29, 30, 31, 33, 34) and the loop to serve as the binding site for a cellular factor designated TRP-185 or TRP-1(32, 35) . Although mutagenesis of the loop or the bulge markedly reduce Tat activation, the mechanism by which the binding of either TRP-185 or Tat to TAR RNA stimulates subsequent transcriptional activation remains to be determined.

Recently, we demonstrated that, in addition to Tat and TRP-185, RNA polymerase II is also able to bind specifically to TAR RNA(36) . HIV-1 templates with wild-type TAR elements that exhibit high in vivo levels of activation in response to Tat bind RNA polymerase II with high affinity, whereas HIV-1 templates with mutated TAR elements that bind RNA polymerase II with lower affinity are not markedly activated in vivo by Tat(36) . During the process of transcriptional elongation in both eucaryotic and procaryotic promoters, RNA polymerase II likely has separate binding sites for both DNA and RNA(37, 38, 39, 40) . Thus, one mechanism to explain the function of TAR in the activation of HIV-1 gene expression would be that the TAR RNA loop and bulge elements bind cellular and viral proteins, respectively, that are each able to help disengage RNA polymerase II bound to TAR RNA. Whether either or both of these factors are able to stably bind to the RNA polymerase II during subsequent transcriptional elongation remains to be determined.

Both RNA polymerase II and TRP-185 by themselves bind weakly to TAR RNA in gel retardation assays(32, 35, 36) . However, the addition of another groups of proteins purified from HeLa nuclear extract markedly stimulates both RNA polymerase II (36) and TRP-185 binding to TAR RNA (32, 35) . These proteins, designated cofactors, are alone unable to stably bind to TAR RNA. The mechanism by which these factors stimulate TRP-185 and RNA polymerase II binding to TAR RNA is unclear. Possibilities include post-translational modification of either the TRP-185 or RNA polymerase II or direct interaction of the cofactors with either TRP-185 or RNA polymerase II bound to TAR RNA . To address these possibilities, we purified proteins in the cofactor fraction and subjected them to amino acid microsequence analysis. We were able to isolate the cDNAs encoding three cellular proteins present in the cofactor fraction. These cDNAs were placed into vaccinia expression systems to produce these recombinant proteins in HeLa cells. Addition of the three purified recombinant cofactor proteins markedly stimulated the binding of both RNA polymerase II and TRP-185 to TAR RNA. Our results suggest that the cofactors function to stimulate TRP-185 and RNA polymerase II binding by both direct interactions with proteins bound to TAR RNA and also by potentially modifying the structure of these proteins. The cofactors potentially have a novel regulatory mechanism which may be involved in the control of HIV-1 gene expression.


EXPERIMENTAL PROCEDURES

Purification of TRP-185 Cofactors

Nuclear extract prepared from 60 liters of HeLa cells as described (Dignam et al., 1983) was applied to a heparin-agarose column (2.5 times 9 cm) equilibrated with buffer A (20 mM Tris-Cl, pH 7.9, 20% glycerol (v/v), 0.2 mM EDTA) containing 0.1 M KCl, 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mM DTT. (^1)The column was washed in the same buffer and then eluted with buffer A with 0.4 M KCl, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT. The 0.4 M KCl fractions were pooled and dialyzed against buffer A with 0.1 M KCl, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT and applied to a HTP Bio-Gel (2.5 times 7 cm) which was equilibrated and washed with the same buffer. The column was then eluted with the same buffer containing 0.1 M potassium phosphate (pH 7.0). These fractions were pooled and precipitated with 70% ammonium sulfate followed by centrifugation at 12,000 rpm for 20 min. The precipitate was resuspended in 6 ml of buffer A with 0.1 M KCl and 1 mM DTT and loaded on a Superdex 200 FPLC (HiLoad 26/60 prep grade) column which was equilibrated in the same buffer. The flow-through fractions containing cofactor activity were pooled and applied to a Q-Sepharose (1.5 times 4 cm) column equilibrated in the same buffer. The flow-through fractions containing cofactor activity were pooled and fractionated further on a Bio-Rex 70 column (1 times 3 cm). Fractions eluted from this column which were able to reconstitute TRP-185 binding activity to HIV-1 TAR RNA were pooled. This preparation gave approximately 20 µg of each of the five cofactor species and was determined to be of approximately 80% purity on a silver-stained SDS protein gel.

Amino Acid Sequence Analysis of Purified Cellular Cofactors

The cofactor fraction from the above preparation was concentrated on a Centricon 10 membrane which was blocked with bovine serum albumin. The concentrated sample (150 µl) was loaded into three lanes of a 10% polyacrylamide, 0.1% SDS protein gel and blotted overnight onto a nitrocellulose membrane (0.45 µM). The proteins were visualized by Ponceau S staining and the NaOH destaining step was omitted(41) . A total of five protein species were excised from the nitrocellulose with approximate molecular masses of 36, 42, 53, 55, and 58 kDa that were designated as CF36, CF42, CF53, CF55, and CF58, respectively. Following Lys C protease digestion of each of the proteins and HPLC separation, the amino acid sequence of each of these proteins was determined by amino-terminal amino acid analysis using Edman degradation(42) .

Isolation of the cDNAs Encoding the Cellular Cofactors

Sequence comparison of these peptides was made using the Intelligenetics program. Sequence homology of several of the peptides with the previously described proteins EF-1alpha and PTB were noted while the peptides obtained from CF58 showed no significant homology with known proteins. To clone the gene encoding CF58, degenerate primers were made to the 5` and 3` amino acid sequences of the 25-mer peptide obtained from the CF58. Polymerase chain reaction analysis was performed with HeLa cDNA, and a 75-base pair fragment was generated which revealed the predicted amino acid sequence from the 25-mer peptide upon analysis of the DNA sequence. This fragment was used as probe to screen a HeLa cDNA library (Clontech) and resulted in the identification of a cDNA of 2 kb which encoded a 539-amino acid open reading frame that we designated SRB. The accession number for SRB is U38846.

Expression and Purification of the Recombinant Cofactors

Polymerase chain reaction primers were made to modify the 5` ATG and 3` end of the EIF1-alpha cDNA into NcoI sites, the PTB cDNA 5` ATG into SphI and the 3` end into EcoRI sites, and the CF58 cDNA 5` ATG into NcoI and the 3` end into BamHI sites. These modified cDNAs were then cloned in a modified pTM1 expression vector (46) with sequences encoding the 12-amino acid influenza hemagglutinin epitope (47) and 6 histidine residues at the carboxyl terminus of the protein coding sequence. Each of the constructs was then transfected onto 20 plates of HeLa plate cells (150 mm) followed by infection with a recombinant vaccinia virus which produced T7 polymerase. The cells were harvested 40 h later, and nuclear and S100 extracts were prepared as described previously. The S100 extract contained most of the overexpressed recombinant proteins as judged by Western blot analysis with the 12CA5 monoclonal antibody. The nuclear or S100 extracts were loaded onto a 2-ml Q-Sepharose column (1.5 times 2 cm) equilibrated with buffer A containing 0.1 M KCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, aprotinin, and 10 mM beta-mercaptoethanol. The columns were washed with the same buffer, and the flow-through fractions were pooled then loaded onto a 1-ml nickel-nitrilotriacetic acid agarose column (Qiagen) equilibrated with the same buffer. The flow-through fractions were reloaded onto the columns a second time, and they were washed with: 1) 20 ml of buffer A, 2) 20 ml of buffer A containing 1.0 M KCl, and 3) 20 ml of buffer A with 0.1 M KCl, respectively. The columns were then eluted with 0.1 M KCl and 60 mM imidazole. The eluted fractions were then dialyzed versus buffer A containing 0.1 M KCl and 1 mM DTT, assayed, and stored at -70 °C. A typical yield of each of the recombinant proteins from these preparations was approximately 100 µg with a purity of 85% as judged by silver staining of the 10% SDS-polyacrylamide gel.

Gel Retardation Assays, with Purified and Recombinant Cellular Cofactors

The binding reactions used in the gel retardation assays were performed as described in (32) . The binding of TRP-185 to HIV-1 TAR RNA was performed with 50 ng of each of the nickel column-eluted recombinant cofactors. The recombinant TRP-185 protein (50 ng) used in these experiments was produced using a pTM1 expression vector, transfected into HeLa cells with a recombinant vaccinia virus producing T7 polymerase, and purified using nickel column chromatography. RNA polymerase II was purified as described(48) , and 50 ng of this protein was used in the binding reactions. The binding reactions with antibodies were performed as described above except that 1 µg of each of the protein A-Sepharose column-purified antibodies was added to the gel retardation assays for 10 min prior to gel electrophoresis. The TRP-185 monoclonal antibody (NK 5.18) used in this study was raised against a GST fusion of TRP-185 corresponding to amino acids 1409 to 1541. The EIF-1alpha antibody was raised against a GST fusion of this protein containing amino acids 1 to 110(43, 44) . The PTB polyclonal antibody was raised against a GST fusion of this protein containing amino acids 291 to 531(45) . The SRB polyclonal antibody was raised against a GST fusion of this protein encoding amino acids 1 to 331. To deplete the endogenous ATP from the recombinant cofactors, a combination of 4.5 units of hexokinase and 250 µM glucose was used(49) .

Northern and Western Blot Analysis of Cellular Cofactors

Northern analysis of the human multiple tissue blot from Clontech was performed using each of the three cofactor cDNAs as probes according to the manufacturer's protocol. This membrane was probed with a nick-translated protein of the CF58 cDNA encoding amino acids 331 to 539 of the PTB cDNA encoding amino acids 291 to 531 and EIF-1alpha full-length cDNA. The probe was also removed following each hybridization, and the filter was stripped of the scanning probe prior to the next hybridization. The nick-translated glyceraldehyde-3-phosphate dehydrogenase cDNA was used as a control for the distribution of poly(A) RNA present in each lane of the tissue blot.

Western blot analysis was performed using the 12CA5 monoclonal antibody (47) and ECL reagents (Amersham). The amounts of protein used in Western blot was 300 ng of each of the recombinant proteins purified using nickel chromatography. Recombinant EF-1alpha, PTB, and CF58 had molecular masses of 54, a doublet of 58, and 62 kDa, respectively.


RESULTS

Purification of Cellular Cofactors

Previously we demonstrated that sucrose gradient ultracentrifugation analysis resulted in the separation of TRP-185, which sedimented at approximately 200 kDa from a group of cellular proteins, designated cofactors, which sedimented at approximately 100 kDa(32) . The sucrose gradient fractions containing either TRP-185 alone or the cofactors alone were each unable to bind to TAR RNA in gel retardation analysis. Addition of the sucrose gradient fractions containing the cofactors in conjunction with fractions containing TRP-185 restored the ability of TRP-185 to bind to TAR RNA (32) . Furthermore, we also recently found that this cofactor fraction was able to stimulate the binding of RNA polymerase II to TAR RNA (36) .

In an attempt to determine the mechanism by which these cofactors stimulated the binding of TRP-185 and RNA polymerase II to TAR RNA, we purified the proteins responsible for this cofactor activity. The ability of the cofactors to stimulate the binding of recombinant TRP-185 to TAR RNA in gel retardation studies was used as our assay. To purify the cellular cofactors, HeLa nuclear extract prepared from 60 liters of cells was applied to a heparin agarose column. The cofactor activity was eluted with 0.4 M KCl, applied to a hydroxylapatite column, and then eluted with 0.1 M potassium phosphate. Following ammonium sulfate precipitation and chromatography on a Superdex 200 column, the active fractions were pooled and applied to a Q-Sepharose column. The flow-through fractions were further fractionated on a Bio-Rex 70 column, and the protein fractions which stimulated the binding of recombinant TRP-185 to TAR RNA were pooled. A flow chart of this purification scheme is shown in Fig. 1.


Figure 1: Purification scheme of cellular cofactors from HeLa cells. The protocol for the fractionation of HeLa cell nuclear extract to purify the cellular cofactors which stimulate TRP-185 binding is shown. The numbers in the figure indicate the concentration of KCl used to elute each column with the exception of the hydroxylapatite Bio-Gel column in which the concentration of potassium phosphate is indicated.



The purified cofactor fraction was then assayed for its ability to stimulate TRP-185 binding to TAR RNA. Addition of increasing amounts of recombinant TRP-185 alone resulted in only minimal binding to TAR RNA (Fig. 2, lanes 2-4). However, upon the addition of the purified cofactors to TRP-185, there was a marked increase in its binding to TAR RNA (Fig. 2, lanes 5-7). There was no binding of the cofactor fraction alone to TAR RNA (Fig. 2, lane 1). The enhancement of TRP-185 binding by the cofactors was not seen with equivalent amounts of other proteins such as GST or albumin (Fig. 2, lanes 8-11). Finally, it was found that increasing the amount of cofactors from 0.1 to 1.0 µg markedly increased the binding of TRP-185 to TAR RNA (Fig. 2, lanes 12-14). These results indicate that the purified cellular cofactors did not bind directly to TAR RNA by themselves, but acted to markedly stimulate the binding properties of TRP-185 to TAR RNA.


Figure 2: Cellular cofactors are required for TRP-185 binding activity to HIV-1 TAR RNA. Gel retardation analysis was performed with a labeled HIV-1 TAR RNA probe (lane 1) in the presence of increasing amounts of purified recombinant TRP-185, 10 ng (lane 2), 25 ng (lane 3), 50 ng (lane 4) either alone or in the presence of 0.4 µg of purified cellular cofactors (lanes 5-7). The enhancement of TRP-185 binding was assayed with wild-type TAR RNA and TRP-185 alone (50 ng) (lane 8) or with 0.4 µg of either cellular cofactors (lane 9), bovine serum albumin (lane 10), or GST (lane 11). TRP-185 binding to TAR RNA was assayed by adding increasing amounts of purified cellular cofactors 0.12 µg (lane 12), 0.4 µg (lane 13), or 1.0 µg (lane 14) and comparing this to the addition of 1 µg of bovine serum albumin (lane 15).



Cloning of the Genes Encoding the Cellular Cofactors

The proteins present in the purified cofactor fraction were subject to electrophoresis on an SDS-polyacrylamide gel. Silver staining of this gel revealed the presence of five species with molecular masses of 58, 55, 53, 42, and 36 kDa, respectively. Approximately 80 µg of these purified cofactors were transferred to nitrocellulose membranes, and each species was excised. The amino acid sequence of each of these proteins was determined following Lys C protease digestion of these species and separation of the peptides by HPLC(41) . These peptides were then compared to known sequences using GenBank. The peptide sequence generated from the 53-, 42-, and 36-kDa proteins, respectively (VETGVLKPGMVVTFAPVNVTTEVKIGGIGTVPVGR, IGGIGTVPVGR, and SVEMHHEALSEALPGDNVGFNVK), were exact matches for elongation factor 1alpha (EF-1alpha)(43, 44) . This is a previously described protein of 462 amino acids which is involved in translational initiation. The 55-kDa protein generated two peptides of 20 and 5 amino acids, respectively, (KLPIDVTEGEVISL-GLPFGK and VSFSK) which were exact matches for the polypyrimidine tract-binding protein(45, 50, 51) . This is a previously described protein of 531 amino acids which is involved in splice junction recognition due to its ability to bind directly to RNA.

The third protein of 58 kDa generated two peptides with the amino acid sequences VVSQYSSLLSPMS and AFADAMEVIPSTLAENAGLNPISTV. Sequence comparison of these two peptides demonstrated no sequence homology with other proteins in GenBank. Degenerate oligonucleotides were synthesized to the 5` and 3` portions of the 25-amino acid peptide obtained from this 58-kDa protein, and these primers were used in polymerase chain reaction analysis with HeLa cDNA. A 75-base pair fragment was obtained and DNA sequence analysis confirmed that the deduced amino acid sequence matched the amino acid sequence obtained by microsequence analysis. This fragment was then used to screen a HeLa cDNA library and resulted in the isolation of a 2.0-kb cDNA which contained a 539-amino acid open reading frame that we designated stimulator of TAR RNA-binding proteins (SRB).

Amino Acid Sequence of SRB

The SRB amino acid sequence was compared for regions of homology with other proteins using the Intelligenetics computer program. SRB had a region located in its amino terminus that had a high degree of homology with the chaperonin family of proteins (52, 53, 54) (Fig. 3A). The highest degree of homology shared by the SRB protein was 55% with the ANC2 protein which is a member of the chaperonin family of proteins (Fig. 3B)(55) . There was also a high degree of homology with another chaperonin, T-complex polypeptide-1, which is a subunit of a heteromeric mammalian cytosolic complex that functions in ATP-mediated folding of actin and tubulin(52, 53) . Chaperonin proteins have been demonstrated to exist frequently in heteromeric complexes and function by ATP binding and hydrolysis to enhance the yield of correctly folded proteins(52, 53) . SRB was also noted to have homology with a so-called A consensus or P loop motif which is a glycine-rich region followed by a conserved lysine and either a serine or threonine that is capable of binding ATP or GTP (56, 57, 58) (Fig. 3A). This domain is also found in a variety of other chaperonin proteins(52, 53) .


Figure 3: Amino acid sequence of SRB. A, the deduced amino acid sequence of the SRB cDNA is indicated with the position of the two peptides generated from the Lys C digestion of the purified SRB protein underlined. The position of a conserved 80-amino acid region in the amino-terminal portion of SRB which is related to the chaperonin family is demarcated by a box as is another seven-amino acid domain which has homology to the P loop sequence which serves as potential binding site for ATP or GTP. B, the homology of the amino terminus of SRB with the previously characterized ANC2 protein is indicated.



Cellular Cofactors Are Ubiquitously Expressed

Next we wanted to compare the RNA expression patterns of the different cofactors. Northern analysis was performed with a blot comprised of multiple tissue RNAs prepared from human tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Fig. 4). There were differences in the amount of RNA loaded in each sample as determined by hybridization to a glyceraldehyde-3-phosphate dehydrogenase probe (Fig. 4D). The EF-1alpha probe hybridized to one transcript of approximately 2.0 kb (43, 44) (Fig. 4A), while the PTB probe hybridized to two transcripts of approximately 5.0 kb and 3.5 kb (45) (Fig. 4B). The SRB probe hybridized to a single transcript of 2.0 kb (Fig. 4C). Thus, each of the mRNAs encoding the cellular cofactors was ubiquitously expressed in a variety of human tissues.


Figure 4: Northern analysis of the expression of cellular cofactors. A blot containing 2 µg of poly(A)-selected RNA from a variety of derived human tissues was analyzed sequentially with cDNA probes derived from either EF-1alpha (A), PTB (B), SRB (C), or glyceraldehyde-3-phosphate dehydrogenase (D) and subjected to autoradiography. The lanes include a size marker (lane M) and RNA isolated from tissues including heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), and pancreas (lane 8).



Expression of Recombinant Cofactor Proteins

To test the role of each of the cofactor proteins on the binding of TRP-185 and RNA polymerase II to TAR RNA, we cloned cDNAs encoding each of the cofactors into the pTM1 expression vector downstream of the T7 RNA polymerase promoter(46) . Following transfection of each of these cDNAs into HeLa cells, their expression was induced by infection with a recombinant vaccinia virus which expressed T7 polymerase. To facilitate the detection of these proteins, the 12 amino acid influenza hemagglutinin sequences was inserted in the carboxyl terminus. In addition, six histidine residues were also inserted in the carboxyl terminus of these proteins to facilitate their purification using nickel chromatography. Following column chromatography, each of these recombinant proteins was eluted from nickel beads with imidazole, and Western blot analysis was performed with the 12CA5 monoclonal antibody which recognizes the influenza hemagglutinin sequences on these proteins. The molecular masses of these three recombinant proteins of 54, 58, and 62 kDa were consistent with predicted molecular masses of EIF-1alpha, PTB, and SRB, respectively (Fig. 5).


Figure 5: Western blot analysis of recombinant cofactor protein. Each of the cellular cofactors was expressed in HeLa cells following transfection of their respective cDNAs cloned into the PTM1 expression vector and infection with recombinant vaccinia virus producing T7 polymerase. Each of these histidine-tagged proteins was purified using nickel affinity chromatography, and Western blot analysis was performed with 300 ng of each protein and detected using 12CA5 monoclonal antibody. This monoclonal antibody recognizes the influenza hemagglutinin epitope inserted on the carboxyl terminus of SRB (lane 1), PTB (lane 2), and EF-1alpha (lane 3). The position of the molecular mass markers (MW) is also indicated.



Recombinant Cofactors Reconstitute TRP-185 Binding to TAR RNA

Next it was important to determine whether the recombinant cofactors were able to reconstitute TRP-185 binding to the same extent as the native cofactors purified from HeLa cells. A mixture of all three of the recombinant cofactors at the highest protein concentration used in these gel retardation studies did not generate a complex which bound to TAR RNA in the absence of added TRP-185 or RNA polymerase II (Fig. 6A, lane 1). Addition of each of the individual cofactors resulted in a low level of TRP-185 binding to TAR RNA as compared to binding performed in the absence of the cofactors (Fig. 6A, lanes 2-5). The addition of SRB resulted in the greatest stimulation of TRP-185 binding (Fig. 6A, lane 5). Addition of the recombinant cofactors in pairs resulted in some increase in the binding of TRP-185 for most of the different combinations assayed (Fig. 6A, lanes 6-8).


Figure 6: Recombinant cofactors stimulate TRP-185 binding to TAR RNA. Gel retardation was performed with vaccinia-produced recombinant TRP-185 in the presence of different recombinant cofactors. A, the wild-type TAR RNA probe was incubated with 50 ng of EF-1alpha, PTB, and SRB (lane 1), with 50 ng of TRP-185 either alone (lane 2) or in the presence of 50 ng of either EF-1alpha (lane 3), PTB (lane 4), or SRB (lane 5) or both 50 ng of EF-1alpha and PTB (lane 6), EF-1alpha and SRB (lane 7), or PTB and SRB (lane 8). B, increasing amounts (10, 20, or 50 ng) of a combination of EF1-alpha, PTB, and SRB (lanes 1-3) were incubated with TRP-185, and the binding was compared to that with 0.4 µg of native cofactors purified from HeLa cells (lane 4).



Finally, we tested whether the addition of all three cofactors was able to increase the binding of TRP-185 to TAR RNA. Addition of increasing amounts of all three cofactors resulted in a marked stimulation of TRP-185 binding to wild-type TAR RNA (Fig. 6B, lanes 1-3). This binding was equivalent to the maximal binding of TRP-185 seen in the presence of native cellular cofactors purified from HeLa cells (Fig. 6B, lanes 3 and 4). These results indicate that the addition of the recombinant cofactors will completely reconstitute TRP-185 binding. Since the native cellular cofactors in HeLa cells exist as a complex during purification, the ability of the individual recombinant cofactors to result in low level stimulation of TRP-185 binding to TAR RNA is likely due to cross-contamination with other copurifying cofactors.

Recombinant Cofactors Stimulate RNA Polymerase II Binding to TAR RNA

Since we demonstrated that the three recombinant cofactors functioned similar to native cellular cofactors to stimulate TRP-185 binding to TAR RNA, we next wanted to determine whether the recombinant cofactors were able to stimulate the binding of RNA polymerase II to wild-type TAR RNA. Unlike TRP-185 which exhibited no detectable binding to TAR RNA in the absence of added cellular cofactors, RNA polymerase II alone exhibited low level binding to TAR RNA (Fig. 7A, lane 2). Addition of each of the recombinant cofactors either alone or in pairs was able to stimulate the binding of RNA polymerase II to wild-type TAR RNA (Fig. 7A, lanes 3-8). The addition of increasing amounts of all three recombinant cofactors was able to markedly stimulate the binding of RNA polymerase II to TAR RNA reaching levels equivalent to that seen with the addition of native cofactors (Fig. 7B, lanes 1-4). Again, there was no binding of either recombinant or purified cofactors to wild-type TAR RNA in the absence of RNA polymerase II (Fig. 7B, lanes 5 and 6). Addition of similar concentrations of other proteins such as albumin or glutathione S-transferase were unable to stimulate the binding of RNA polymerase II to wild-type TAR RNA (data not shown). Thus, the recombinant cofactors were also able to markedly stimulate the binding of RNA polymerase II to TAR RNA.


Figure 7: Recombinant cofactors stimulate RNA polymerase II binding to TAR RNA. A, gel retardation was performed with wild-type TAR RNA (lane 1) and 50 ng of purified RNA polymerase II alone (lane 2) or in the presence of 50 ng of either EIF-1alpha (lane 3), PTB (lane 4), or SRB (lane 5) or both EF-1alpha and PTB (lane 6), EF-1alpha and SRB (lane 7), or PTB and SRB (lane 8). B, increasing amounts (10, 20, or 50 ng) of a combination of each of the three cofactors EIF-1alpha, PTB, and SRB (lanes 1-3) were incubated with RNA polymerase II, and the binding to TAR RNA was compared with that of 0.4 µg of native cofactors (lane 4). The binding properties of a combination of 50 ng of EF-1alpha, PTB, and SRB (lane 5) or 0.4 µg of purified cellular cofactors in the absence of added RNA polymerase II is also shown (lane 6).



PTB in the Presence of TRP-185 Associates with TAR RNA

The mechanism by which the cofactors were able to stimulate the binding of TRP-185 and RNA polymerase II to TAR RNA was investigated next. Based on the function of the procaryotic chaperonin ClpX(59) , it seemed possible that SRB may function as a potential chaperonin to modify RNA polymerase II and TRP-185 protein folding such that they were able to bind more efficiently to TAR RNA. However, it was not clear how EF-1alpha and PTB might function to stimulate the binding of TRP-185 and RNA polymerase II to TAR RNA. One possibility was that EF-1alpha and PTB might associate directly with TRP-185 or RNA polymerase II and either remain associated or disassociate during gel electrophoresis.

To address whether the cofactors directly associated with proteins bound to TAR RNA during gel retardation analysis, we raised rabbit polyclonal antibodies to glutathione S-transferase protein fusions comprised of portions of either EF-1alpha, PTB, or SRB. Western blot analysis was performed with each of these polyclonal antibodies, and they each reacted specifically with vaccinia-produced recombinant cofactors (data not shown). Each of these antibodies was then affinity-purified using protein A-Sepharose columns and tested for its ability to supershift a complex comprised of TRP-185 or RNA polymerase II and cofactors in gel retardation analysis. Such a result would indicate that the individual cofactors were present in the TAR RNA complex with either TRP-185 or RNA polymerase II.

Gel retardation analysis was performed with recombinant TRP-185 and either the native cofactors (Fig. 8A, lanes 1-6) or the recombinant cofactors (Fig. 8A, lanes 7-11). Gel retardation analysis indicated that a complex formed by the addition of TRP-185 and purified cellular cofactors was supershifted by a monoclonal antibody directed against TRP-185 (Fig. 8A, lane 3). Antibody directed against either EF-1alpha (Fig. 8A, lane 4) or SRB (Fig. 8A, lane 6) did not result in a similar supershifted complex. However, the addition of antibody directed against PTB resulted in a shift of the complex bound to TAR RNA (Fig. 8A, lane 5) similar to that seen with TRP-185 monoclonal antibody. Addition of the PTB antibody alone with wild-type TAR RNA did not result in a detectable complex in gel retardation analysis (data not shown).


Figure 8: PTB associates with TRP-185 but not RNA polymerase II in a complex with HIV-1 TAR RNA. A, gel retardation assays were performed with wild-type TAR RNA (lane 1) and recombinant TRP-185 reconstituted with either the native cofactors purified from HeLa cells (lanes 2-6) or the recombinant cofactors (lanes 7-11). TRP-185 with either cellular cofactors (lane 2) or recombinant cofactors (lane 7) in the absence of added antibody or in the presence of 1 µg of affinity-purified rabbit polyclonal antibody directed against either TRP-185 (lanes 3 and 8), EF-1alpha (lanes 4 and 9), PTB (lanes 5 and 10), or SRB (lanes 6 and 11) is shown. B, gel retardation assays were performed with wild-type TAR RNA (lane 1) and purified RNA polymerase II (50 ng) in the presence of 0.4 µg of native cofactors alone (lane 2) or in the presence of 1 µg of affinity-purified antibodies directed against the carboxyl-terminal domain of RNA polymerase II (lane 3), EF-1alpha (lane 4), PTB (lane 5), or SRB (lane 6).



These gel retardation experiments were then repeated using recombinant cofactors and TRP-185. Antibodies directed against either TRP-185 (Fig. 8A, lane 8) or PTB (Fig. 8A, lane 10) were again able to supershift the complex bound to TAR RNA, while antibodies directed against either EF-1alpha or SRB (Fig. 8A, lanes 9 and 11) were unable to supershift this complex. These results indicated that PTB was able to associate directly in a complex comprised of TAR RNA and TRP-185. We cannot definitively rule out that either EF-1alpha or SRB may also associate directly with this complex. This is because only a subset of antibodies directed against the same protein are able to alter the mobility of the gel-retarded complex resulting in either a loss or a shift in the complex. This was true with a panel of rabbit polyclonal antibodies directed against TRP-185 of which only two of five antibodies was able to supershift this protein in gel retardation analysis with TAR RNA (data not shown).

Cofactors Do Not Directly Associate with RNA Polymerase II

It was next important to determine whether any of the cofactor proteins were able to associate in a complex comprised of TAR RNA and RNA polymerase II. Gel retardation analysis was performed with purified RNA polymerase II and native cofactors purified from HeLa cells. The addition of antibody directed against the carboxyl-terminal domain of RNA polymerase II disrupted the binding of RNA polymerase II to TAR RNA (Fig. 8B, lane 3). However, the addition of antibodies directed against either EF-1alpha, PTB, or SRB did not result in a super-shifted species or disrupt the binding of the gel-retarded complex (Fig. 8B, lanes 4-6). Similar results were seen in gel retardation assays by adding these antibodies with recombinant cellular cofactors and RNA polymerase II (data not shown). Thus, differences in the nature of the TAR RNA complex comprised of cofactors and RNA polymerase II as compared to the cofactors and TRP-185 does not permit recognition following the addition of cofactor antibodies.

ATP Inhibits TRP-185 Binding to TAR RNA

Since SRB has marked homology with chaperonin proteins, we wished to address whether the presence of ATP enhanced the ability of SRB in the context of the other cofactors to increase the binding of TRP-185 to TAR RNA. This would be expected if the role of SRB was to function as a chaperonin to alter the protein conformation of TRP-185 in an ATP-dependent manner(52, 53) . To address this point, we depleted the endogenous ATP present in the recombinant cofactors by treatment with a combination of hexokinase and glucose(49) . First, we demonstrated that the binding of TRP-185 alone in the absence of added cofactors was not affected by the addition of hexokinase and glucose (Fig. 9, lane 1). The addition of untreated TRP-185 and a combination of the three recombinant cofactors markedly stimulated TRP-185 binding to wild-type TAR RNA (Fig. 9, lane 2). Treatment of the cofactors with hexokinase and glucose prior to the addition of TRP-185 resulted in a slight stimulation of TRP-185 binding (Fig. 9, lane 3) while the addition of ATP to the cofactors prior to the addition of TRP-185 markedly decreased TRP-185 binding to TAR RNA (Fig. 9, lane 4). The addition of hexokinase and glucose or ATP to the cofactor fraction (Fig. 9, lanes 5 and 6) in the absence of TRP-185 did not result in its binding to TAR RNA. These results suggest that binding sites for ATP on either TRP-185 or the cofactors may inhibit TRP-185 binding to TAR RNA. One potential explanation for these results is that ATP binding sites on EF-1alpha and SRB may function to remove ATP bound to TRP-185 to increase its ability to bind to TAR RNA. Mutagenesis of potential ATP binding sites on TRP-185, EF-1alpha, and SRB will be needed to address such a possibility.


Figure 9: ATP inhibits TRP-185 binding to TAR RNA. Gel retardation analysis was performed with TRP-185 and a mixture of 50 ng of the three recombinant cofactors EF-1alpha, PTB, and SRB and a wild-type HIV-I TAR RNA probe. The gel retardation assays included TRP-185 alone (lane 1) or TRP-185 in the presence of either untreated recombinant cofactors (lane 2), these same cofactors treated with hexokinase (4.5 units) and glucose (250 µM) (lane 3), or ATP (2 mM) (lane 4) prior to incubation with TRP-185. Cofactors alone in the absence of added TRP-185 which were treated with either hexokinase and glucose (lane 5) or ATP (2 mM) (lane 6) were also tested in the gel retardation assay.




DISCUSSION

The TAR element is critical for the activation of HIV-1 gene expression by the transactivator protein Tat although the mechanism by which TAR functions in conjunction with Tat remains open to question (9-13, 15, 18, 30, 32, 60-62). However, it is clear that the structure of nascent TAR RNA is critical for Tat function(60) . A number of previous studies have demonstrated that the TAR RNA bulge element (15, 28, 29, 30, 31) is important for Tat activation and that the Tat protein is capable of binding to the HIV-1 TAR RNA bulge element (28, 29, 30, 31, 33, 34) . However, mutation of the TAR RNA loop sequences also markedly decrease Tat activation(10, 11, 15, 30, 32) , and, yet, these mutations have little or no effect on Tat binding, suggesting a critical role for cellular factors that bind to TAR RNA loop sequences (28, 29, 30, 31, 33, 34, 43) . We were interested in defining cellular proteins that bind to TAR RNAs transcribed from HIV-1 templates that were activated in vivo by Tat but did not bind efficiently to HIV-1 templates that were not activated in vivo by Tat. Using a biochemical fractionation of HeLa nuclear extract, we determined that only two proteins, TRP-185 and RNA polymerase II, met this criteria(36) .

While characterizing the binding of TRP-185 and RNA polymerase II to TAR RNA, we found that their binding was stimulated greatly by the addition of a group of cellular factors designated cofactors(32, 36) . Although the cofactors by themselves did not exhibit any binding to TAR RNA, the addition of these factors with TRP-185 or RNA polymerase II markedly stimulated their binding to TAR RNA. Previous data suggested that TRP-185 and the cofactors remained stably associated following column chromatography(32) . The ability of the three cofactor proteins to remain associated in the purification scheme used in the current study further suggest that these proteins may exist in a complex within the cell. Whether any cellular RNAs may be associated with this complex remains to be determined. It will be critical to determine whether the cofactors modulate HIV-1 gene expression. However, given the high endogenous levels of these proteins in the cell, transfection assays did not allow us to ascertain the effects of these factors on HIV-1 gene expression.

The purification of the cofactor proteins and subsequent microsequence analysis revealed that the cofactors were comprised of at least three proteins. These included EF-1alpha, PTB, and SRB. The potential relationship of these proteins remains unclear at this time. EIF-1alpha is a 53-kDa cytosolic protein which is important in initiating eucaryotic translation(43, 44) . EF-1alpha has been demonstrated to bind to aminoacyl-tRNA and also bind and hydrolyze GTP. Whether the hydrolysis of GTP is critical for its ability to stimulate TRP-185 or RNA polymerase II binding to TAR RNA remains to be determined. It will also be critical to determine how this primarily cytoplasmic localized protein is able to be transported and function in the nucleus. It has been demonstrated previously that several translation factors including eIF-4F, eIF-4E, and eIF-2 can be detected in the nucleus(63) .

The PTB protein was first purified from nuclear extract and demonstrated to bind to the polypyrimidine tract present in the 3` portion of RNA splice sites(45) . The PTB gene gives rise to alternatively spliced mRNAs which encode proteins with domains homologous to previously described ribonucleoprotein binding domains that mediate binding RNA(45) . It has also been demonstrated that PTB is able to bind directly to the bulge region of the poliovirus RNA leader sequence which is involved in facilitating internal ribosomal initiation(50) . It is interesting to note that this poliovirus RNA sequence contains several three-nucleotide bulge sequences which are identical with the TAR RNA bulge sequences. Using an RNA selection procedure to determine the optimal binding sites for PTB, it has been demonstrated recently that PTB binds preferentially to uridine-rich RNA sequences(51) . Since we have demonstrated that PTB associates with TRP-185 in a complex bound to TAR RNA, it is possible that PTB may in fact be in contact with the TAR RNA bulge. Such a possibility may be one explanation for the failure of TRP-185 to bind to TAR RNA which contains a deleted TAR RNA bulge. However, we have been unable to detect direct binding of PTB to TAR RNA in the absence of either TRP-185 or RNA polymerase II.

The SRB protein appears to have the greatest effect of the three cofactor proteins on the binding of TRP-185 and RNA polymerase II to TAR RNA. SRB has homology to both eucaryotic and procaryotic chaperonin proteins (52, 53, 54, 55) and contains a so-called P-loop domain (56, 57, 58) which in a variety of proteins including chaperonins has been demonstrated to bind ATP or GTP. However, we detected decreased binding of TRP-185 in the presence of cofactors when either ATP or GTP was added to the gel retardation assay. It is interesting to note that in contrast to a variety of other chaperonin proteins which are exclusively cytoplasmic (52, 53) SRB is present in both the nucleus and cytoplasm. Given the demonstration that the procaryotic chaperonin protein ClpX is able to stimulate the binding properties of 0 to its DNA recognition site (59) , it seems likely that other nuclear chaperonin proteins will be identified that are able to stimulate the binding of either DNA or RNA-binding proteins to their cognate binding sites. Since chaperonin proteins exist in heteromeric complexes with other proteins(53) , it is possible that SRB forms a hetero-oligomeric complex associated with PTB and EF-1alpha.

The question arises how the three cofactors are able to stimulate the binding of TRP-185 and RNA polymerase II to TAR RNA. Both PTB (45, 50, 51) and EF-1alpha (64) have been demonstrated to bind directly to other RNAs. The demonstration that PTB was present in a complex with TRP-185 bound to TAR RNA suggests that the cofactors may be able to interact either directly with TRP-185 or bind weakly to a portion of TAR RNA in conjunction with TRP-185. We have been unable to demonstrate direct interactions between TRP-185 and any of the cofactors using the mammalian two-hybrid system so that we would favor the possibility that PTB binds weakly to TAR RNA in conjunction with TRP-185. The inability to detect PTB in a complex bound to TAR RNA with RNA polymerase II may be due to the fact that domains of PTB are no longer accessible to the antibody. It is also possible that PTB may have a different mechanism of action to stimulate TRP-185 binding to TAR RNA as compared to that of RNA polymerase II. The role of EF-1alpha on stimulating the binding of TRP-185 and RNA polymerase II to TAR RNA remains unclear. EF-1alpha like PTB may potentially bind weakly to TAR RNA in the presence of TRP-185 or RNA polymerase II but fail to be recognized in a complex bound to TAR RNA by the antibodies that we have generated. Since EF-1alpha has been demonstrated to bind to aminoacyl-tRNA, it seems possible that it may be capable of binding to TAR RNA in conjunction with other proteins. Whether the ability of EF-1alpha to hydrolyze GTP may be important in either TAR RNA binding or transcriptional regulation remains to be determined. It is possible that the function of SRB to stimulate protein binding to TAR RNA (45, 50, 51) is due to its ability to result in optimal folding of TRP-185 and RNA polymerase II. However, our results using ATP addition and depletion to the cofactor fraction would also be consistent with a model in which EF-1alpha and SRB function to remove ATP which is potentially bound to TRP-185 to increase its binding to TAR RNA. Mutagenesis of potential binding ATP sites in TRP-185, SRB, and EF-1alpha will be required to address more carefully the role of the cofactors in the stimulation of TRP-185 and RNA polymerase II binding to TAR RNA.

In summary, we have isolated the cDNAs encoding three cellular factors that copurify from HeLa nuclear extract and facilitate the binding of TRP-185 and RNA polymerase II to TAR RNA. These cofactor proteins are important in regulating the binding of cellular factors to TAR RNA. Whether these factors have a role on the regulation of HIV-1 gene expression in either the presence or absence of Tat remains to be determined. In vitro transcription studies using extracts depleted of individual or multiple cellular cofactors will be required to address the role of these factors on modulating both basal and Tat-induced levels of HIV-1 gene expression.


FOOTNOTES

*
This work was supported by grants from the National Institutes of Health and the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38846[GenBank].

§
To whom correspondence should be addressed: Division of Molecular Virology, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235-8594. Tel.: 214-648-7570; Fax: 214-648-8862.

(^1)
The abbreviations used are: DTT, dithiothreitol; HPLC, high performance liquid chromatography; EF-1alpha, elongation factor 1alpha; PTB, polypyrimidine tract-binding protein; SRB, stimulator of TAR RNA-binding proteins; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We thank Susan Vaughn for the preparation of the artwork and Sharon Johnson for typing this manuscript.


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