©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cellular C1 Factor of the Herpes Simplex Virus Enhancer Complex Is a Family of Polypeptides (*)

(Received for publication, November 9, 1994)

Thomas M. Kristie (2) (1)(§) Joel L. Pomerantz (2) (3)(¶) Teresa C. Twomey (4) Stephen A. Parent (4) Phillip A. Sharp (2)

From the  (1)Laboratory of Viral Diseases, National Institutes of Health, Bethesda, Maryland 20892, the (2)Center for Cancer Research and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the (3)Harvard University-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, and (4)Merck Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alpha/immediate early genes of herpes simplex virus are regulated by the specific assembly of a multiprotein enhancer complex containing the Oct-1 POU domain protein, the viral alpha-transinduction factor alphaTIF, (VP16, ICP25), and the C1 cellular factor. The C1 factor from mammalian cells is a heterogeneous but related set of polypeptides that interact directly with the alpha-transinduction factor to form a heteromeric protein complex. The isolation of cDNAs encoding the polypeptides of the C1 factor suggests that these proteins are proteolytic products of a novel precursor. The sequence of the amino termini of these polypeptide products indicate that the proteins are generated by site-specific cleavages within a reiterated 20-amino acid sequence. Although the C1 factor appears to be ubiquitously expressed, it is localized to subnuclear structures in specific cell types.


INTRODUCTION

The regulation of the expression of the herpes simplex virus alpha/immediate early genes by multiprotein assemblies has provided a model system for analysis of DNA-protein and protein-protein interactions involved in the modulation of homeodomain function. The enhancer elements of these genes nucleate the assembly of transcriptional regulatory complexes (C1 and C2 complexes) containing Oct-1, a member of the POU domain family, alphaTIF (^1)(VP16, ICP25, VMW65), the HSV-encoded alpha gene transactivator, and C1 (HCF), a novel cellular factor(1, 2, 3, 4, 5, 6, 7, 8, 9) .

The POU-specific and Pou-homeo subdomains of the Oct-1 protein cooperatively recognize a homolog of the octamer element (ATGCAAAT) in the 5` domain of the alpha gene enhancer element (a response element, ATGCTAATGATATTCTTTGG)(9, 10, 11) . This protein has also been implicated in the regulation of the expression of small nuclear RNA genes(12, 13, 14, 15, 16, 17, 18, 19) , the cell cycle expression of the histone H1 and H2B genes(20, 21, 22) , the expression of lymphoid-specific genes(23, 24, 25, 26, 27, 28, 29, 30, 31) , and the stimulation of DNA replication(32, 33, 34) . Although the promoters of these genes bind the Oct-1 protein, they are each regulated in a distinct manner. Therefore, the specificity of the transcriptional regulation is likely to be determined by the combined interactions of assembled regulatory complexes in a manner that is analogous to the formation of the HSV-specific C1 complex.

The HSV alpha transactivator, alphaTIF, contributes to the specificity of the C1 complex in two manners. As a DNA-binding protein, it recognizes sequences in the 3` domain of the alpha response element (9, 35) and is stabilized by a cooperative DNA-binding interaction with the Oct-1 homeodomain(9, 35, 36) . In addition, alphaTIF discriminates between the homeodomains of Oct-1 and the highly related Oct-2 through selective protein-protein interactions(36, 37) . However, the interactions of the Oct-1 homeodomain and alphaTIF are insufficient to form a stable regulatory complex, thus requiring the presence of an additional cellular factor (C1 factor; Refs. 2, 6, 9, 38, and 39).

The highly conserved C1 factor activity has been detected in insect and mammalian cells (6, 9) and interacts with high affinity with alphaTIF to form a heteromeric protein complex(9, 39) . The C1 factor does not appear to bind DNA independently, but rather, interacts with the components of the C1 regulatory complex(9, 38, 39) . Purification of this activity from mammalian cells has shown that it is a heterogeneous set of related polypeptides, ranging from 100 to 230 kDa(40) . The presence of stoichiometric quantities of the 100- and 123-135-kDa proteins in the purified preparations suggested that these polypeptides were subunits of the factor(40) . In addition, a subset of the purified proteins specifically interacted with alphaTIF in a phosphorylation-dependent manner(40) .

cDNAs have been isolated which encode the polypeptides of the C1 factor. Furthermore, the use of specific antiseras has shown that these proteins are components of the C1 factor and are present in the assembled C1 complex. The analyses of the cDNAs as well as amino-terminal sequencing of the purified proteins indicate that a novel 230-kDa precursor is proteolytically processed to yield the family of C1 factor polypeptides.


MATERIALS AND METHODS

Electrophoretic Mobility Gel Shift Assays

The purification of the Oct-1 protein from HeLa cells and the alphaTIF protein from AcNPV-alphaTIF infected SF9 cells was as described previously(6, 9) . The HSValpha0 DNA probe is equivalent to pRB608(41) . Electrophoretic mobility shift assay reaction and electrophoresis conditions were as described (40) and included 1.0 µl of affinity purified Oct-1, 30 ng of purified alphaTIF, and 0.2 µl of purified C1 factor (8 saturation units/MSD15 preparation) in a total of 10 µl. For antibody supershifts, the reactions were incubated at 30 °C for 10 min before the addition of affinity purified antibodies from Ab2125 or Ab2126 seras and continued incubation at 30 °C for 10 min.

SDS-PAGE and Western Blots

Parallel aliquots of extracts and chromatographically purified C1 factor were resolved in 8% denaturing SDS-polyacrylamide gels (acrylamide:bisacrylamide, 30:0.8). One set of resolved polypeptides was stained with silver and the remaining sets were transferred to nitrocellulose as described(40) . The nitrocellulose blots were blocked in phosphate-buffered saline + 5% nonfat dry milk for 30 min and incubated with the appropriate preimmune or immune sera in phosphate-buffered saline + 1% bovine serum albumin for 3 h. The Western blots were visualized using the protoblot alkaline phosphatase system (Promega) according to the manufacturer's recommendations.

Purification and Sequencing of the C1 Factor Polypeptides

Purification of the mammalian C1 factor from HeLa cell nuclear extracts and the isolations of tryptic peptides from the 100-, 125-135-, and 68-kDa proteins were as described(40) . Selected tryptic peptides were subjected to sequence analysis on an Applied Biosystems model 477A protein sequencer with an on-line model 120 phenylthiohydantoin-derivative analyzer. For amino-terminal sequencing of the 100-kDa and 123-135-kDa polypeptides, 40 pmol of purified C1 factor activity were precipitated with acetone, resuspended in 30 µl of SDS-PAGE loading buffer, and resolved in a 7.5% (acrylamide:bisacrylamide 30:0.08) SDS denaturing gel. The resolved polypeptides were transferred to Bio-Rad polyvinylidene difluoride membrane and stained with Coomassie Blue as described(40) . The protein bands representing the 100- and 123-135-kDa polypeptides were excised and subjected to amino-terminal sequence analysis as described above. The tryptic peptide and amino-terminal sequence analyses were done by R. Cook in the MIT Center for Cancer Research Biopolymers Laboratory.

Generation of a Nondegenerate cDNA Probe Encoding the Internal Sequences of the C1 Factor Tryptic Peptide A-34

Peptide sequence derived from A-34 (18 amino acids) of the 100- and 123-135-kDa proteins was used to synthesize two oligonucleotides A34-opt2 (5`-GGATTCGCTGTGACCACAGTG-3`) and A34-opt1 (5`-CGGATCCGGCACAGAGGGGCCAGG-3`) which represent the most frequently utilized codons for the NH(2)-terminal 5 and COOH-terminal 6 amino acids of the peptide, respectively. Two µg of HeLa cell poly(A) mRNA was primed with 25 ng of A34-opt1 at 55 °C for 3 h followed by the reverse transcription of the mRNA at 52 °C as described(42) . Aliquots of the cDNA reaction were added to a polymerase chain reaction containing 1 µM A34-opt1, 1 µM A34-opt2, 100 µCi of [alpha-P]dCTP (3000 Ci/mM), 200 µM of each deoxynucleotide, and 1 µl of AmpliTaq DNA polymerase (Perkin Elmer). The reaction was denatured at 94 °C, annealed at 45 °C, and extended at 72 °C for a total of 45 cycles. The expected 60-base pair cDNA product was purified and the nucleotide sequence was determined(43) .

Screening Libraries for cDNAs Encoding the C1 Factor Polypeptides

C1.probe A oligonucleotide (5`-GGGACCGGTGTGGACTGCGTCAC-3`) encoded 7 amino acid residues of the tryptic peptide A-34 which were internal to the A34-opt1 and A34-opt2 oligonucleotide primers. C1.probe A was end-labeled with [-P]ATP (6000 Ci/mM) and T4 polynucleotide kinase (U. S. Biochemical Corp.). Hybridization probes derived from H10, H5c, H302, and H303 were prepared by isolation of the appropriate cDNA clone DNA fragment and labeling with [alpha-P]dCTP (3000 Ci/mM) using the Klenow fragment of Escherichia coli DNA polymerase I (Boehringer Mannheim) and random hexamer primers (Pharmacia Biotech Inc.) according to the manufacturer's recommendations. Probes derived from FL150.2 were prepared by polymerase chain reaction using primers which were specific for the 300-base pair 5` terminus of the cDNA clone.

cDNA clones H10 and H40 were isolated from an oligo(dT) and random primed HeLa cell cDNA library in gt11 (Clontech). Nytran (Schleicher and Schuell) filter lifts were prepared for hybridization according to the manufacturer's recommendations. The filters were subsequently irradiated in a Stratagene UV-Linker; baked for 30 min at 80 °C; rinsed in 3 times SSC (SSC; 150 mM NaCl, 17 mM Na citrate), 0.1% SDS at 25 °C for 45 min and at 55 °C for 60 min; prehybridized in 6 times SSC, 5 times Denhardt's solution, 0.5% SDS, 0.05% Na(2)HPO(4), 100 µg/ml salmon sperm DNA at 55 °C for 20 h; and hybridized in 6 times SSC, 1 times Denhardt's solution, 0.05% Na(2)HPO(4), 100 µg/ml yeast tRNA, 2 times 10^6 cpm/ml of C1.probe A at 55 °C for 20 h. Following hybridization, the filters were washed twice in 6 times SSC, 0.05% Na(2)HPO(4) at 25 °C for 1 h, once in 2 times SSC, 0.05% SDS at 25 °C for 30 min, and twice in 6 times SSC, 0.05% Na(2)HPO(4) at 55 °C for 30 min. Both strands of the cDNA inserts were sequenced by the dideoxynucleotide method at 50 °C in reactions containing either deoxyguanosine or deoxyinosine according to the manufacturer's recommendations (Sequenase 2.0, Stratagene).

cDNA clones PB5c, PB6e, and PB7e were isolated from a subsequent screening of nytran filter lifts (gift of J. Lees) prepared from a human pre-B cell cDNA library (gift of A. Bernards). The filters were rinsed as above, prehybridized in 5 times SSC, 50% formamide, 5 times Denhardt's solution, 0.5% SDS, 125 µg/ml salmon sperm DNA at 42 °C for 12 h and hybridized in 5 times SSC, 50% formamide, 1 times Denhardt's solution, 0.5% SDS, 125 µg/ml salmon sperm DNA, 75 µg/ml yeast tRNA, 2.0 times 10^6 cpm/ml of H10 cDNA insert at 42 °C for 12-16 h. The filters were subsequently washed twice in 2 times SSC, 0.1% SDS at 25 °C for 30 min, and three times at 68 °C for 30 min. cDNA clones H302 and H303 were isolated from a custom cDNA library (Stratagene) produced by the specific priming of HeLa cell poly(A) mRNA with a mixture of the oligonucleotides (5`-GCAGCGGTGCTGACCGCATGG-3`) and (5`-GCACAGCAGTGCCTCCAGG-3`). cDNA clones designated FR were isolated from a human fetal retina cDNA library (gift of M. McDonald) while those designated FL were isolated from a human fetal liver cDNA library (Clontech) using probes generated from the 5`-termini of cDNAs H5c, H302, H303, and FL150.2 in successive screenings. Both strands of the cDNA inserts were sequenced as described above.

Data Base Comparisons

The nucleotide and amino acid sequences of the C1 factor clones were compared to available data bases using the BLAST network service at the National Center for Biotechnology Information(44) . Additional analyses were done using FASTA, tFASTA, and MacVector software (IBI-Kodak).

Northern Blot Analysis

Total cytoplasmic and poly(A) mRNA was isolated from HeLa cells as described previously(45) . 10 µg of each mRNA preparation were resolved in a 0.8% denaturing formaldehyde-agarose gel and transferred to a nylon membrane(46) . The membrane was baked at 80 °C for 1 h, prehybridized in 5 times SSC, 50% formamide, 1 times Denhardt's, 0.02 M NaHPO(4) (pH 6.8), 150 µg/ml salmon sperm DNA, 75 µg/ml yeast tRNA at 42 °C for 12 h, and hybridized with the addition of 1.0 times 10^6 cpm/ml of randomly primed H10 cDNA insert probe at 42 °C for 16 h. The blot was subsequently washed twice with 2 times SSC, 0.1% SDS at 25 °C for 30 min and twice with 0.1 times SSC, 0.1% SDS at 68 °C for 45 min.

Production and Purification of Antiseras

pGST-PB5c and pGST-PB5cDeltaT were produced by the insertion of cDNA sequences encoding C1 ORF amino acids 1176-1787 and 1176-1488, respectively, in the appropriate pGEX bacterial expression vector (Pharmacia). Extracts from induced cells were prepared by sonication of the cells in lysis buffer (20 mM sodium phosphate (pH 7.2), 500 mM NaCl, 10 mM EDTA, 0.05% Tween 20, 10 mM beta-mercaptoethanol, 5 mM phenylmethylsulfonyl fluoride); addition of Triton X-100 to 1.0% (v/v); and centrifugation at 25,000 times g for 20 min. The fusion proteins were purified by affinity chromatography on glutathione-Sepharose (Pharmacia) as recommended by the manufacturer.

For the production of antigen, the purified fusion proteins were further resolved by preparative SDS-PAGE, visualized by staining with KCl, and excised(47) . Peptide antigens (CPEELQVSPGPRQQLPPRQ (amino acids 1501-1518) and CTSKDSSGTKPANKRPMS (amino acids 2003-2019)) were synthesized by J. Coligan in the NIAID Biological Resources Branch and were conjugated to keyhole limpet hemocyanin (Pierce) according to the manufacturer's recommendations. New Zealand White rabbits were innoculated with GST-PB5c (Ab2125), GST-PB5cDeltaT (Ab2131), or a mixture of the conjugated peptides (Ab2126) according to standard procedures (48) .

Glutathione-Sepharose purified GST-PB5c, GST-PB5cDeltaT, and control GST proteins were further purified by Superose 12 gel filtration chromatography, dialyzed against 100 mM Hepes (pH 7.5), and coupled to Affi-Gel 10 or Affi-Gel 15 (5.0 mg of protein/1.5 ml of matrix) according to the manufacturer's recommendations. Ab2125 and Ab2131 seras were diluted 1:5 (v/v) with 10 mM Tris (pH 7.5), passed through the control GST protein column, and adsorbed to the appropriate fusion protein matrix. The matrices were washed sequentially with 15 ml each of 10 mM Tris (pH 7.5) and 10 mM Tris (pH 7.5), 500 mM NaCl. Antibodies were eluted from the columns with 15 ml of 100 mM glycine (pH 2.5). Ab2126 was similarly purified by direct absorption to a peptide affinity matrix produced by the coupling of the peptide antigens to Sulfolink (Pierce) according to the manufacturer's recommendation.

Immunofluorescence

HeLa, MRC-5, HepG2, 143, Hep-2, K-562, WIL2-NS, Raji, Nalm6, C4II, SiHa, C33A, Caski, and Me180 cells were kindly provided by L. Wolffe, J. Bennink, J. Yewdell, J. McKnight, L. Staudt, and A. McBride, respectively. 2 times 10^5 cells were fixed with 4.0% paraformaldehyde for 20 min at 25 °C, permeabilized with 1.0% Triton X-100 in phosphate-buffered saline for 2 min, incubated with the appropriate affinity purified antisera (5 µg ml in phosphate-buffered saline + 3% bovine serum albumin) for 2 h at 4 °C and stained with rodamine or fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Boehringer Mannheim) for 1 h at 4 °C(49) . The mounted coverslips were viewed using either a Zeiss Axiophot fluorescent microscope or a Zeiss Axioplan confocal microscope.


RESULTS

The Polypeptides of the Purified C1 Factor

As described previously, the purification of the C1 factor activity from mammalian HeLa cells resulted in chromatographic fractions containing a polypeptide of 100 kDa and a cluster of proteins of 123-135 kDa(40) . In addition, preparations of the C1 factor activity contained less abundant polypeptides of 68, 155-180, and 230 kDa. However, only the 100- and 123-135-kDa proteins were present in all preparations of the C1 activity, thus suggesting that this factor is composed of one or more of these polypeptides(40) . Subsequent tryptic digestion of the 100- and 123-135-kDa proteins and analysis of the high performance liquid chromatographic patterns of the resulting tryptic peptides indicated that these proteins were nearly identical and may be variant forms of a single polypeptide(40) . To confirm this, primary amino acid sequence was obtained from two corresponding tryptic peptides, designated A-33 and A-34, of the 100- and 123-135-kDa proteins. In each case, identical amino acid sequence was obtained for the equivalent peptides derived from the 100- and 123-135-kDa proteins.

Isolation of cDNAs Encoding the C1 Factor Polypeptides

Primary amino acid sequence was obtained from multiple tryptic peptides derived from the 100-, 123-135-, and 68-kDa proteins present in the various purified C1 factor preparations. The amino acid sequence of one peptide (A-34, AVTTVTQSTPVPGSVPP) of the 100/123-135-kDa proteins was used to synthesize oligonucleotides which represented the most frequently utilized codons for residues 1-5 (AVTTV) and 13-18 (PGSVPP). These oligonucleotides were used in a reverse transcriptase-polymerase chain reaction with HeLa cell poly(A) mRNA to generate products whose sequence contained the authentic codons for the internal 6 residues of the peptide (TQSTPV). An oligonucleotide of this internal sequence was then used to screen a HeLa cell cDNA library, resulting in the isolation of the H10 and H40 cDNA clones. These cDNAs encoded the carboxyl terminus of the C1 factor ORF ( Fig. 1and 2). Subsequent rounds of screening of human pre-B cell, human fetal retina, human fetal liver, and specifically primed HeLa cell cDNA libraries produced the overlapping cDNA clones illustrated in Fig. 1.


Figure 1: The cDNAs encoding the polypeptides of the C1 factor. The structure of the assembled cDNA encoding the C1 factor polypeptides is schematically illustrated with the positions of the 5`-untranslated domain (nucleotides 1-349), C1 open reading frame (C1 ORF, nucleotides 350-6455), and the 3`-untranslated domain (nucleotides 6456-8210). The overlapping clones that were isolated in this study are positioned above the complete cDNA and are designated according to the cDNA library from which they were derived as follows: FL, human fetal liver; FR, human fetal retina; H, HeLa cell; and PB, human pre-B cell.



Surprisingly, the assembled cDNA sequence (Fig. 2) contains a single ORF which is predicted to encode a polypeptide of 209 kDa. All of the tryptic peptides derived from the 100- and 123-135-kDa are found within the carboxyl-terminal portion of the ORF, whereas the peptides derived from the 68-kDa protein are clustered within the amino-terminal sequences, suggesting that the final C1 factor polypeptides are processed from a larger precursor protein. The 155-180- and 230-kDa proteins which are detected in lower abundance in preparations of the C1 factor (40) are likely to be additional products or intermediates of the complete C1 factor ORF.


Figure 2: The amino acid sequence encoded by the complete C1 factor ORF. The complete amino acid sequence of the C1 factor ORF is listed. The sequence of the tryptic peptides derived from the 68 kDa (C-8, 22, and 17) and the 100 or 123-135 kDa (A-34, 50, 35, 11, and 15) polypeptides of the purified C1 factor are underlined. The bold type designates the core of the six C1 factor amino acid reiterations while the positions of the amino termini of the 100- and 123-135-kDa C1 factor polypeptides are indicated with closed or open triangles, respectively. The portions of the ORF used to produce the Ab2125 (amino acids 1176-1787) and Ab2131 (amino acids 1176-1488) antiseras are indicated by the arrows, while the peptides used to generate the Ab2126 antisera are denoted with hatched lines.



Antisera Produced against Domains of the C1 Factor ORF React with the Purified C1 Factor Polypeptides

The set of proteins present in the chromatographic fractions of the purified C1 activity are likely to represent polypeptides of the C1 factor as they were present in sufficient quantities to account for the C1 factor activity. In addition, when incubated with labeled alphaTIF protein, the immobilized 123-135-, 155-180-, and 230-kDa proteins specifically interacted with the protein probe(40) . To determine if these polypeptides are, in fact, the polypeptides of the C1 factor and are present in the assembled C1 complex, antiseras were produced against various domains of the C1 factor ORF (Fig. 2).

HeLa cell nuclear extract and chromatographic fractions containing purified C1 factor were resolved by SDS-PAGE (Fig. 3, center). Parallel aliquots were transferred to nitrocellulose and reacted with either preimmune (lanes 4 and 5) or immune seras (Ab2125 (ORF amino acids 1176-1787), lanes 6 and 7). As shown, the immune sera specifically reacted with the 100-, 123-135-, 155-180-, and 230-kDa polypeptides of the purified C1 factor (compare Fig. 3, left and center). Similarly, the sera specifically reacted with the corresponding proteins in the HeLa cell nuclear extract, indicating that the heterogeneity of the family of polypeptides was not a result of the purification protocol.


Figure 3: Western blot of the C1 factor polypeptides with anti-C1 seras. Left panel, a silver-stained SDS-denaturing gel shows the resolution of the polypeptides of the chromatographically purified mammalian C1 factor (lane 3, (40) ). MW1 and MW2 (lanes 1 and 2) are protein markers whose molecular weight, in thousands, are indicated at the left. Center and rightpanels, Parallel aliquots of HeLa cell nuclear extract (lanes 4, 6, and 8-10) and purified C1 factor (lanes 5 and 7) were resolved in an SDS-denaturing gel, transferred to nitrocellulose, and probed with the antisera indicated at the top of the lane. Ab2125 preimmune and immune indicate the use of seras while Ab2125, Ab2126, and Ab2131 represent affinity purified antibodies.



The relationship of the various polypeptides of the purified C1 factor to the cloned C1 factor ORF was further investigated by Western blot of the purified factor with antisera produced against central and carboxyl-terminal domains of the C1 ORF. As illustrated in Fig. 3(right), antiseras Ab2125 and Ab2131 representing amino acids 1176-1787 and 1176-1487, respectively, reacted in a similar fashion with the 100-, 123-135-, 155-180-, and 230-kDa polypeptides of the C1 factor preparation (lanes 8 and 10). In contrast, Ab2126, produced against amino acids 1501-1518 and 2003-2119, reacted with a subset of the proteins which included the 100 and several of the 123-135-kDa polypeptides. In addition, this sera reacted weakly with the 230-kDa polypeptide (lane 9). Thus, the 100-, 123-135-, 155-180-, and 230-kDa polypeptides contain common determinants from the central region of the C1 factor ORF whereas the 100, a subset of the 123-135, and the 230-kDa proteins contain determinants derived from the carboxyl-terminal domain of the ORF. Furthermore, as expected, the antiseras did not react with the 68-kDa polypeptide as this protein is derived from the amino terminus of the ORF (Fig. 2, peptides C-8, C-22, and C-17) and thus would not contain determinants common to these antigens (Fig. 3, lanes 6-8).

Anti-C1 Factor Sera Specifically Supershifts the Assembled C1 Complex

As Ab2125 and Ab2126 specifically reacted with distinct subsets of the purified C1 factor proteins, affinity purified antibodies or control anti-GST antibodies were added to reactions containing the Oct-1 protein or the assembled C1 complex (Fig. 4). The addition of the affinity purified Oct-1 protein to a DNA binding reaction containing the HSValpha0 DNA probe resulted in the formation of the Oct-1-DNA complex (lane 1). A reaction containing Oct-1, alphaTIF, and the purified C1 activity generated the characteristic C1 complex (lane 2). Addition of either Ab2125 or Ab2126 to these reactions specifically retarded the mobility of the C1 complex (lanes 4, 5, 7, and 8) without affecting the migration of the Oct-1-DNA complex (lanes 3 and 6). In contrast, the addition of control anti-GST antibodies did not affect the mobility of the Oct-1-DNA or C1 complex (data not shown). Thus, the specific reactivity of the immune seras to the various polypeptides of the purified C1 factor demonstrates that these proteins are components of the C1 factor.


Figure 4: Supershift of the assembled C1 complex with anti-C1 antibodies. Protein-DNA binding reactions were done as described under ``Materials and Methods.'' The positions of the Oct-1-DNA, C1, and C1-antibody complexes are indicated at the right. Reactions 1, 3, and 6 contained only the Oct-1 protein while reactions 2, 4, 5, 7, and 8 contained Oct-1, alphaTIF, and the purified C1 activity. In addition, the reactions in lanes 3, 5, 6, and 8 contained 0.25 µg, while lanes 4 and 7 contained 0.1 µg of the affinity purified antibody that is indicated at the top of the gel.



Characteristics of the C1 Factor ORF

Comparison of the predicted amino acid and nucleic acid sequences of the C1 factor to the available data bases suggested that this factor represents a novel class of protein. Structurally, the cDNA has a high G + C content (61%) and the ORF encodes a protein which is highly enriched for threonine (13.1%), serine (8.9%), proline (9.5%), and alanine (11.5%) residues. The predicted polypeptide of the C1 factor contains numerous sites for potential post-translational modifications, including phosphorylation by cAMP protein kinase, protein kinase C, casein kinase, and tyrosine kinase. These modifications may be of significant interest as phosphorylation of the C1 factor polypeptides is important for the interactions of these proteins with alphaTIF(40) .

However, the most striking feature of the C1 factor ORF is the presence of 6 reiterations of a novel amino acid repeat ( Fig. 2and 5). As demonstrated below, this repeat represents a site of specific proteolytic processing of the C1 factor precursor to generate members of the family of C1 factor polypeptides.

The Amino Termini of the 100- and 123-135-kDa Polypeptides

One or more of the purified polypeptides are clearly contained within the C1 complex. In addition, several of these proteins directly interact with alphaTIF(40) . Therefore, to more clearly define the relationship of these proteins to the 230-kDa precursor, the amino termini of the 100- and 123-135-kDa proteins were sequenced and positioned within the larger C1 factor ORF (Fig. 2).

Amino-terminal sequence of the 100-kDa polypeptide generated a mixture of two peptide sequences which were resolved to THETGTTHTATTVTSNM and PPPAASDQGEVE. These two positions correspond to sequences within and directly adjacent to the sixth amino acid repeat, respectively ( Fig. 2and Fig. 5). Similarly, amino-terminal analysis of the 123-135-kDa polypeptides generated peptide sequences containing TH(E/V)TGTT(H/N)TATTA(T/M)S and THETGTTHTATTATSNGG. These sequences represent a mixture of polypeptides whose amino termini lie within the second, third, and fifth repeat. Therefore, it is likely that these members of C1 factor polypeptides result from the specific proteolytic processing of the larger 230-kDa precursor precisely between positions 8 and 9 of the amino acid reiterations (Fig. 5).


Figure 5: The C1 factor amino acid repeats. The sequences of the six C1 factor amino acid repeats are aligned above the derived consensus. In each case, the highly conserved core sequence is set apart from the more divergent flanking residues. RPT-D represents an additional repeat, located between RPT-5 and RPT-6 in the C1 factor ORF, whose sequence diverges from the conserved core. The proposed site of the proteolytic cleavage(s) that generate the polypeptides of the C1 factor is indicated below the consensus (C1-PPS, C1 proteolytic processing site).



The 230- and 155-180-kDa proteins which are detected in lower abundance in preparations of the C1 factor probably represent full-length products and intermediates of the complete ORF. In support of this, an mRNA of >7 kilobases, sufficient to encode the precursor polypeptide, was detected in Northern blot analyses of HeLa cell poly(A) mRNA (data not shown).

The C1 Factor Is Expressed in Many Cell Types But Is Present in Unique Subnuclear Structures in HeLa Cells

The C1 factor activity has been detected in nuclear extracts from several human cell lines. (^2)In addition, Western blots with anti-C1 sera specifically detects polypeptides similar to the family of HeLa cell proteins in human cells of lymphoid origin, neural origin, lung, liver, kidney, and muscle, indicating that the factor may be ubiquitously expressed.^2 Furthermore, a homologous activity has been detected in cells derived from organisms as diverse as mouse and insects (SF9 and Schneider), suggesting that the C1 factor is highly evolutionarily conserved(6, 9) .^2 As shown in Fig. 6, the location of the C1 factor was determined in a variety of cell types by immunofluorescence using affinity purified Ab2125, Ab2131, or Ab2126 antibodies. Fluorescent staining of HeLa (cervical carcinoma, panel A), MRC-5 (normal lung, panel B), HepG2 (hepatocellular carcinoma, panel C), and 143 (osteosarcoma, panel D) with Ab2125 clearly indicates that the factor is diffusely localized throughout the nucleus. Identical patterns were also obtained using several human lymphoid cell lines (K-562, human erythroleukemia; WIL2-NS, mature human B lymphocyte; Raji, human lymphoblast; and Nalm6, human pre-B lymphocyte (data not shown)). In contrast, cells stained with control antibodies which were derived from the same seras did not exhibit any significant fluorescence (data not shown).


Figure 6: Immunofluorescent localization of the C1 factor. The preparation, fixation, and staining of cells was done as described under ``Materials and Methods.'' In each case, cells were stained with affinity purified Ab2125. The cells in panels A-D are as follows: A, HeLa; B, MRC-5; C, HepG2; and D, 143.



Surprisingly, in addition to the diffuse nuclear staining, HeLa cells specifically exhibited localization of the C1 factor to subnuclear punctate structures (Fig. 6, panel A). These structures were evident in all of the cells and were present in a relatively constant number per nucleus. Hep-2 cells, a clonal derivative of the HeLa line, exhibited an identical pattern while several other human papilloma virus positive and negative cervical carcinoma lines (C4II, SiHa, C33A, Caski, and ME180) exhibited only the typical diffuse nuclear fluorescence (data not shown). In addition, neither fixation of the cells with organic solvents nor infection of the cells with HSV-1(F) for 2 h altered the localization of the C1 factor in these cells (data not shown). Finally, identical patterns were obtained using affinity purified Ab2131 and Ab2126 antibodies. Therefore, although the C1 factor appears to be highly conserved and widely expressed in many cell types, the specific nuclear pattern observed in the HeLa cells suggests that the function(s) of this protein complex may differ in various cell types.


DISCUSSION

The Polypeptides of the C1 Factor Activity

The C1 factor is a required component of the multiprotein C1 regulatory assembly. In this context, the factor interacts directly with alphaTIF to form a heteromeric protein complex that recognizes the Oct-1 homeodomain and sequences of the HSV alpha/immediate early response element. The purification of the mammalian C1 activity indicated that the factor was a heterogeneous set of polypeptides. The more abundant 100- and 123-135-kDa proteins were proposed to be components of the factor based upon their quantity and stoichiometry in the fractions containing the purified C1 activity(40) . Consistent with this, the 123-135-, 155-180-, and 230-kDa polypeptides interacted directly with alphaTIF in protein blotting analyses. The tryptic digestions of the 100- and 123-135-kDa proteins indicated that they are highly related and the primary amino acid sequences of peptides derived from these proteins confirmed that they are products of a single gene. In addition, antisera derived against the C1 factor ORF reacted with the 100-, 123-135-, 155-180-, and 230-kDa polypeptides. Furthermore, these sera supershifted the assembled C1 complex, clearly indicating that one or more of these proteins is present in this complex. It is likely, therefore, that the heterogeneity exhibited by the assembled C1 complex reflects the heterogeneity of the C1 factor polypeptides(40) .

The C1 Factor Polypeptides Are Products of a Single Precursor Protein

The cDNA encoding the tryptic peptides derived from the 68-, 100-, and 123-135-kDa polypeptides contains a single ORF that encodes a novel polypeptide of 209 kDa. Tryptic peptide sequences derived from the 68-kDa protein are clustered within the amino terminus while those derived from the 100- and 123-135-kDa proteins are found within the carboxyl terminus of the predicted product of the ORF. In addition, antiseras directed against the central domain of the C1 ORF reacted with the 100-, 123-135-, 155-180-, and 230-kDa proteins while sera derived from the carboxyl-terminal domain of the ORF only reacted with a subset of these polypeptides. In each case, these seras did not react with the amino-terminal 68-kDa protein. Thus, it is likely that the 230-kDa protein represents the complete product of the ORF. The 110 and a subset of the 123-135-kDa proteins represent carboxyl-terminal products, while the remainder of the polypeptides (123-135 and 155-180) represent amino-terminal products.

Interestingly, the amino termini of the 100- and 123-135-kDa polypeptides lie within or directly adjacent to one of the six C1 factor amino acid reiterations, strongly suggesting that this family of polypeptides are derived by specific proteolytic processing of the larger precursor protein. In support of this, Wilson et al.(50) have also purified the mammalian C1 factor (HCF) and have obtained cDNA clones which contain the identical C1/HCF ORF. Upon expression of an epitope-tagged form of the complete ORF in mammalian cells, the entire set of C1/HCF polypeptides were detected by immunoblot. These results suggest that the processing of the C1 (HCF) precursor occurs in vivo.

The proteolytic processing of nuclear proteins is unusual. In the case of the C1 factor, the function of the cleavage is not apparent as the resulting products appear to remain complexed. The native factor has a gel permeation size of 10^6 daltons and sediments at 4-5 S (50) , suggesting that it is of high molecular mass with an extended structure. Thus, it is likely that several of the proteolytic cleavage products of the 230-kDa precursor remain associated with one another in a fibrous structure. Furthermore, as the C1 factor was purified in the presence of 3 M urea, the association of the polypeptide constituents is unusually stable(40) . These characteristics are distinct from those of other transcription factors which are proteolytically processed such as the large polypeptide of the basal transcription factor IIA (51, 52, 53) and the sequence specific factors NF-kappaB (54) and sterol response element-binding protein(55, 56) . In the latter examples, the precursor polypeptides are cleaved in response to cytoplasmic regulatory signals before transport to the nucleus. Whether the site of the C1 factor processing is cytoplasmic or nuclear is unclear. However, it is interesting that this cleavage occurs within a sequence that is reiterated six times in the precursor polypeptide. This repeated structure may be important for the frequency and efficiency of the cleavage, or alternatively, may be utilized to generate products which have distinct specific activities.

The C1 Factor

The C1/HCF factor represents a novel protein involved in the specific regulation of transcription by RNA polymerase II. The apparent ubiquitous expression of the C1 polypeptides as well as the evolutionary conservation of the factor suggest that it probably participates in basic, critical cellular processes that are independent of its specific role in the regulation of HSV gene expression. Consistent with a role in gene regulation, the factor is diffusely localized in the nucleus but additionally is concentrated in a few subnuclear foci in HeLa or HeLa derived cell lines. The unique localization in these cells does not appear to correlate with the origin of these cells from cervical carcinomas or with the presence of the human papilloma virus genome. However, this variation in subcellular localization may suggest that the factor participates in specific activities in a cell-dependent manner. Similar subnuclear structures have been detected in many cells and have been associated with both basic cellular processes such as pre-mRNA splicing(57, 58, 59) and DNA replication (60, 61, 62) as well as with specific mutations resulting from chromosomal translocations(63, 64) .

The isolation of a cDNA encoding the C1 proteins will permit further analyses of the interactions which dictate the regulated assembly of a multiprotein transcription complex. Specifically, the interaction of this protein with the HSV immediate early gene transactivator (alphaTIF, VP16, ICP25, VMV65) probably represents a critical determinant in the expression of the alpha/immediate early genes and the lytic/latent cycle of HSV. In this respect, it is of interest that the 123-135-, 155-180-, and 230-kDa proteins of the purified C1 factor specifically interacted with alphaTIF in a phosphorylation-dependent manner. In contrast, the 68-kDa protein which is derived from the amino terminus and the 100 kDa which is derived from the carboxyl terminus of the C1/HCF ORF did not participate in this interaction(40) . Therefore, the specific processing, modification, or selective representation of the various C1 factor polypeptides may be critical determinants in mediating the function of this factor. The characterization of the composition and subcellular localization of the C1 polypeptides may provide further insights into the proteolytic mechanisms of regulation.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant PO1-CA42063, partially by Cancer Center Support (core) Grant P30-CA14051 from the National Cancer Institute, by Cooperative Agreement CDR-8803014 from the National Science Foundation (to P. A. S.), by the Merck Research Laboratories, and by the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Viral Diseases. 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.

§
Leukemia Society of America Special Fellow and is presently an Investigator at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Viral Diseases. Present address and to whom correspondence should be addressed: National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Viral Diseases, Bldg. 4, Rm. 133, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-3854; Fax: 301-480-1560.

Sterling Winthrop Research Fellow in Health Sciences and Technology.

(^1)
The abbreviations used are: alphaTIF, alpha-transinduction factor; HSV, herpes simplex virus; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

(^2)
T. Kristie, unpublished data.


ACKNOWLEDGEMENTS

We thank S. Ludmerer and R. Wobbe of Merck Research Laboratories for their continued support; A. Bernards, M. McDonald, and J. Lees for the generous gifts of human pre-B cell cDNA library, human fetal retina cDNA library, and prepared library filters, respectively; M. C. Justice for assistance in the identification of C1 sequences in cDNA libraries; A. McBride for the human papilloma virus positive and negative cervical carcinoma cell lines; L. Staudt for the lymphoid cell lines; J. Yewdell and J. Bennink for their guidance and expertise in the immunofluorescence studies; D. Chasman for computer analyses; J. L. C. McKnight, C. Query, and D. Munroe, for helpful discussions; J. L. C. McKnight for the critical review of this manuscript; R. Issner, Y. Qiu, M. Duarte, and R. Dashner for technical assistance; R. Cook (MIT CCR Biopolymers Laboratory) for the tryptic peptide and amino-terminal sequence analyses; S. Schultz and J. Sisler for the synthesis of oligonucleotides; R. Coligan for the synthesis of C1 factor peptides; A. Wilson, K. LaMarco, M. Peterson, and W. Herr for the communication of their data; M. Siafaca, the members of the Sharp laboratory, and the Laboratory of Viral Diseases for their interest and support.


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