The Mouse Homologue of the Human Transcription Factor C1 (Host Cell Factor)

The assembly of the herpes simplex virus (HSV) α/IE gene enhancer complex is determined by the interactions of the Oct-1 POU domain protein, the viral αTIF (α-trans-induction factor, VP16, ICP25, VMW65), and the C1 factor (host cell factor, HCF). A unique transcription factor, C1 consists of a family of polypeptides derived from a common precursor by site-specific proteolytic processing. To analyze the role of this factor in the determination of HSV lytic-latent infection, cDNAs and genomic DNAs encoding the mouse homologue have been isolated. This factor is nearly identical to the human protein, contains multiple consensus proteolytic processing sites, and functions efficiently in the assembly of a specific HSV enhancer complex. Interestingly, the differential expression of the C1 factors in both human and mouse tissues may be important for the determination of HSV tissue tropism in these two organisms.


From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892
The assembly of the herpes simplex virus (HSV) ␣/IE gene enhancer complex is determined by the interactions of the Oct-1 POU domain protein, the viral ␣TIF (␣-trans-induction factor, VP16, ICP25, VMW65), and the C1 factor (host cell factor, HCF). A unique transcription factor, C1 consists of a family of polypeptides derived from a common precursor by site-specific proteolytic processing. To analyze the role of this factor in the determination of HSV lytic-latent infection, cDNAs and genomic DNAs encoding the mouse homologue have been isolated. This factor is nearly identical to the human protein, contains multiple consensus proteolytic processing sites, and functions efficiently in the assembly of a specific HSV enhancer complex. Interestingly, the differential expression of the C1 factors in both human and mouse tissues may be important for the determination of HSV tissue tropism in these two organisms.
The genes of herpes simplex virus (HSV) 1 form three major regulatory classes. The ␣/immediate early (␣/IE) genes are expressed immediately upon infection, and the products of these genes are required for progression to the later stages of viral gene expression (␤ and ␥), resulting in viral DNA replication and capsid assembly (reviewed in Refs. 1 and 2). The expression of the ␣/IE genes is regulated by ␣TIF (VP16), a transactivator that is packaged in the tegument structure of the virus (3)(4)(5)(6), released into the cell upon infection, and assembled into a specific transcription enhancer complex (7)(8)(9)(10)(11)(12)(13)(14)(15)(16).
The assembly of the multiprotein ␣/IE enhancer complex is mediated by a complex set of protein-DNA and protein-protein interactions. The cellular POU domain protein, Oct-1, recognizes the octamer motif (ATGCTAAT) within the enhancer element via binding of both the POU-specific subdomain (ATGC) and POU-homeo subdomain (TAATGA) (16 -19). ␣TIF recognizes sequences in the 3Ј domain of the enhancer element and cooperatively interacts with helix 1 of the Oct-1 POUhomeo subdomain (16,20).
The stable enhancer complex further requires the C1 factor (HCF) (9,13,16,21,22), an unusual cellular protein, which consists of a family of polypeptides (68, 100, 123-135, and 155-180 kDa) that are derived from a common 230-kDa precursor by site-specific proteolysis (23)(24)(25). This protein does not contain any apparent specific DNA binding activity but interacts, in a phosphorylation dependent manner, with ␣TIF (26,27) and the POU domain of Oct-1. 2 Since the C1 factor is a highly conserved protein (13,16,28) and has been implicated in the control of cell cycle (29), it is likely that this protein participates in a number of basic processes in addition to its role in the regulation of viral gene expression. Furthermore, the interaction of the C1 factor with a number of cellular proteins and transcription factors suggests that it is a significant component of the regulation complexes of cellular genes. 3 One of the key issues in the regulation of HSV gene expression concerns the biochemical mechanism(s) which determine the lytic-latent life cycle of the virus. Following a primary infection, an individual harbors the viral genome in the neurons of the sensory (trigeminal/sacral) ganglia where it remains in a latent state until complex stimuli induce reactivation and viral replication (reviewed in Ref. 2). Although little is known about the factors involved in determining the latentlytic states of the virus, the expression of the ␣/IE genes may represent a critical regulatory point in this process. Specifically, C1-dependent modulation of the viral cycle is an attractive model as the factor consists of a set of proteolytic forms and modification states that would enable it to effectively regulate the latent-lytic switch, it has a critical role in the regulation of HSV IE gene expression, and it is involved in basic cellular processes.
As no tissue culture system faithfully represents a latent HSV infection, studies have focused upon the use of recombinant viruses and transgenes in the mouse model system (reviewed in Ref. 2). To develop a genetic system to analyze the role of the C1 factor in cellular processes and in the regulation of the latent-lytic cycle of HSV, the forms and function of the mouse C1 homologue have been characterized. cDNAs and genomic clones which encode this protein were isolated and demonstrate that the mouse homologue is nearly identical to the human factor. Interestingly, in contrast to the ubiquitous expression of the C1 factor in cultured cells (24), the protein is not expressed in all human tissues. Furthermore, the differential expression of the C1 polypeptide(s) in human and mouse tissues may account for the tissue tropism of HSV in these two organisms.

EXPERIMENTAL PROCEDURES
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assay reaction and electrophoresis conditions were as described previ-* This work was supported by the National Institutes of Health, NIAID, 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 be 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 /EBI Data Bank with accession number(s) U53925 and U80821. ously (26) and included 15-fm HSV␣0 DNA probe, 20 ng of affinitypurified Oct-1, 30 ng of purified ␣TIF, and 50 ng of purified human C1 factor or 0.5 l (3.5 g of protein) of nuclear extract in a total of 10 l. For antibody supershifts, the reactions were incubated at 30°C for 10 min before the addition of 175 ng of affinity-purified anti-human C1 antibody (Ab2125) (24) for an additional 10 min.
Extracts and Western Blots-Nuclear extracts from HeLa, NA-AJ11, RMA, and P815 cells were prepared as described previously (31). 20 g of each extract were resolved in a 7.5% denaturing SDS-polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8), transferred to nitrocellulose, and probed with affinity-purified anti-human C1 antibody (AB2125) as described elsewhere (24). Protein extracts from tissues of 7-week-old BALBc mice were prepared by homogenization of 0.5 g of tissue in 2 ml of t-ext buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5 mM EDTA, 20 g ml Ϫ1 RNase A, "complete" protease inhibitor (aprotinin, leupeptin, Pefabloc, EDTA; Boehringer Mannheim) in a tissue grinder using a Teflon pestle attached to a variable speed drill. The homogenates were incubated on ice for 15 min and subsequently cleared by centrifugation at 20,000 ϫ g for 20 min. Human tissue extracts were purchased from CLONTECH. As various tissues contain a disproportionate level of specific proteins (i.e. actin in muscle tissues, neurofilaments in neural tissues), protein extracts were equalized according the representation of several common proteins. Replicate sets of equivalent amounts of protein from mouse or human tissues were resolved by SDS-PAGE, transferred to Immobilon, and probed with affinity-purified antibodies directed against the amino terminus (Ab2157 and Ab2159), the PPS domain (Ab2131) (24), or the carboxyl terminus (Ab2126) (24) of the human C1 factor. The blots were developed for chemiluminescent detection according to the manufacturer's recommendations (Pierce). Transfer efficiency was monitored by staining the membrane with Coomassie Brilliant Blue prior to blotting as described previously (32).
Screening Libraries for cDNAs Encoding the C1 Factor Polypeptide-Hybridization probes derived from the amino terminus (FL29), middle (FL53, FL150.2, and FL191.1), or carboxyl terminus (PB5c and H10) of the human C1 factor were prepared by isolation of the appropriate cDNA clone DNA fragments and labeling with [␣-32 P]dCTP (3000 Ci/mM) using the Klenow fragment of Escherichia coli DNA polymerase I (Boehringer Mannheim) and random hexamer primers (Pharmacia) according to the manufacturer's recommendations.
Overlapping cDNAs were isolated by successive screenings of a mouse fetal liver gt10 cDNA library (CLONTECH) using the appropriate human cDNA probe mixture. Nytran (Amersham Corp.) filter lifts were prepared for hybridization according to the manufacturer's recommendations. The filters were irradiated in a Stratagene UVlinker; baked for 30 min at 80°C; rinsed in 3 ϫ SSC (SSC, 150 mM NaCl, 17 mM sodium citrate), 0.1% SDS at 25°C for 45 min and at 55°C for 60 min; prehybridized in 5 ϫ SSC, 50% formamide, 5 ϫ Denhardt's solution, 0.5% SDS, 125 g/ml salmon sperm DNA at 42°C for 12 h and hybridized in 5 ϫ SSC, 50% formamide, 1 ϫ Denhardt's solution, 0.5% SDS, 125 g/ml salmon sperm DNA, 75 g/ml yeast tRNA, 2.0 ϫ 10 6 cpm/ml of the appropriate probe mixture at 42°C for 12-16 h. The filters were subsequently washed twice in 2 ϫ SSC, 0.1% SDS at 25°C for 30 min and three times at 68°C for 30 min. cDNA inserts from positive phage were subcloned into pBSK (Stratagene), and both strands were sequenced by the dideoxynucleotide method (33) at 45°C (Sequenase 2.0, Amersham Corp.). The nucleotide and amino acid sequences of the C1 factor clones (GenBank TM accession no. U53925) were compared with the human homologue using the BLAST network service (34) at the National Center for Biotechnology Information and MacVector software (IBI-Kodak).
Fluorescent in Situ Hybridization Analysis-DNAs from the mouse genomic clones 5341 and 5342 were labeled with digoxigenin dUTP by nick translation, combined with sheared mouse genomic DNA, and hybridized to male embryonic stem cell metaphase chromosomes in 2 ϫ SSC, 50% formamide, 10% dextran sulfate (Genome Systems). Hybridization signals were detected by incubation in fluoresceinated antidigoxigenin antibodies followed by 4,6-diamidino-2-phenylindole counterstain. Eighty metaphase chromosomes were analyzed with 72 exhibiting specific labeling. The specific chromosomal location was determined by cohybridization of 5341/5342 DNA probes with a probe that is specific for the centromeric region of the X chromosome. Ten specifically hybridized X chromosomes were measured to determine the position of the 5341/5342 signal from the telomere/heterochromatic-euchromatic boundary. RESULTS C1 (HCF) is a unique transcription factor due both to the unusual specific proteolytic processing that gives rise to the family of polypeptides as well as its participation in the regulation of processes such as RNA polymerase II-directed transcription and cell cycle control. However, little is known concerning the biochemical role of this protein in these processes due to the ubiquitous expression of the factor in cultured cells and to the lack of an appropriate genetic system. To develop a genetic system to analyze the role of the C1 factor in cellular functions and in the lytic-latent cycle of herpes simplex virus infection, cDNAs and genomic DNAs that encode the mouse C1 factor have been isolated and characterized. The Forms and Function of the Mouse C1 Factor-The human C1 factor consists of a family of polypeptides ranging from 68 to 230 kDa that are derived from a common precursor protein (23)(24)(25)(26). To determine if these polypeptides are conserved in the mouse, nuclear extracts of HeLa (human cervical carcinoma), NA-AJ11 (mouse neural), RMA (mouse lymphoid), and P815 (mouse lymphoid) cells were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified anti-human C1 sera (Fig. 1, left panel). As shown, the sera specifically reacted with the family of C1 polypeptides in the HeLa (lane 1) and mouse cell extracts (lanes 2-4), illustrating the conservation of the processed C1 factor forms.
The function of the mouse C1 factor in the assembly of an ␣/IE enhancer complex was determined by electrophoretic mobility shift assay in the presence of the HSV ␣TIF and the human Oct-1 proteins. As illustrated in Fig. 1 (right panel), the addition of the affinity-purified Oct-1 protein to a DNA binding reaction containing the HSV␣0 DNA probe resulted in the FIG. 2. cDNAs and amino acid sequence of the mouse C1 factor. Top, the assembled cDNA encoding the mouse C1 factor polypeptides is schematically illustrated with the positions of the 5Ј-untranslated sequence (nucleotides 1-54), ORF (nucleotides 55-6192), and 3Ј-untranslated domain (nucleotides 6193-7879). The cDNA clones that were isolated from a mouse fetal liver gt10 library are shown above and below the complete gene. Bottom, the amino acid sequence of the C1 factor ORF is shown with the eight conserved proteolytic processing sites in bold type.
formation of the Oct-1-DNA complex (lane 1), while a reaction containing Oct-1, ␣TIF, and the purified human C1 factor generated the characteristic C1 complex (H-C1, lane 2). Similarly, the addition of mouse nuclear extract derived from P815 cells to a reaction containing the human Oct-1 and ␣TIF proteins generated a stable C1 complex (M-C1, lane 3). The formation of the M-C1 complex was strictly dependent upon the inclusion of ␣TIF in the reaction and was not formed using HSV␣0 mutant probes (data not shown). Furthermore, addition of Ab2125 to the M-C1 reaction specifically retarded the mobility of the C1 complex (lane 4), in contrast to the addition of control anti-GST antibodies (data not shown). It should be noted that the difference in mobility between the human and mouse C1 complexes (lanes 2 and 3) is due to the particular C1 factor cleavage products that are present in the two preparations. Whereas the mouse P815 nuclear extract contains the complete family of proteins (68 -230 kDa), the purified human C1 factor consists primarily of the 68-and 100 -123-kDa C1 polypeptides (24,26).
The Mouse C1 Factor ORF-cDNAs encoding the mouse C1 factor were isolated from a gt10 mouse fetal liver library by hybridization with the human C1 factor cDNA (Fig. 2, top) (23,24). Assembly of the complete 7879-nucleotide cDNA reveals an ORF of 2045 amino acids (Fig. 2, bottom). As illustrated in Fig.  3, the predicted amino acid sequence of the mouse C1 factor is nearly identical (96% amino acid identity) to the human with the exception of a region of relatively high divergence within the central domain of the protein. Most significantly, the mouse ORF contains eight reiterations of the 20-amino acid sequence that represent the sites of specific proteolytic processing that generates the family of C1 factor polypeptides (PPS) (24). As shown, the consensus sequence for the mouse PPS is identical to that which was derived from the six human processing sites (23,24).
The Genomic Locus and Organization of the Mouse C1 Factor Clones containing the mouse C1 factor genomic sequences were hybridized to immobilized metaphase mouse chromosomes. The arrow designates the location of the C1 (HCF) gene in band C1 of the X chromosome, a domain that is equivalent to the human C1 factor chromosomal locus (36).
FIG. 6. Expression of the C1 factor polypeptides in human and mouse tissues. Equivalent amounts of protein extract of human (top right panel), or mouse tissues (left panels) were resolved in an SDSdenaturing gel, transferred to Immobilon, and probed with affinitypurified antibodies directed against the amino terminus (NH 2 ), proteolytic processing domain (PPSD), and carboxyl terminus (COOH) of the human C1 factor. The tissue type is identified at the top of the gel as: HL, control nuclear extract of HeLa cells; H, heart; B, brain; LV, liver; K, kidney; S, spleen; L, lung; T, thymus; and Y, thymus. Bottom right panel, duplicate blots of increasing amounts of mouse and human brain extracts were probed with anti-neurofilament P200 sera (N200) or with affinity purified anti-C1 factor antibodies. The molecular weights of the C1 factor polypeptides, in thousands, are indicated at the left of the panels.
Gene-As previously discussed, the development of a genetic system for the analysis of the C1 factor would significantly enhance the understanding of the role of this unique protein in cellular functions as well as the lytic/latent cycle of herpes simplex virus. Therefore, P1 phage containing the mouse genomic C1 gene were identified in an arranged library by polymerase chain reaction. Two recombinant phage (5341 and 5342) were subcloned and sequenced. The 25-kilobase pair deduced genomic structure of the C1 gene ORF (Fig. 4) is comprised of 26 exons with an unusually large single exon (501-amino acid/1503-nucleotide) encoding the entire C1 factor proteolytic processing domain.
The P1 genomic clones subsequently were used to determine the chromosomal location of the C1 factor gene by fluorescent in situ hybridization analysis. As shown in Fig. 5, hybridization of mouse metaphase chromosomes with 5341/5342 resulted in specific labeling of the third largest chromosome (X chromosome). The identity of the chromosome was confirmed and the location of the C1 gene within band XC1 was determined by cohybridization of 5341/5342 with a probe that was specific for the centromeric region of the X chromosome.
Expression of the C1 Factor in Human and Mouse Tissues-The C1 factor has been detected by immunofluorescence and Western blot in nuclei of many cultured or transformed human cell lines 2 (24). However, it is not clear that the polypeptides are stably expressed in all primary tissue types (35). Therefore, to investigate the expression of the C1 factor in human tissues, equivalent amounts of protein from total extracts of representative human tissues (heart, brain, liver, kidney, skeletal muscle, lung, and testis) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with a mixture of affinity-purified anti-C1 antiseras (Fig. 6, top right panel). Although the factor was detected in extracts of human heart, liver, kidney, and testis, the polypeptides were not found in extracts of brain, skeletal muscle, or lung, suggesting that the protein is not normally expressed ubiquitously. It should be noted that only a subset of the processed C1 factor polypeptides (100-and 125-135-kDa forms) are detected in these particular tissue extracts, likely due to the manner in which they were prepared. 4 The mouse represents an important animal model system for human infection by herpes simplex virus (reviewed in Ref. 2). However, there are distinct differences in the infection tropism between these two organisms (see "Discussion) (reviewed in Refs. 2 and 37). To determine if the expression of the C1 factor polypeptides correlates with this difference, replicate extracts from select mouse tissues (heart, brain, lung, spleen, and thymus; Fig. 6, left panels) were blotted with antisera directed against the amino terminus (NH 2 ), the proteolytic processing domain (PPSD), or the carboxyl terminus (COOH) of the human C1 factor. In contrast to the human, the C1 factor polypeptides were detected in all of the tested mouse extracts.
To control for differences in the mouse and human extracts, duplicate titrations of both brain extracts were resolved by SDS-PAGE and probed with anti-C1 or with anti-neurofilament p200 sera. While the human extract clearly contains a 4 T. Kristie, unpublished observations. significantly higher level (Ͼ10 fold) of the neurofilament p200, the C1 factor polypeptides are detected only in the mouse extract (Fig. 6, bottom right panel). This expression difference is further emphasized by quantitative Northern blot analyses of mouse and human tissue mRNA where C1 mRNA was significantly more abundant in mouse but not human neural tissues (Fig. 7, left). Interestingly, quantitative comparison of fetal and adult C1 mRNA levels suggests that the C1 factor mRNA is more highly expressed in early developmental stages (Fig. 7, right). DISCUSSION The Human, Mouse, and Caenorhabditis elegans C1 Factors-C1 (HCF) is a unique transcription factor that consists of a family of polypeptides which are involved in the regulated assembly of the herpes simplex virus ␣/IE gene enhancer complex by direct interaction with ␣TIF and Oct-1. In addition, the protein has recently been implicated in the control of cell cycle suggesting that C1 is involved in a number of basic cellular processes. To develop a mouse genetic system for the analysis of the role of C1 (HCF) in cell and viral functions, cDNAs and genomic DNAs have been isolated which encode the mouse C1 factor homologue.
As suggested by the conservation of epitopes and functional assembly of a C1 complex, the mouse C1 factor is nearly identical to the human (94% amino acid identity/96.0% amino acid similarity). Most significantly, the mouse factor contains the identical 20 amino acid repeats which are the sites of specific proteolytic processing to generate the family of C1 polypeptides. Although the actual function of the processing of the C1 factor remains unknown, the series of amino-and carboxylterminal products remain tightly associated, 2 suggesting that the processing is a novel mechanism by which the function or activity of the protein is regulated. The high level of conservation of these sites in the mouse C1 factor suggests that this processing is critical to the function of the factor. In support of this, data from two-hybrid analyses have indicated that the PPS are not simply sites of specific proteolytic processing but are also sites for protein interactions with other cellular proteins. 3 As illustrated in Figs. 3 and 8, C. elegans also contains a gene that is homologous to the human and mouse C1 factors. This homologue has a striking alignment to the amino-terminal and the carboxyl-terminal portions of the mammalian factors. It, however, is a significantly smaller protein that lacks the central region and the processing domain of the human and mouse factors, suggesting that its functions may be more limited than the mammalian counterparts. Interestingly, the elegans homologue retains the amino-terminal domain of the C1 factor which has been implicated in the control of cell cycle (29) but lacks the mammalian C1 factor domains which are involved in the interaction with several transcription factors, 3 suggesting that these functions are not interdependent.
The Mouse C1 Factor and Herpes Simplex Virus-After a primary infection with herpes simplex virus, the virus remains in a latent state in the sensory ganglia (trigeminal, sacral) until stimulated to enter a reactivated replication mode (reviewed in Ref. 2). However, the biochemical mechanism(s) by which the latent/lytic cycle is regulated are largely unknown, primarily due to the fact that HSV does not establish latent infections in tissue culture systems. As the ␣/IE genes of herpes simplex virus encode factors that are required for the progression of the viral lytic cycle, the regulation of this gene class could represent a significant point in the lytic/latent decision.
The C1 factor provides a striking key target for regulation of the viral lytic/latent cycle due to its regulated interaction with the components of the HSV ␣/IE enhancer complex and its role in the control of cell cycle. Furthermore, the difference in the expression of the C1 factor in human and mouse brains correlates with the observed ability of the virus to productively replicate in this tissue type as evidenced by the significantly different frequency of viral encephalitis in these organisms following central nervous system infection (reviewed in Refs. 2 and 37). Most significantly, studies of the expression and localization of the C1 factor polypeptides in mouse trigeminal ganglia indicate that the protein is specifically activated by stimuli which induce the reactivation of herpes simplex virus from the latent state. 5 As studies of the events which determine latency or reactivation of HSV are restricted to animal model systems, the characterization of the mouse C1 factor allows for the development of a genetic system to investigate the role of this protein in viral as well as cellular processes.