EPSTEIN-BARR VIRUS NUCLEAR ANTIGEN 1 PROTEIN AND ITS EXPRESSION AND RECOVERY

Abstract: A process for expressing and recovering Epstein-Barr nuclear antigen 1 (EBNA1) protein or polypeptide treats cells having a nucleus containing expressed EBNA1 protein or polypeptide to recover the nucleus containing the expressed EBNA1 protein or polypeptide. The nucleus containing the expressed EBNA1 protein or polypeptide is then separated into a liquid fraction containing the expressed EBNA1 protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus. The liquid fraction is separated from the solid fraction, and EBNA1 protein or polypeptide is recovered from the liquid fraction. Also encompassed by the present invention is an EBNA1 protein or polypeptide having substantially no components which generate false positive readings when used to detect Epstein-Barr virus in human serum, the DNA molecule encoding it, and recombinant expression of the protein. The protein is useful in a method for detection of Epstein-Barr virus.

Description: EPSTEIN-BARR VIRUS NUCLEAR ANTIGEN 1 PROTEIN AND ITS EXPRESSION AND RECOVERY The subject matter of this invention was developed with the support of the United States Government (NIH Grant Nos. R0I-GM38839 and ROI-CA531-01) . FIELD OF THE INVENTION The present invention relates to Epstein-Barr virus nuclear antigen 1 (EBNAl) protein and its expression and recovery. More particularly, the present invention relates to a process for recovering EBNAl protein or polypeptide from cells having a nucleus containing expressed EBNAl protein or polypeptide. BACKGROUND Epstein-Barr virus ("EBV"), a human herpesvirus, is one of the most common viruses infecting man, and antibodies to EBV proteins are present in greater than 80% of human serum samples. Milman et al. , "Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man," Proc. Natl. Acad. Sci. USA, 82:6300-04 (1985), which is hereby incorporated by reference. EBV was discovered during the course of attempts to learn the cause of lymphoma that was the most common tumor affecting children in certain parts of East Africa. The clinical syndrome, which was described in detail by Dennis Burkitt in 1958, had, in retrospect, been known to clinicians and pathologists since the beginning of the 20th century. However, through Burkitt's efforts, the disease was unified into a clearly delineated entity with characteristic clinical, pathological, and epidemiological features. See Burkitt D., "A sarcoma involving the jaws in African children," Br. J. Surq. , 46:218-223 (1958) , which is hereby incorporated by reference. In 1964, Epstein and Barr reported the first successful attempt to establish continuous lymphoblastoid cell lines from explants of Burkitt's lymphoma ("BL") , which were eventually found to be infected with EBV by . and G. Henle in 1966. Epstein et al . , "Cultivation in vi tro of human lymphoblasts from Burkitt's malignant lymphoma," Lancet, 1:252-53 (1964) and Henle et al. , "Immunofluorescence in cells derived from Burkitt's lymphoma, " J. Bacteriol .. 91:1248-1256 (1966) , which are hereby incorporated by reference. In addition to its involvement in BL, EBV is the etiological agent of infectious mononucleosis and has been implicated in the pathogenesis of nasopharyngeal carcinoma. EBV can also induce fatal lymphoproliferative disease, sometimes with the features of frank lymphoma, in certain patients with global immunodeficiency that is either congenital (such as severe combined immunodeficiency or ataxia telangiectasia) or acquired as the result of immunosuppression for organ or tissue transplantation or due to AIDS. Kieff et al . , "Epstein-Barr Virus and Its Replication," Chapter 67, pp. 1889-1920 and Miller, "Epstein-Barr Virus: Biology, Pathogenesis, and Medical Aspects," Chapter 68, pp. 1921-1958, in Virology. Second Edition, edited by B. N. Fields, D. M. Knipe et al . , Raven Press, Ltd., New York, 1990, which are hereby incorporated by reference. It has been determined that the principal biological activity of EBV that underlies its role in the pathogenesis of lymphoproliferative diseases is the ability of the virus to cause indefinite in vi tro proliferation of lymphocytes, a process termed "immortalization." The sequence of several events in the process of immortalization has been defined. The process is thought to consist of two phases: (i) an initial phase of B-cell activation, triggered by virus binding to the cell surface, and (ii) a subsequent phase of permanent blastogenesis which requires the expression of 10 EBV gene-encoded products - the primary of which is EBV nuclear antigen 1 ("EBNAl") . The 172, 000-base-pair ("bp") DNA genome of EBV is found in all "immortalized" permanent B-cell lyτnphoblast lines as multicopy latent extrachromosomal circular DNA plasmids or episomes. Only EBNAl is essential for the replication of these EBV plasmids. The EBNAl protein comprises 641 amino acids ("aa") . One-third of EBNAl (aa 90 to 325) consists of a repetitive array of glycine ("Gly") and alanine ("Ala") amino acid residues. Shah et al. , "Binding of EBNA-1 to DNA Creates Protease-Resistant Domain That Encompasses the DNA Recognition and Dimerization Functions," Journal of Virology. 66:6:3355-62 (1992) and Yates et al . , "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells," Nature, 318:812-15 (1985), which are hereby incorporated by reference. The size of the repeat array varies among different EBV isolates and the EBNAl polypeptide shows corresponding size variations ranging from 68 kDa to 84 kDa. Milman et al . , "Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man, " Proc. Natl. Acad. Sci. USA. 82:6300-04 (1985) , which is hereby incorporated by reference. Additionally, it has been reported that the Gly- Ala repeat sequence has homology to cellular DNA, and antisera to Gly-Ala repeat-containing peptides also react with cellular proteins, e.g., E. coli , mammalian or baculovirus cellular proteins containing glycine plus alanine-rich regions. Id. EBNAl protein binds in trans to the latent origin of replication, oriP, at multiple sites present in the two regions of oriP which were found to be necessary and sufficient for origin function. One of these regions is composed of 20 tandem copies of a 30-bp sequence (i.e., family of repeats) , each of which contains an EBNAl binding site . The other region includes four EBNAl binding sites (dyad symmetry element) , two of which are located within a 65-bp region of dyad symmetry. The interaction of EBNAl with oriP occurs mainly through the carboxyl-terminal third of the protein. It has been theorized that EBNAl activates oriP to function not only as an origin of replication but also as a plasmid maintenance element and a transcriptional enhancer. Frappier et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus," The Journal of Biological Chemistry, 266 (12) :7819-26, (1991) , and Yates et al. , "Dissection of DNA Replication and Enhancer Activation Functions of Epstein-Barr Virus Nuclear Antigen 1," Cancer Cells 6/Eukaryotic DNA Replication, pp. 197-205, Cold Spring Harbor Laboratory, 1988, which are hereby incorporated by reference. Unfortunately, the production of biochemical assays to analyze the mechanism by which EBNAl activates oriP to function as the origin of replication, a plasmid maintenance element, and a transcriptional enhancer has been difficult due to the lack of efficient systems for production of the virus and the very low amounts of gene products in transformed cells. Further, low protein expression has hindered the application of cell-derived EBNAl protein as an antigen in a detection immunoassay for EBV. Initially, researchers utilized E. coli-based expression systems in an attempt to produce the EBNAl protein. For example, Orlowski et al . , "Inhibition of Specific Binding of EBNAl to DNA by Murine Monoclonal and Certain Human Polyclonal Antibodies," Virology, 176:638-42 (1992) , expressed EBNAl as a non-fusion protein in E. coli under control of the lac promoter. Milman et al. , "Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man," Proc. Natl. Acad. Sci. USA. 82:6300-04 (1985) , synthesized the carboxyl-terminal one-third of EBNAl encoded by the BamHI restriction fragment K in E. coli by use of the expression plasmid pHE6. Expression of the EBNAl fusion polypeptide was poor, i.e., only approximately 1.3 μg was recovered. In Chen et al. , "Separation of the Complex DNA Binding Domain of EBNA-1 into DNA Recognition and Dimerization Subdomains of Novel Structure, " Journal of Virology. 67:8:4875-85 (1993) , and Shah et al . , "Binding of EBNA-1 to DNA Creates Protease-Resistant Domain That Encompasses the DNA Recognition and Dimerization Functions," Journal of Virology, 66:6:3355-62 (1992) , the DNA binding and dimerization functions of EBNAl were studied by creating a series of deletions and point mutations in the region of that protein spanning amino acids 408 to 641. Genes encoding for these modified forms of EBNAl were cloned into plasmids and transformed into E. coli . Expression was poor in both studies. The use of mammalian cell expression systems has also been described. For example, Yates et al. , "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells," Nature, 318:812-15 (1985) , studied the functions of various segments of the gene encoding EBNAl by deletion analysis. As shown in FIG. 5 of Yates et al. , several of these deletions involve removal of the Gly-Ala repeat amino acid sequence. The genes encoding for these proteins were cloned into plasmids which were used to trar.sfect human cells. Middleton et al . , "EBNAl Can Link the Enhancer Element to the Initiator Element of the Epstein-Barr Virus Plasmid Origin of DNA Replication, " Journal of Virology. 66(l) :489-95 (1992), which is hereby incorporated by reference, expressed EBNAl in CV-lp cells by using an infectious simian virus (SV) 40 vector containing the EBNAl gene. Expression was quite poor. Hammarskjδld et al. , "High-level expression of the Epstein-Barr virus EBNAl protein in CV1 cells and human ly phoid cells using a SV40 late replacement vector, " Gene. 43:41-50 (1986) , which is hereby incorporated by reference, inserted the EBNAl gene-containing EBV BamHI-K fragment (B95-8 strain) into an expression vector composed of SV40 and pBR322 DNA. The vector was transfected into CV1 monkey cells and yielded EBNAl protein (which included the entire Gly-Ala repeat unit) in 40-50% of the transfected cells. Unfortunately, protein contaminants produced by current E. coli or mammalian cell expression systems can contribute to false positive readings when E. coli or mammalian cell-derived EBNAl protein is used as an antigen in a detection immunoassay for EBV. This is due to crossreactivity of the detecting antibodies with E. coli or mammalian cellular contaminant proteins containing glycine plus alanine-rich regions, i.e., the antibodies could bind with the Gly-Ala repeat portion of the contaminant proteins thereby incorrectly indicating the presence of EBV. See Milman et al . , "Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man, " Proc. Natl. Acad. Sci. USA. 82:6300-04 (1985) , which is hereby incorporated by reference. Several researchers have attempted to express EBNAl protein utilizing baculovirus expression systems, but with limited success with regard to quantity and purity of recovered protein. For example, Hearing et al. , "Interaction of Epstein-Barr Virus Nuclear Antigen 1 with the Viral Latent Origin of Replication, " Journal of Virology. 66 (2) :694-705 (1992), which is hereby incorporated by reference, described a process for expression and purification of EBNAl using a baculovirus expression system. The gene encoding the protein contained the entire EBNAl open reading frame of the B95-8 virus isolate, including the entire Gly-Ala repeat amino acid sequence. As in the E. coli and mammalian cell expression systems discussed above, the presence of the Gly-Ala repeat in baculovirus cell-derived EBNAl protein could contribute to false positive readings when such an EBNAl protein is used as an antigen in a detection immunoassay for EBV. Hearing et al. also achieved poor expression levels. Frappier et al. , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus," The Journal of Biological Chemistry. 266:12:7819-26 (1991) , which is hereby incorporated by reference, expressed and purified the EBNAl protein using a baculovirus expression system. In this process, a portion of the EBNAl protein was expressed with an undefined deletion of amino acids in its Gly-Ala repeat amino acid sequence. As a result, due to the reference's failure to define the nature of the Gly-Ala repeat amino acid sequence deletion, its work cannot be repeated. Approximately 1.4 mg (representing a yield of 33%) of homogeneous 50-kDa baculovirus-derived EBNAl protein was recovered. In addition, only a low yield of EBNAl was recovered, because, after disruption of the nucleus, the EBNAl protein and nuclear DNA were not adequately separated. Instead, there was a substantial gelatinous fraction containing both the protein and DNA. The EBNAl protein in this fraction could not be recovered. Since the above-described expression systems only teach how to produce small amounts of relatively impure EBNAl protein, the use of the EBNAl protein continues to be hampered. Further, the currently expressed EBNAl proteins are susceptible to false positive readings when used as an antigen in a detection immunoassay for EBV. There thus remains a need to achieve improved EBNAl protein expression. SUMMARY OF THE INVENTION One aspect of the present invention relates to a process for recovering EBNAl protein or polypeptide. In this process, cells having a nucleus containing expressed EBNAl protein or polypeptide are treated to recover the nucleus containing the expressed EBNAl protein or polypeptide. The nucleus containing the expressed EBNAl protein or polypeptide is then separated into a liquid fraction containing the expressed EBNAl protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus. The liquid fraction is separated from the solid fraction, and EBNAl protein or polypeptide is recovered from the liquid fraction. This process produces abundant quantities of purified EBNAl protein or polypeptide useful for diagnosis of EBV. The present invention also relates to an isolated EBNAl protein or polypeptide formulation having substantially no components which generate false positive readings when used to detect EBV in human serum. This isolated EBNAl protein or polypeptide formulation can be utilized for detection of EBV in a sample of human tissue or body fluids. This detection process involves providing the isolated EBNAl protein or polypeptide formulation as an antigen, contacting the sample with the antigen, and detecting any reaction which indicates that EBV is present in the sample using an assay system. Additionally, the present invention provides an isolated DNA molecule encoding EBNAl protein or polypeptide, a recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA molecule encoding EBNAl protein or polypeptide, and a host cell incorporating a heterologous DNA molecule encoding EBNAl protein or polypeptide, all of which have substantially no components which generate false positive readings when used to detect EBV in human serum. The present invention also provides a process of expressing an EBNAl protein coding sequence in a cell. In this process, an EBNAl protein coding sequence is cloned into a baculovirus transfer vector. The baculovirus transfer vector and Autographica calif ornica nuclear polyhedrosis genomic DNA are then co-transfected into insect cells, and recombinant baculoviruses are recovered. Cells are then infected with the recombinant baculovirus under conditions facilitating expression of isolated EBNAl protein or polypeptide in the cell. In this process, the EBNAl protein coding sequence includes no more than 90% of the Gly-Ala repeat amino acid sequence present in the naturally- occurring EBNAl protein coding sequence which spans the Gly- Ala repeat amino acid sequence. By utilizing this process, EBNAl protein or polypeptide is expressed in quantities sufficient for the production of a detection immunoassay for EBV which provides few false positive readings. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the construction of the EBNAl baculovirus transfer vector pVL941-EBNAl. The sequence of the oligonucleotide linkers inserted in the polyhedrin gene of the baculovirus transfer vector pVL941-S is shown above the plasmid. The underlined ATG is the only ATG sequence in the 5' region of the polyhedrin gene and was used as the start codon for translation of the EBNAl gene. After linearization of pVL941-SW with Ncol and digestion with bacterial alkaline phosphatase, the 3' -recessed ends were extended with the Klenow fragment of DΝA polymerase I. The EBNAl gene was excised from p205 with Rsal and Ball enzymes, which remove the first seven codons of the gene, and ligated into pVL941-SW to form pVL941-EBNAl . Hygromycin phosphotransferase (hph) and δ-lactamase (amp) genes are also shown. FIG. 2 shows a modified protocol for improved yield and purity of bEBNAl. This is a Coomassie Blue stained SDS-polyacrylamide gel analysis of each step in the new purification scheme. The lanes read from right to left instead of from left to right. Starting at the far right the lane marked "EBNAl" is a lane of bEBNAl protein purified by this procedure (i.e. it is the same as the lane on the far left) as verified by ability to bind to oriP. Cells - are whole SF-9 cells infected with the recombinant bEBNAl recombinant baculovirus and the bEBNAl band is visible. Cytoplasm - is the cytoplasmic supernatant after lysing the cells and spinning down the nuclei. Nuclei - is the whole nuclei after cell lysis and separating out nuclei from cytoplasm by centrifugation. PolyminP - is the supernatant after lysis of the nuclei and pelleting the DNA by PolyminP and centrifugation. 30% A.S. - is the pellet that forms upon adding ammonium sulfate to the PolyminP supernatant (no significant bEBNAl present) . 45% A.S. is the pellet that forms upon adding ammonium sulfate to the 30% A.S. supernatant to a final concentration of 45% (contains enriched bEBNAl) . 60% A.S. - is the pellet that forms upon adding ammonium sulfate to the 45% supernatant to a final concentration of 60% (no bEBNAl present due to its being present only in the 30-45% cut) . Heparin - bEBNAl after chro otography of the 45% A.S. pellet over the Heparin Sepharose column. Oligo. Aff. - bEBNAl after chromatography of the Heparin fraction over the oriP oligonucleotide affinity column. FIG. 3 shows the phosphate labeling and phosphatase digestion of bEBNAl. Sf-9 cells were infected with the AcMNPV-EBNAl baculovirus and labeled with [ 32 P] orthophosphate as described in the Examples. Labeled cells were separated into cytoplasmic (cyt) and nuclear inuc) fractions, and bEBNAl was purified to homogeneity from the nuclear extract. Pure [ 32 P] EBNAl was incubated at 25°C for 1 h either with (+) or without (-) CIP. Samples were subjected to electrophoresis on 12% SDS-polyacrylamide gels and 32 P-labeled proteins were detected upon autoradiography of wet gels. FIG. 4 shows the phosphoamino acid analysis of bEBNAl. Pure [32P]bEBNAl was hydrolyzed in 6 N HC1 (constant boiling) at 110°C for 1 or 2 h, then combined with unlabeled phosphoserine ( Ser-P) , phosphothreonine ( Thr-P) , and phosphotyrosine ( Tyr-P) markers. The mixture of hydrolyzed amino acids and phosphoamino acid standards was separated by high voltage paper electrophoresis. Positions of phosphoamino acid markers were visualized by ninhydrin staining and are indicated by dotted circles . Positions of 32 P-labeled amino acids and nonhydrolyzed bEBNAl (0-h data lane) were identified by autoradiography. FIGS. 5A and B show the native aggregation state of bEBNAl. bEBNAl was combined with the protein standards apoferritin { apo; 440 kDa) , IgG (158 kDa) , bovine serum albumin (BSA; 66 kDa) , ovalbumin { ova; 45 kDa) and myoglobin {myo; 17 kDa) , then analyzed by glycerol gradient sedimentation (A) or gel filtration on Superose (B) as described herein. bEBNAl was identified in column fractions by the nitrocellulose filter binding assay. The sedimentation coefficient (s) and Stokes radius of bEBNAl were determined by comparison to the positions of protein standards of which the s values and Stokes radii are known. FIG. 6 shows the stoichiometry of [ 35 S]bEBNAl bound to oriP DNA. [ 35 S] bEBNAl was incubated with pGEMoriP7, then gel-filtered to separate [ 35 S] bEBNAl bound to pGEMoriP7 in the excluded fractions from unbound bEBNAl in the included fractions as described in the Examples. Fractions were analyzed for DNA and [ 35 S] bEBNAl. FIG. 7 shows the salt dependence of bEBNAl binding to the family of repeats and the dyad symmetry element . bEBNAl (50 ng) was incubated with 40 fmol of 32 P-end-labeled DNA containing either the dyad symmetry element { closed circles) or the family of repeats { open circles) in the presence of 2.5 μg of calf thymus DNA and various concentrations of NaCl . After 10 min at 23°C, the reaction mixture was filtered through nitrocellulose, and the DNA retained on the filters was quantitated by liquid scintillation. FIG. 8 shows the effect of the family of repeats on binding of bEBNAl to the dyad symmetry element. Top, diagram of oriP showing the disposition of EBNAl binding sites [boxes) . Bottom, 10 fmol of 32 P-labeled DNA fragment containing either the family of repeats ( open circles) , the dyad symmetry element ( closed circles) , or the complete oriP ( closed triangles) were incubated with various amounts of bEBNAl (shown as fmol dimers) in 50 mM HEPES (pH 7.5) , 300 mM NaCl, 5 mM MgCl 2 for 10 min at 23°C. Reactions containing the family of repeats or dyad symmetry element were then filtered through nitrocellulose. Reactions containing the complete oriP ( closed triangles) were treated with 50 units of BcoRV for 3 min at 37°C to separate the family of repeats from the end-labeled dyad symmetry element (see scheme, top) prior to filtration through nitrocellulose. FIGS. 9A and B show the protection of the Aval site in the dyad symmetry element by bEBNAl. In FIG. 9A, the 300-bp DNA fragment containing the dyad symmetry element, 32 P-end-labeled at one end only, was incubated with various amounts of bEBNAl (shown as fmol dimers) prior to digestion with Aval and electrophoresis on a 6% polyacrylamide gel. The DNA was visualized by autoradiography of dried gels. Scheme of DNA fragment (top) shows EBNAl consensus binding sites (boxes) . In FIG. 9B, the Aval-protected bands in the autoradiograph in A were quantitated by a laser densitometer (LKB Bromma Ultroscan XL) . FIG. 10 is cloning scheme for preparation of a vector for expression in E. coli of EBNAl. FIG. 11 is a map for the plasmid p291. The Hindlll fragment contains the eEBNAl gene's nucleotides 107930-110493 (2.563kb) from the strain EBV B93-8, with the eEBNAl gene itself spanning nucleotides 107950 to 109872 (1.922kb) . The PCR product of the eEBNAl gene between the N and C terminii PCR primers is shown in the upper right . FIGS. 12A-C show the full double stranded DNA PCR product of the eEBNAl gene with restriction endonuclease sites. The upper strand corresponds to SEQ. ID. No. 3. DETAILED DESCRIPTION The present invention relates to a process for recovering EBNAl protein or polypeptide having the following steps: providing cells having a nucleus containing EBNAl protein or polypeptide; recovering the nucleus containing expressed EBNAl protein or polypeptide from the cells; separating the nucleus containing expressed EBNAl protein or polypeptide into a liquid fraction containing the expressed EBNAl protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus; separating the liquid fraction from the solid fraction; and recovering EBNAl protein or polypeptide from the liquid fraction. In this process, the nucleus is separated by centrifugation where the liquid fraction is a supernatant and the solid fraction is a pellet. After centrifugation, the supernatant contains less than 5% of DNA. The process further provides subjecting the liquid fraction to a first ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing contaminant proteins and a liquid phase containing EBNAl protein or polypeptide, followed by subjecting the liquid phase containing EBNAl protein or polypeptide to a second ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing EBNAl protein or polypeptide and a liquid phase containing contaminant proteins and then finally separating the solid phase containing EBNAl protein or polypeptide and the liquid phase containing contaminant proteins. The first ammonium sulfate treatment is at a >0 to 30%, preferably 30%, ammonium sulfate concentration and the second ammonium sulfate treatment is at a 30 to 45%, preferably 45%, ammonium sulfate concentration. The solid phase containing EBNAl protein or polypeptide is then purified, after separation, by affinity column chromatography, such as agarose-heparin column chromatography or oligonucleotide affinity column chromatography. By utilizing this purification process, it is believed that the recovered EBNAl protein is folded in its natural conformation. This process produced abundant quantities of purified EBNAl protein or polypeptide useful for diagnosis of EBV. According to one embodiment, insect cells, preferably Sf-9 insect cells, are grown and infected with EBNAl-containing recombinant baculovirus, then harvested after a sufficient amount of time has passed to allow for protein expression. The cytoplasmic membrane is disrupted and the nuclei containing expressed baculovirus-derived EBNAl protein or polypeptide ("bEBNAl") are pelleted to remove cytoplasm. The nuclei are lysed, producing a viscous solution ("nuclear extract") due to the presence of DNA. The DNA is then removed by sonication which shears the DNA and partially reduces the viscosity of the nuclear extract. A chromatography preparation solution is then added to the nuclear extract which is incubated and then centrifuged. This packs the DNA down tight into a small pellet, leaving most of the solution free of DNA. The solution is decanted and then treated according to the above-described two-step ammonium sulfate precipitation procedure. The centrifugation procedure after the second ammonium sulfate precipitation step produced a supernatant which is discarded and a pellet with bEBNAl. The pellet containing bEBNAl is dissolved in a buffer and then dialyzed against the buffer. This dialyzed preparation is loaded onto an ion exchange chromatography column and eluted from it with a salt gradient and then purified using affinity column chromatography. In another embodiment, E. coli cells, rather than insect cells, are used as host cells. The present invention also relates to an isolated EBNAl protein or polypeptide formulation having substantially no components which generate false positive readings when used to detect EBV in human serum. Furthermore, wherein naturally-occurring EBNAl protein or polypeptide spans a Gly-Ala repeat amino acid sequence, the isolated EBNAl protein or polypeptide of the present invention includes no more than 90%, preferably no more than 94%, of the Gly-Ala repeat amino acid sequence. Additionally, the present invention provides an isolated DNA molecule encoding EBNAl protein or polypeptide, a recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA molecule encoding EBNAl protein or polypeptide, and a host cell, such as an insect cell, incorporating a heterologous DNA molecule encoding EBNAl protein or polypeptide, all of which have substantially no components which generate false positive readings when used to detect EBV in human serum. The heterologous DNA molecule encoding the bEBNAl protein or polypeptide of the present invention comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: ATG ACA GGA CCT GGA AAT GGC CTA GGA GAG AAG GGA GAC ACA TCT GGA CCA GAA GGC TCC GGC GGC AGT GGA CCT CAA AGA AGA GGG GGT GAT AAC CAT GGA CGA GGA CGG GGA AGA GGA CGA GGA CGA GGA GGC GGA AGA CCA GGA GCC CCG GGC GGC TCA GGA TCA GGG CCA AGA CAT AGA GAT GGT GTC CGG AGA CCC CAA AAA CGT CCA AGT TGC ATT GGC TGC AAA GGG ACC CAC GGT GGA ACA GGA GCA GGA GCA GGA GCG GGA GGG GCA GGA GCA GGA GGT GGA GGC CGG GGT CGA GGA GGT AGT GGA GGC CGG GGT CGA GGA GGT AGT GGA GGC CGC CGG GGT AGA GGA CGT GAA AGA GCC AGG GGG GGA AGT CGT GAA AGA GCC AGG GGG AGA GGT CGT GGA CGT GGA GAA AAG AGG CCC AGG AGT CCC AGT AGT CAG TCA TCA TCA TCC GGG TCT CCA CCG CGC AGG CCC CCT CCA GGT AGA AGG CCA TTT TTC CAC CCT GTA GGG GAA GCC GAT TAT TTT GAA TAC CAC CAA GAA GGT GGC CCA GAT GGT GAG CCT GAC GTG CCC CCG GGA GCG ATA GAG CAG GGC CCC GCA GAT CAC CCA GGA GAA GGC CCA AGC ACT GGA CCC CGG GGT CAG GGT GAT GGA GGC AGG CGC AAA AAA GGA GGG TGG TTT GGA AAG CAT CGT GGT CAA GGA GGT TCC AAC CCG AAA TTT GAG AAC ATT GCA GAA GGT TTA AGA GCT CTC CTG GCT AGG AGT CAC GTA GAA AGG ACT ACC GAC GAA GGA ACT TGG GTC GCC GGT GTG TTC GTA TAT GGA GGT AGT AAG ACC TCC CTT TAC AAC CTA AGG CGA GGA ACT GCC CTT GCT ATT CCA CAA TGT CGT CTT ACA CCA TTG AGT CGT CTC CCC TTT GGA ATG GCC CCT GGA CCC GGC CCA CAA CCT GGC CCG CTA AGG GAG TCC ATT GTC TGT TAT TTC ATG GTC TTT TTA CAA ACT CAT ATA TTT GCT GAG GTT TTG AAG GAT GCG ATT AAG GAC CTT GTT ATG ACA AAG CCC GCT CCT ACC TGC AAT ATC AGG GTG ACT GTG TGC AGC TTT GAC GAT GGA GTA GAT TTG CCT CCC TGG TTT CCA CCT ATG GTG GAA GGG GCT GCC GCG GAG GGT GAT GAC GGA GAT GAC GGA GAT GAA GGA GGT GAT GGA GAT GAG GGT GAG GAA GGG CAG GAG TGA The amino acid sequence, corresponding to the DNA molecule of SEQ. ID. No. 1, is SEQ. ID. No. 2 as follows: Met Thr Gly Pro Gly Asn Gly Leu Gly Glu Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gin Arg Arg Gly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro Arg His Arg Asp Gly Val Arg Arg Pro Gin Lys Arg Pro Ser Cys lie Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly Gly Ala Aly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro Ser Ser Gin Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu Tyr His Gin Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly Ala lie Glu Gin Gly Pro Ala Asp His Pro Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gin Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gin Gly Gly Ser Asn Pro Lys Phe Glu Asn lie Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala lie Pro Gin Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala Pro Gly Pro Gly Pro Gin Pro Gly Pro Leu Arg Glu Ser lie Val Cys Tyr Phe Met Val Phe Leu Gin Thr His lie Phe Ala Glu Val Leu Lys Asp Ala lie Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn lie Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gin Glu OPA Production of this isolated protein or polypeptide is preferably carried out using recombinant DNA technology. Furthermore, the isolated DNA molecule is isolated from any other DNA molecule which expresses protein that generates false positive readings when the EBNAl protein or polypeptide is used to detect EBV in human serum. Additionally, the heterologous DNA molecule encoding the E. coli expression system-derived EBNAl protein or polypeptide ("eEBNAl") of the present invention comprises the nucleotide sequence corresponding to SEQ. ID. No. 3 as follows : ATG GGA GAA GGC CCA AGC ACT GGA CCC CGG GGT CAG GGT GAT GGA GGC AGG CGC AAA AAA GGA GGG TGG TTT GGA AAG CAT CGT GGT CAA GGA GGT TCC AAC CCG AAA TTT GAG AAC ATT GCA GAA GGT TTA AGA GCT CTC CTG GCT AGG AGT CAC GTA GAA AGG ACT ACC GAC GAA GGA - 19 - ACT TGG GTC GCC GGT GTG TTC GTA TAT GGA GGT AGT AAG ACC TCC CTT TAC AAC CTA AGG CGA GGA ACT GCC CTT GCT ATT CCA CAA TGT CGT CTT ACA CCA TTG AGT CGT CTC CCC TTT GGA ATG GCC CCT GGA CCC GGC CCA CAA CCT GGC CCG CTA AGG GAG TCC ATT GTC TGT TAT TTC ATG GTC TTT TTA CAA ACT CAT ATA TTT GCT GAG GTT TTG AAG GAT GCG ATT AAG GAC CTT GTT ATG ACA AAG CCC GCT CCT ACC TGC AAT ATC AGG GTG ACT GTG TGC AGC TTT GAC GAT GGA GTA GAT TTG CCT CCC TGG TTT CCA CCT ATG GTG GAA GGG GCT GCC GCG GAG GGT GAT GAC GGA GAT GAC GGA GAT GAA GGA GGT GAT GGA GAT GAG GGT GAG GAA GGG CAG GAG CTG CGT CGT GCT TCT GTT GGT TAA The amino acid sequence, corresponding to the DNA molecule of SEQ. ID. No. 3, is SEQ. ID. No. 4 as follows: Met Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gin Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gin Gly Gly Ser Asn Pro Lys Phe Glu Asn lie Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala lie Pro Gin Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala Pro Gly Pro Gly Pro Gin Pro Gly Pro Leu Arg Glu Ser lie Val Cys Tyr Phe Met Val Phe Leu Gin Thr His He Phe Ala Glu Val Leu Lys Asp Ala He Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn He Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gin Glu Leu Arg Arg Ala Ser Val Gly OCH The DNA molecule encoding the EBNAl protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present) . The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus. Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtll, gt ES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUCIS, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference) , pQE, pIH821, pGEX, pET series (see F. . Studier et . al. , "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990) , which is hereby incorporated by reference) and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al . , Molecular Cloning: A Laboratory Manual. Cold Springs Laboratory, Cold Springs Harbor, New York (1982) , which is hereby incorporated by reference. A variety of host-vector systems may be utilized to express the protein-encoding sequence (s) . Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus (e.g., baculovirus) . The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation) . Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells. Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribcsomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzvmology. 68:473 (1979) , which is hereby incorporated by reference. Promotors vary in their "strength" (i.e. their ability to promote transcription) . For the purposes of expressing a cloned gene, it is desirable to use strong prorr.otors in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable prorr.otors may be used. For instance, when cloning in E. coli , its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA pror.otor, ribosomal RNA promotor, the P R and P L promotors of coliphage lambda and others, including but not limited, to 2acUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Addi ionally, a hybrid trp-lacUV5 ( tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside) . A variety of other operons, such as trp, pro, etc., are under different controls. Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DΝA or other techniques involving incorporation of synthetic nucleotides may be used. Once the isolated DΝA molecule has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, insect, virus, yeast, mammalian cells, and the like. The present invention also provides a method of expressing an EBΝA1 protein coding sequence in a cell . In this expression process, an EBΝA1 protein coding sequence is cloned into a baculovirus transfer vector. The baculovirus transfer vector and Autographica californica nuclear polyhedrosis genomic DNA are then co-transfected into insect cells, and recombinant baculoviruses are recovered. Cells are then infected with the recombinant baculovirus under conditions facilitating expression of isolated EBNAl protein or polypeptide in the cell. In this process, the EBNAl protein coding sequence includes no more than 90%, preferably no more than 94%, of the Gly-Ala repeat amino acid sequence present in the naturally-occurring EBNAl protein coding sequence which spans the Gly-Ala repeat amino acid sequence. The isolated EBNAl protein or polypeptide formulation of the present invention can be utilized for detection of EBV in a sample of human tissue or body fluids. This detection process involves providing the isolated EBNAl protein or polypeptide formulation as an antigen, contacting the sample with the antigen, and detecting any reaction which indicates EBV is present in the sample using an assay system. More specifically, this technique permits detection of EBV in a sample of the following tissue or body fluids: blood, spinal fluid, sputum, pleural fluids, urine, bronchial alveolor lavage, lymph nodes, bone marrow, or other biopsied materials. In one embodiment, the assay system has a sandwich or competitive format. Examples of suitable assays include an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, or an immunoelectrophoresis assay. EXAMPLES Example 1 - Cells and Virus The wild-type baculovirus, Autographica californica nuclear polyhedrosis virus (AcMNPV) , and Spodoptera frugiperdi (Sf-9) cells used to propogate the baculoviruses, were kindly provided by Dr. Ora Rosen (Sloan- Kettering Cancer Center) , with permission from Dr. Max D. Summers (Texas A & M University) . Sf-9 cells were grown as monolayer cultures in Grace's medium (Gibco Laboratories) with 0.33% yeastolate and 0.33% lactalbumin hydrolysate (Difco) supplemented with 10% fetal bovine serum. Example 2 - Plasmids pVL941-SW (see Figure l)was constructed from pVL941 by Dr. Susan Wente in Dr. Ora Rosen's laboratory, by insertion of an Ncol / Xbal / Spel linker into the BamHI site of the polyhedrin gene in pVL941. As shown in plasmid p205, containing the EBNAl gene with a 700 bp (± 20 bp) deletion in the Gly-Ala repeat region, was kindly provided by Dr. Bill Sugden. Plasmid pGEMoriP7 was constructed by ligating Rsal/Hindlll DNA linkers to the ends of the i?sal fragment of p220.2 (kindly provided by Dr. Bill Sugden) containing oriP and the EBNAl gene and inserting this DNA fragment into the HindiII site of pGEMII (Promega Biotec, Madison, I) . pGEMcriP was constructed from pGEMoriP7 using AccI to excise 2 kilobase pairs of DNA containing the EBNAl gene followed by religation to give pGEMoriP, which contains the entire orii? sequence. Example 3 - Construction of the EBNAl Recombinant Baculovirus (AcMNPV-EBNAl) The EBNAl gene was excised from p205 using i?sal and Ball, which remove the first seven codons. The initiating methionine was regenerated upon ligation into the baculovirus transfer vector pVL941-S to yield pVL941/EBNAl (Fig. 1) . pVL941/EBNAl and AcMNPV DNA were cotransfected into Sf-9 insect cells by the calcium phosphate precipitation method as described by Summers et al. , Tex. Agric. Exp. Stn. Bull., 1555:27-31 (1987), which is hereby incorporated by reference. Five days post-transfection, serial dilutions of the medium from the transfected cells were plated with Sf-9 cells in 96-well plates. After amplification of the virus for 4 days, the cells were screened for the presence of virus containing the EBNAl gene by dot blot analysis. The medium from a positive well was then used in a plaque assay according to Summers et al. , Tex. Agric. EXP. Stn. Bull.. 1555:27-31 (1987) , which is hereby incorporated by reference, and a recombinant (non- occluded) plaque was picked, analyzed for the presence of the EBNAl gene by dot blots, and subjected to one more round of plaque purification. Virus from one of the resulting recombinant plaques was amplified in Sf-9 cells. Total DNA was prepared from these cells, digested with restriction enzymes, and analyzed by Southern blot hybridizations to verify the presence of the complete EBNAl Rsal -Ball fragment in the recombinant virus. Example 4 - DNA Oligonucleotide Affinity Column The oligonucleotide affinity column used in the procedure of Frappier, et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus," The Journal of Biological Chemistry, 266 (12) :7819-26 (1991) was very difficult to synthesize and to use. It had very low binding capacity and each prep, needed to be run over the column in several batches. The bEBNAl that eluted was thus quite dilute and needed to be concentrated using either a heparin column or a MonoQ column. The following is an account of how to synthesize this improved column. The oligonucleotide sequences were: OLIGOl 5'Biotin-GGGAAGCATATGCTACCC-3' (SEQ. I.D. No. 5) ; and OLIGO2 5' -GGGTAGCATATGCATATGCTTCCC-3' (SEQ. I.D. No. 6) . 350 nmole of oligol and 440 nmole of oligo2 were mixed in 20ml of 10 mM Tris-HCl (pH 7.2), 0.3 M NaCl, and 0.03 M sodium citrate (final pH 8.5) . The reaction was heated to 95°C for two minutes and allowed to cool to room temperature. The annealed oligonucleotide was incubated with 20 ml of a 1:1 slurry of strepavadin beads (Sigma Chemical Company) and rotated end over end for 12 hours at 4°C. The solution was then placed into a glass column, the beds were allowed to settle, followed by an extensive wash to remove unreacted oligonucleotide with 20 mM Hepes (pH 7.5), 0.5 mM EDTA, 10% glycerol, and 350 mM NaCl. This column had a capacity of approximately 0.7 mg of bEBNA-1 per ml of packed beads. Example 5 - Nitrocellulose Filter Binding Assays During purification, bEBNAl was followed and quantitated by its ability to specifically retain a 900-bp fragment of oriP containing 20 copies of the 30-bp repeated sequence (family of repeats fragment) onto nitrocellulose filters. The family of repeats fragment was excised from pGEMoriP with EcoRI and Ncol , purified by agarose gel electrophoresis followed by electroelution, and quantitated by measuring the absorbance at 260 nm. The oriP repeat fragment was end-labeled by filling in the 3 ' -recessed ends using the Klenow fragment of DΝA polymerase I with four dΝTPs and [α- 32 P]TTP. Assays for bEΝBAl were performed by incubating an aliquot (20-200 ng of protein) of each fraction with 10-100 fmol of the end-labeled family of repeats fragment for 10 min at 23°C, in 25μl of 50 mM HEPES (pH 7.5) , 5 mM MgCl 2 , and 300 mM NaCl containing 2.5-5 μg of calf thymus DNA. Reaction mixtures were then diluted with 900 μl of 50 mM HEPES (pH 7.5) , 5 mM MgCl 2 and immediately filtered through 0.45-μm HA filters (Millipore) . The filters were dried and counted by liquid scintillation. In nitrocellulose filter binding assays of bENBAl with the dyad symmetry element of oriP, a 300-bp fragment of oriP containing the dyad and its four associated EBNAl binding sites was incubated with bENBAl as described for the family of repeats. This dyad symmetry element fragment was excised from pGEMoriP with Hindlll and EcόKV , gel-purified, quantitated by absorbance at 260 nm, and end-labeled as described for the family of repeats fragment . In assays in which bENBAl was incubated with the complete oriP sequence, a 2-kilobase pair DNA fragment containing oriP was prepared from pGEMoriP and end-labeled near the dyad. This fragment was prepared by linearizing pGEMoriP with Hindlll, filling in the 3 ' -recessed ends with [α- 3 P]TTP using the Klenow fragment of DNA polymerase I, then digesting with BamHI . The Hindlll to BamHI fragment containing the complete oriP sequence was gel-purified and incubated with bEBNAl as described for the family of repeats fragment . Example 6 - Protein Determinations Protein concentration was determined by the method of Bradford, Anal. Biochem.. 72:248-254 (1976) , which is hereby incorporated by reference, using bovine serum albumin as a standard. The concentration of pure bEBNAl was determined by amino acid analysis (Sloan-Kettering Institute, Microchemistry Laboratory) . Example 7 - Phosphate and Methionine Labeling of bEBNAl Sf-9 cells (2.8 x 10 s cells, 10 x 150-cm 2 flasks) were infected with recombinant EBNAl baculovirus as described herein. Twenty-four hours post-infection, the media was replaced with phosphate-free or methionine-free Grace's media (Gibco) supplemented with 0.33% lactalbumin hydrolysate and 1 Ci of [ 32 P] orthophosphate or [ 35 S]methionine (Du Pont-New England Nuclear) . Cells were labeled for 18 h before nuclei were prepared. Labeled bEBNAl was purified as described herein. Example 8 - Phosphoamino Acid Analysis of bEBNAl Acid hydrolysis of bEBNAl and resolution of phosphoamino acids was performed according to the method of Cooper et al . , Methods Enzvmol .. 99:387-402 (1983) , which is hereby incorporated by reference. Four-microgram samples of pure [ 32 P]bEBNAl (4 μl) were added to 50 μl of 6 N constant boiling HCl (Pierce Chemical Co.) and heated to 110°C in a screw-cap 1.5-ml Eppendorf tube for 1, 2, or 4 h. The samples were lyophilized and resuspended in 2 μl of distilled water containing 4 μg each of phosphoserine, phosphothreonine, and phosphotyrosine markers. One microliter of each sample (2 μg of hydrolyzed bEBNAl) was spotted onto a strip of Whatman No. 3MM paper and subjected to electrophoresis in 0.5% pyridine, 5% acetic acid for 10 min at 2000 V. The paper was then dried and stained with ninhydrin to visualize the phosphoamino acid markers. 32 P- Labeled amino acids of bEBNAl were identified by autoradiography. Example 9 - Phosphatase Treatment Complete dephosphorylation of 32 P-labeled bEBNAl was achieved by treating 0.25 μg of [ 32 P] bEBNAl with 9 units of CIP (i.e., alkaline phosphatase from calf intestine) (Sigma) in 20 μl of lOmM Tris-HCl (pH 8.0) , 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 300 mM NaCl for 1 h at 25°C. Example 10 - Native Molecular Weight Determinations The sedimentation coefficient of bENBAl was measured by layering 40 μg of bEBNAl either alone or along with 60 μg of molecular weight standards (apoferritin, IgG, bovine serum albumin, avalbumin, and myoglobin) in 200 μl of 25 mM Tris-HCl (pH 7.5) , 300 mM NaCl, 0.5 mM EDTA, 10% glycerol onto 12-ml 10-30% glycerol gradients containing 25 mM Tris-HCl (pH 7.5) , 300 mM NaCl, 0.5 mM EDTA. Gradients were spun for 40 h at 270,000 x g at 5°C in a TH-641 rotor. After centrifugation, fractions of 160 μl were collected from the bottom of each tube. The Stokes radius of bENBAl was determined by injecting 40 μg of bEBNAl along with 60 μg of molecular weight standards in 200 μl of 25 mM Tris-HCl (pH 7.5) , 300 mM NaCl, 0.5 mM EDTA, 10% glycerol onto a 30-ml fast protein liquid chromatography Superose 12 gel filtration column. The column was developed in the same buffer. Fractions of 160 μl were collected. Two microliters of each fraction from the glycerol gradients and gel filtration columns were assayed for the presence of bEBNAl using the nitrocellulose filter binding assay described above. bENBAl and the molecular weight standards were visualized after SDS- polyacrylamide gel electrophoresis analysis by staining with Coomassie Blue. Example 11 - Stoichiometry of bEBNAl on oriP Thirty-five micrograms (7.7 pmol as plasmid circles) of pGEMoriP7 were incubated with excess 35 S-labeled bEBNAl (56 μg, 1.1 nmol as monomer) for 10 min at 37 °C in 200 μl of 20 mM HEPES (pH 7.5), 5 mM MgCl 2 , 300 mM NaCl, 40% glycerol. The reaction was gel-filtered over a 5 ml Bio-Gel A-5m column at 4 °C in the same buffer, and 140-μl fractions were collected. [ 35 S]bEBNAl in each fraction was quantitated by counting 30 μl in a scintillation counter. The molar quantity of DNA in each fraction was measured upon diluting 100 μl of column fraction with 400 μl of column buffer and measuring the absorbance at 260 nm (assuming 1 absorbance unit equals 50 μl/ml DNA) . Approximately 90% of the radioactivity and absorbance at 260 nm was recovered after gel filtration. Example 12 - Aval Endonuclease Protection Assay The 300-bp Hindlll to BcoRV fragment of pGEMoriP containing the dyad symmetry element was end-labeled using the Klenow fragment of DNA polymerase I and [α- 32 P]TTP to fill in the Hindlll end of the fragment. bEBNAl was incubated with 10 fmol of the 32 P-labeled dyad fragment in a 20-μl reaction containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM MgCl 2 for 10 min at room temperature. The reactions were then diluted to 50 mM NaCl and incubated with 30 units of Aval at 37 °C for 3 min. Digestions were stopped by the addition of SDS to 1%. Half of each reaction was then subjected to electrophoresis on a 6% polyacrylamide gel, which was dried prior to autoradiography. Example 13 - Expression of EBNAl in Baculovirus The EBNAl gene was excised from plasmid p205 and inserted into the pVL941-SW baculovirus transfer vector as described more fully above and as shown in Fig. 1. The resulting plasmid, pVL941-EBNAl, contained the EBNAl gene, which translates into a 50 kDa protein lacking six amino- terminal amino acids and approximately 232 contiguous Gly- Ala residues of the Gly-Ala repeat region. Of these 232 amino acid residues, 6 were downstream of the Gly-Ala repeat such that there are still 13 of the 239 Gly-Ala residues remaining, representing 5.44%. Neither of these regions was essential for EBNAl-dependent replication in vivo when tested separately. Recombination of pVL941 with AcMNPV wild-type baculovirus DNA resulted in a recombinant baculovirus (AcMNPV-EBNAl) containing the EBNAl gene controlled by the strong polyhedrin gene promoter. The EBNAl protein or polypeptide produced by Ac-MNPV-EBNAl is bENBAl. bEBNAl is not a fusion protein, as the ENBA1 gene was placed directly adjacent to the only ATG sequence present in the 5' region of the polyhedrin gene in pVL941-SW (Fig. 1) . Initially, Sf-9 monolayers were infected with AcMNPV-EBNAl and harvested at 24-h intervals to determine the time course of bEBNAl expression. bEBNAl protein levels peaked approximately 48 h post-infection as determined by the ability of whole cell extracts to specifically retain the oriP repeat fragment on a nitrocellulose filter. The level of oriP binding activity correlated with the appearance on Coomassie Blue-stained SDS-polyacrylamide gels of a 50 kDa protein that was not present in Sf-9 cells infected with wild-type baculovirus (data not shown) . Example 14 - Purification of bEBNAl from Insect Cells Sf-9 cells were seeded into 16 150-cm 2 culture flasks (3 x 10 7 cells/flask) (Corning) , allowed to attach, then infected with AcMNPV-EBNAl at a multiplicity of infection of three. The cells were harvested 46 h post- infection, washed in 250 ml of ice-cold phosphate-buffered saline, and resuspended on ice in 70 ml of hypotonic buffer (20 mM HEPES (pH 7.5), 1 mM MgCl 2 , 1 mM PMSf) using a Dounce homogenizer with pestle B. Nuclei were collected upon centrifugation at 1000 x g for 10 min at 5°C, washed in 70 ml of cold hypotonic buffer, and resuspended with the Dounce homogenizer and pestle B in 20 ml of 20 mM HEPES (pH 7.5) , 1 M NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM MgCl 2 , 1 mM PMSF, followed by incubation for 1 h on ice. This nuclear extract is sonicated for 2 minutes to shear the DNA and partially reduce the viscosity. A solution of 5% Polymin P * (Poiyethylenimine, average molecular weight 50,000, Sigma Chemical Co., St. Louis, Mo.) of molecular weight 5000 daltons was prepared in 20 mM Tris-HCl (pH 7.5) and then added to the nuclear extract to a final concentration of 0.25%, incubated for 30 minutes on ice and then spun for 30 minutes at 18,000 rpm in the SS-34 rotor (Sorvall) . This packs the DNA down tight into a small pellet thereby leaving most of the solution approximately 95% free of DNA. After removing the DNA, the supernatant is adjusted to 30%, saturation of ammonium sulfate (e.g., adding 8.6 volume of 100% saturated ammonium sulfate solution) to precipitate contaminant proteins but not bEBNAl. After slowly stirring for 1 hour at 4 °C, the preparation is spun at 15,000 rpm for 30 minutes at 4 °C. The supernatant is decanted and then ammonium sulfate is added to a final saturation of 45% (e.g., adding 7.8 ml of 100% saturated ammonium sulfate solution) in order to bring down the bEBNAl, yet leave other contaminants in solution. After slowly stirring for 1 hour at 4 °C, the preparation is spun for 30 minutes at 15,000 rpm at 4 °C. After discarding the supernatant, the bEBNAl protein-containing pellet is then dissolved in buffer A (20 mM Hepes (pH 7.5) , 0.5 mM EDTA, 2 mM DTT, 1 mM PMSF, 20% glycerol) and dialyzed against 2 liters of buffer A for 4 hours at 4 °C and then against another 2 liters of buffer A overnight before loading onto a 30-ml heparin-agarose column (Bio-Rad) . All column chromatography procedures to follow were at 4°C. The heparin-agarose column was washed with 60 ml cf buffer A containing 500 mM NaCl at 0.27 ml/min; then bEBNAl was eluted with buffer A containing 1 M NaCl (see FIG. 2) . Fractions of the 1 M NaCl eluate containing oriP binding activity were pooled (26 ml) , diluted to 350 mM NaCl with buffer A, then loaded onto 9 ml of the DNA oligonucleotide affinity column. The DNA affinity column was washed with 18 ml of 350 mM NaCl, and then bEBNAl was eluted using buffer A containing 2 M NaCl . If necessary, to concentrate bEBNAl, the 2 M NaCl eluate containing 33% of the oriP binding activity (50 ml) was dialyzed against 500 mM NaCl, diluted with buffer A to a conductivity equivalent to 260 mM NaCl (105 ml) , and loaded onto a 1-ml Mono Q column. bEBNAl was eluted with buffer A containing 500 mM NaCl. Aliquots of active fractions (20 μl/tube) were stored at -70°C. Alternatively, the bEBNAl can be concentrated by diluting the preparation with buffer A to a conductivity in the range of 250-300 mM NaCl and loaded onto a 1 ml Heparin Agarose column followed by elution using buffer A containing 1M NaCl. Fraction Protein Activity Specific Purification Yield Activity mg units units/mg -fold % Cytoplasm 129 Nuclear/Polyp 64 31,050 470 1 100 0-30% AS 23.9 30-45% AS 16.6 22,500 1355 2.9 73 Heparin 11. 18,100 1631 3.5 58 oligo-affinity 7.3 11,800 1616 3.4 38 This modified protocol gives about a 5-fold higher amount of the bEBNA-1 at the end of the procedure. The greater amount is probably due to recovery of more bEBNAl from the nucleus due to the elimination of DNA using Polyamine P instead of high speed centrifugation. In effect, one obtains much more solution phase due to tight compaction of the DNA by Polyamine P. The purity at the end is undoubtedly better than in the previous protocol due to the ammonium sulfate cut, but it cannot be detected by specific activity, because the difference is only between 95% and 98% (or greater) purity. However, for use of this product in an ELISA assay, one never knows when a very small level of impurity will invalidate the assay. Thus, the more pure - the better - even if it is a difference in going from 98 to 99 percent. Example 15 - Biochemical Assays of bEBNAl Homogeneous bEBNAl was assayed for the ability to hydrolyze ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and TTP in 1, 3, and 10 mM MgCl 2 , in the absence of DNA and in the presence of either oriP-containing duplex DNA or single- stranded DNA. Nucleotide hydrolysis assays were performed by incubating 200 ng of bEBNAl with 50 μM [α- 32 P] - or [γ- 32 P] nucleoside triphosphate and deoxynucleoside triphosphate in 10 μl of 20 mM Tris-HCl (pH 7.5) and 1, 3, or 10 mM MgCl 2 for 30 min at 37°C. Additional assays for nucleoside triphosphate and deoxynucleoside triphosphate hydrolysis were performed in the presence of 50 ng of bacteriophage M13 single-stranded DNA at the three MgCl 2 concentrations, as well as in the presence of 75 ng of pGEMoriP at the three MgCl 2 concentrations. ATPase activity was also tested in the presence of 2 and 8 mM sodium acetate. Samples (0.5 μl) of reaction mixtures were spotted on polyethyleneimine cellulose thin layer chromatography plates and developed in 0.8 M acetic acid, 0.8 M LiCl (when γ- 32 P-labeling was used) . Reaction products were identified by autoradiography. The τ subunit of Escherichia coli DNA polymerase III holoenzyme was used as a positive control for ATP hydrolysis according to the method of Tsuchihashi et al . , J. Biol . Chem.. 264:17790-17795 (1989) , which is hereby incorporated by reference. No hydrolysis of any nucleoside triphosphate by bEBNAl was detected (data not shown) . Although all known helicases are ATPases, bEBNAl was tested in the standard oligonucleotide displacement type of helicase assay according to Matson, J. Biol . Chem.. 261:10169-10175 (1986) , which is hereby incorporated by reference. bEBNAl was examined for an ability to displace, from single-stranded circular bacteriophage <>X174 DNA, a 32 P- end-labeled flush DNA 30-mer, a 5' -tailed DNA 30-mer, and a 3 '-tailed DNA 46-mer. In separate experiments three different synthetic DNA oligonucleotides were hybridized to bacteriophage X174 single-stranded DNA to give either 1) flush (30-mer), 2) 5' -tailed (30-mer with 20 nucleotides annealed), or 3) 3' -tailed (46-mer with 30 nucleotides annealed) helicase substrates. The annealed oligonucleotides were 3 ' -end-labeled using either [α- 32 P] dCTP and the Klenow fragment of DNA polymerase I (flush and 5'- tailed substrates) or using terminal transferase (3' -tailed substrate) . Each helicase substrate was then purified from unhybridized oligonucleotide by gel filtration on Bio-Gel A- 1.5m Helicase assays were performed by incubating 400 ng of bEBNAl with 9 fmol of DNA substrate in 30 mM HEPES (pH 7.5) , 4 mM ATP, 7 mM MgCl 2 , 1 mM dithiothrietol for 30 min at 37°C. Positive control reactions contained 400 ng of SV40 large T antigen. Reaction products were analyzed for oligonucleotide displacement on a 15% polyacrylamide gel. The SV40 large T antigen was used as a positive control according to the method of Goetz et al. , J. Biol . Chem. , 263:383-392 (1988) , which is hereby incorporated by reference. Although the SV40 T antigen displaced each of these DNA oligonucleotides, no helicase activity was detected for baculoEBNAl, consistent with its lack of ATPase activity. Also tested were bEBNAl for DNA polymerase, DNA ligase, endonuclease, exonuclease, and topoisomerase activities without positive results (not shown) . Note that in the following Examples 16-20 the bEBNAl protein was purified by the process disclosed in L. Frappier, et.al., "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Espstein-Barr Virus," J. Biol. Chem. 766 (12) :7819-26 (1991) rather than by the process of Example 14. Example 16 - Phosphoamino Acid Analysis of bEBNAl bEBNAl was labeled in vivo with [ 32 P]orthophosphate and purified to homogeneity. bEBNAl was the major 32 P- labeled protein in the nuclear extract and was not detected in the cytoplasm (Fig. 3) . Treatment of pure [ 32 P]bEBNAl with CIP resulted in loss of all detectable radioactive phosphate from bEBNAl (Fig. 3) . Since CIP has previously been shown to dephosphorylate serine residues only, Shaw et al . , Virology, 115:88-96 (1981) and Klausing et al, Virol.. 62:1258-1265 (1988), which are hereby incorporated by reference, bEBNAl is presumably phosphorylated only on serine. Further identification of phosphorylated residues in bEBNAl was performed by acid hydrolysis of [ 32 P]bEBNAl and separation of the phosphoamino acids by high voltage paper electrophoresis (Fig. 4) . Samples of [ 32 P] bEBNAl hydrolyzed for 1, 2, and 4 h were analyzed to ensure identification of any [ 32 P]phosphothreonine, which requires longer hydrolysis times, or [ 32 P]phosphotyrosine, which is less stable to acid hydrolysis according to the method of Cooper et al . , Methods Enzvmol .. 99:387-402 (1983) , which is hereby incorporated by reference. Upon electrophoresis, all the radioactive phosphate in bEBNAl migrated in the position of phosphoserine; no radioactivity was detected at positions of phosphothreonine or phosphotyrosine (Fig. 4) . After 4 h of acid hydrolysis, most of the radioactive phosphate was detected as free phosphate (data not shown) . Example 17 - Native Molecular Mass bEBNAl was analyzed by glycerol gradient sedimentation; an s value of 4.6 was obtained by comparison with protein markers with known s values (Fig. 3A) . A Stokes radius of 50 A for bENBAl was determined by gel filtration analysis and comparison with protein standards of known Stokes radius (Fig. 3B) . In both glycerol gradient and gel filtration analyses, oriP binding activity co-eluted with the bEBNAl protein visualized in SDS-polyacrylamide gel analysis of the column fractions (data not shown) . The s value and Stokes radius were combined in the equation of Siegel et al . , Biochim. Biophvs. Acta, 112:346-362 (1966) , which is hereby incorporated by reference, to calculate a native molecular mass of 94 kDa for bEBNAl. The amino acid sequence of EBNAl deduced from the DNA sequence of the EBNAl gene predicts a molecular mass of about 41,309 kDa for a bEBNAl monomer. Hence, the native molecular mass of bEBNAl indicates that bEBNAl is a dimer. Example 18 - Stoichiometry of bEBNAl Binding to oriP 35 S-Labeled bEBNAl protein was prepared in vivo by metabolic labeling using [ 35 S]methionine followed by purification to homogeneity. The [ 35 S] bEBNAl was used to measure the number of bEBNAl molecules bound to oriP under conditions of saturating bEBNAl. A plasmid containing the complete oriP sequence was incubated with increasing amounts of [ 35 S] bEBNAl then gel-filtered to separate [ 35 S] bEBNAl bound to DNA in the excluded fractions from the unbound [ 35 S] bEBNAl in the included fractions. Upon saturation of oriP with bEBNAl, indicated by the appearance of bEBNAl monomers per oriP DNA which comigrated in the excluded fractions was 56 to 1 (Fig. 6) . Since there are 24 EBNAl binding sites in oriP, the stoichiometry of 2.3 bEBNAl monomers per EBNAl binding site indicates that bEBNAl bound its site as a dimer, consistent with the native molecular weight of bEBNAl and the palindromic structure of the consensus EBNAl binding site. Example 19 - Effect of Salt on Binding of bEBNAl to the Family of Repeats and Dyad Symmetry Element The effect of NaCl concentration on bEBNAl binding to the family of repeats or the dyad symmetry element of oriP was studied using the nitrocellulose filter binding assay. bEBNAl (50 ng) was incubated with 40 fmol of 32 P- labeled dyad fragment or 32 P-labeled repeat fragment in various concentrations of NaCl and in the presence of excess (2.5 μg) calf thymus DNA (Fig. 7) . The binding profile indicates that the specific interaction of bEBNAl with the dyad symmetry element was maximum at 250-300 mM NaCl and dropped off sharply at higher NaCl concentrations. Binding of bEBNAl to the family of repeats, however, remained stable up to 500 mM NaCl. Hence, the relative binding strength of bEBNAl for the family of repeats versus the dyad symmetry element depended on the salt concentration. The apparent requirement of high salt for binding bEBNAl to labeled DNA in these experiments may be attributed to efficient competition by nonspecific calf thy us DNA at low NaCl concentration. Example 20 - bEBNAl Binding to the Dyad Symmetry Element The interaction of bEBNAl with the family of repeats and dyad symmetry element of oriP was also assessed by examining the amount of bEBNAl required to retain each element on nitrocellulose filters. Increasing amounts of bEBNAl were incubated with 10 fmol of 32 P-end-labeled repeat or dyad DNA fragment in 20 μl of buffer containing 300 mM NaCl and no calf thymus DNA. Retention of the dyad symmetry element onto nitrocellulose appeared to have a threshold where significant retention was not observed below 20 bEBNAl dimers per dyad fragment (200 ng in Fig. 8, closed circles) , but full retention was achieved at 50 bEBNAl dimers per dyad (500 ng in Fig. 8) . It would seem from this behavior that bEBNAl must reach a critical concentration before it binds the dyad symmetry element. The apparent K d for bEBNAl binding to the dyad symmetry element calculated from these data is 2 nM (assuming four bEBNAl dimers were bound per dyad symmetry element) . The family of repeats was retained onto nitrocellulose at lower levels of bEBNAl than required for binding the dyad symmetry element (Fig. 8, open circles) . An apparent K d for bEBNAl binding to the family of repeats was calculated to be 0.2 nM (assuming four bEBNAl dimers were bound per family of repeats) . The binding of bEBNAl to the dyad symmetry element was further examined by an Aval endonuclease protection assay. An Aval site was present at the junction of two of the four EBNAl binding sites in the dyad symmetry element (Fig. 9) . Increasing amounts of bEBNAl were incubated with 10 fmol of the dyad symmetry element, end-labeled with 32 P at one end only. The reaction was then treated with sufficient Aval to completely digest the DNA within 3 min at 37°C. Digestions were stopped with SDS and subjected to polyacrylamide gel electrophoresis to separate DNA fragments cut by Aval from uncut (Aval-protected) DNA (Fig. 9) . As in the nitrocellulose binding assay, the Aval protection analysis showed that a 20-fold molar excess of bEBNAl dimers (200 ng in Fig. 9) was required over the dyad fragment to detect protection of the Aval site, followed by a very sharp increase in protection against Aval at levels above 20 bEBNAl dimers per dyad symmetry element. The small difference between the Aval protection assay (Fig. 9) and the nitrocellulose filter binding assay (Fig. 8) showed approximately 1.5 times more bEBNAl was needed to bind the dyad symmetry element onto a nitrocellulose filter relative to the amount of bEBNAl needed to protect the Aval site. This may be due to the requirement for bEBNAl to bind to only one particular site in the dyad symmetry element to protect it from Aval, whereas retention of the dyad onto nitrocellulose may require bEBNAl bound to another site or multiple bEBNAl molecules bound to multiple sites in the dyad symmetry element . In vivo the dyad symmetry element is accompanied by the family of repeats within oriP which may affect the interaction of EBNAl with the dyad symmetry element in the complete oriP sequence. bEBNAl was incubated with oriP labeled with 32 P at the end near the dyad. Just prior to filtration through nitrocellulose, the family of repeats was separated from the dyad symmetry element by digestion with EcoRV (Fig. 8) for each assay an aliquot was removed prior to filtration, quenched with SDS (i.e., sodium dodecyl sulfate) , and analyzed in an agarose gel to confirm that BcoRV had completely separated the dyad from the oriP DNA. The results showed significant amounts of dyad symmetry element were retained onto nitrocellulose at lower levels of bEBNAl (200 fmol and less) in the presence of the family of repeats than in their absence (Fig. 8, closed triangles) . However, further along in the titration, more bEBNAl was required to bind the dyad on oriP than to bind the dyad alone. Complete retention onto nitrocellulose of the isolated family of repeats and dyad symmetry fragments required 300 and 500 fmol of bEBNAl, respectively. Hence, it seems a paradox that even 800 fmol of bEBNAl was not sufficient to retain onto nitrocellulose more than half of the dyad fragment when it was within the context of oriP. Possible explanations include the following. The presence of the family of repeats may destabilize the interaction of bEBNAl with the dyad. A less stable complex of bEBNAl with the dyad may assemble in the presence of the family of repeats. The nonessential region of oriP between the family of repeats and dyad symmetry element may influence the nitrocellulose binding assay, or the presence of the dyad may cause more cooperative binding of bEBNAl to the family of repeats, effectively decreasing the availability of bEBNAl for binding the dyad. The above examples describe the overproduction of EBNAl, the viral encoded protein which binds the latent phase origin ( oriP) of EBV, in the baculovirus system and its purification of homogeneity. Like EBNAl from latently infected B cell lines (see Jones et al . , J. Virol . , 63:101- 110 (1989) ; Hearing et al . , Virology. 145:105-116 (1985) ; and Gahn et al . , Cell, 58:527-535 (1989) , which are hereby incorporated by reference) the bEBNAl bound tightly to oriP, arrested replication forks within or near the oriP family of repeats and was phosphorylated on serine residues. Since phosphorylation can modulate protein function (see Donaldson et al., Proc. Natl. Acad. Sci. U.S.A.. 84:759-763 (1987) ; Gould et al . , Nature, 342:39-45 (1989) ; and McVey et al . , Nature. 341:503-507 (1989), which are hereby incoporated by reference) , it seems likely that initiation of replication from oriP will be regulated by phosphorylation of EBNAl. The palindromic nature of each EBNAl consensus site suggests that EBNAl binds its DNA site as a dimer. Indeed bEBNAl appeared to be a dimer in solution and the stoichiometry of 56 bEBNAl molecules per 24 EBNAl binding sites in the oriP sequence was consistent with EBNAl binding its site as a dimer as predicted. See Ambinder et al . , J^ Virol . , 64:2369-2379 (1990) , which is hereby incorporated by reference. Increasing evidence suggests replication initiates within the dyad symmetry element of oriP. See Gahn et al . , Cell, 58:527-535 (1989) and Wysokinski et al. , J. Virol.. 63:2657-2666 (1989), which are hereby incorporated by reference. Replication initiation in the dyad is greatly stimulated by the family of repeats. I_d. One mechanism by which the repeats might activate the dyad is by altering the interaction of EBNAl with the dyad symmetry element. The nitrocellulose filter binding assay suggested that the family of repeats reduced the concentration of bEBNAl required to initiate binding to the dyad of bEBNAl required to initiate binding to the dyad symmetry element. If the interaction of EBNAl with the dyad symmetry element is important for the initiation of replication from oriP, then the stimulation of dyad binding by the family of repeats at low EBNAl concentration may be one mechanism by which the repeats enhance replication from oriP. EBNAl is essential for latent EBV replication, yet the precise biochemcal function of EBNAl remains elusive. The bEBNAl protein should prove useful in biochemical assays to analyze the mechanism by which EBNAl activates oriP to function as an origin of replication, a plasmid maintenance element, and a transcriptional enhancer. See Yates et al . , Cancer Cells. 6:197-205 (1988) , which is hereby incorporated by reference. Applicant finds no ATPase (or other nucleoside triphosphatase) , helicase, ligase, topoisomerase, DNA polymerase, oxonuclease, or endonuclease activities associated with bEBNAl. The absence of ATPase and helicase activity suggests EBNAl plays a different role in replication than the large T antigen of SV40. It is always possible, however, that the true activity of EBNAl will only be revealed upon binding other proteins or by modification at a specific site(s) . Furthermore, the possibility cannot be excluded that, although the six amino-terminal amino acids and glycine-alanine repeat region of EBNAl, lacking in bEBNAl, are nonessential for EBNAl function in vivo, id.. they may affect the biochemical activity of EBNAl in vi tro . Elucidation of the precise role of EBNAl in replication and the mechanism(s) of replication control at oriP would be greatly facilitated by development of an in vi tro system capable of initiating replication from oriP. Example 21 - Determination of Nucleotide and Amino Acid Sequences The DNA template used for the sequence analysis of the GlyAla deletion was the 10.6 kb bEBNAl baculovirus transfer vector, called pVL941-EBNAl, the construction of which was described in L. Frappier, et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus, " J. Biol. Chem. 766(12) :7819-26 (1991) . The sequencing primer used in this analysis was positioned 187 nucleotides in from the A of the ATG start codon of EBNAl; the sequence of the sequencing primer was 5'AAAAACGTCCAAGTTGCATTG-3 ' (SEQ. ID. No. 7) . Sequencing was performed using the Sequenase based protocol and version 2 kit of United States Biochemical, Cleveland, Ohio according to the manufacturers specifications. Example 22 - Expression and Purification of eEBNAl The gene and expression plasmid were constructed by PCR using the following primers: N - terminus - 5' - GAT CGG CAT ATG GGA GAA GGC CCA AGC ACT GGA - 3' (the underline is the Met for the first amino acid, and the GGA that follows encodes amino acid 442 of EBNAl) (SEQ. ID. No. 8) ; and C - terminus - 5' - CT GGT GGA TCC TTA ACC AAC AGA AGC ACG ACG CAG CTC CTG CCC TTC CTC AC - 3' (the underlined codcn encodes the last amino acid of the eEBNAl) (SEQ. ID. No. 9) . The template used in the PCR reaction was p291 (FIG. 11) , a plasmid containing the entire EBNAl gene (see FIGS. 12A-C) . The cycling conditions were 94 °C, 30 sec./ 60 °C, 30 sec./ 72 °C, 60 sec. This cycle is repeated 30 times in 100 μliters of 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl : , 200 μ olar each dATP, dCTP, dGTP, dTTP, 0.01% gelatin, 2.5 units TagI polymerase (Perkin-Elmer Cetus) , 1 μmolar of each primer (described above) , and 1 ng of plasmid p291. After the PCR reaction, the 641 bp fragment was purified by phenol extraction in 2% SDS followed by sequential digestion with 10 units of Ndel (New England Biolabs) and then 10 unir.3 of BamHI (New England Biolabs) . The Ndel/BamHI 624 bp fragment (see SEQ. ID. NO. 3) was purified from an agarose gel and ligated into pET3c (digested with Ndel and BamHI) to yield pET-eEBNAl, as shown in Figure 10. Sequence analysis confirmed that no errors had been introduced by PCR amplification. To express eEBNAl, the pET-eEBNAl plasmid was transformed into E. coli strain BL21 (DE3)pLysS and the cells were grown at 37 °C in 4 liters of LB medium (per liter: lOg Bac o-tryptone, 5g Bacto-yeast, lOg NaCl, pH 7.5) supplemented with 1% glucose, 10 μg/ml thiamine, 50 μg/ml thy .ine, 100 μg/ml ampicillin, and 30 μg/ml chloramphenicol . Upor. reaching an absorbance at 600 nm of 0.8, IPTG was added 46 to 0.4 mM, and after 2 hours at 37 °C, the cells were harvested by centrifugation (15g net weight) . The cells were frozen at -70 °C and then thawed to 4 °C, and then resuspended in 40 ml of 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 50 mM glucose. At this point, the cells lyse due to the lysozyme produced by the pLysS plasmid and the freeze-thaw procedure. The volume was brought to 100 ml using solution I and the DNA removed by precipitation by adding 10 ml NaCl, 1.4 ml of 5% Polymin P * (50 kDa) dissolved in 20 mM Tris-HCl (pH 7.5) . After stirring slowly for 30 minutes at 4 °C, the precipitation was spun at 18,000 rpm at 4 °C. The supernatant (82 ml) was adjusted to 70% ammonium sulfate by adding 191 ml of 100% saturated ammonium sulfate to precipitate the eEBNAl protein. The eEBNAl- containing precipitate was then pelleted by centrifugation for 30 minutes at 1,000 rpm in the GSA rotor at 4 °C. The pellet was dissolved in 40 ml of buffer B (20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride (i.e., PMSF) ) and loaded onto a 330 ml column of Bio-Gel P-6 equilibrated in buffer B. Fractions of 8 ml were collected at a flow rate of 3 ml/minute and assayed for total protein by the Bradford reagent (Bio-Rad) . Peak fractions (11-29) are pooled (700 mg protein) . The 700 mg protein pool was loaded onto a 320 ml column of Heparin-Agarose (Bio-Rad) equilibrated in buffer B. The column was eluted with a 3.2 liter linear gradient of buffer B from 0 mM NaCl to 800 mM NaCl. Fractions of 26 ml were collected and assayed for total protein and for eEBNAl. The eEBNAl eluted in fractions 60-96 and these were pooled (39 mg) and precipitated by adding 434g solid ammonium sulfate (70% saturation) . The protein precipitate was collected by centrifugation, resuspended in 20 ml buffer B, and dialyzed against 2 liters of buffer B for 4 hours and then against another 2 liters of buffer B overnight. The dialysate was loaded onto a 40 ml column of Q Sepharose (Pharmacia) equilibrated in buffer B. The eEBNAl was eluted with a linear gradient of 400 ml of 0 mM NaCl to 800 ml NaCl in buffer B. Fractions of 5 ml were collected at a flow rate of 1 ml/minute and the fractions were assayed for eEBNAl. Fractions containing eEBNAl were pooled (fractions 34-44, 20 mg total) . This eEBNAl-containing pool had a conductivity equal to 386 mM NaCl and was diluted with buffer B to a conductivity equal to 48 mM NaCl, then loaded onto a 4 ml column of CM Sepharose (Pharmacia) equilibrated in buffer B. The eEBNAl was eluted using a 40 ml linear gradient of 0 mM NaCl to 700 mM NaCl in buffer B and the fractions containing eEBNAl were pooled (fractions 24-34, 18 mg total) and dialyzed against buffer B and stored frozen at -70 °C. Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Cornell Research Foundation, Inc. (ii) TITLE OF INVENTION: Epstein-Barr Virus Nuclear Antigen 1 Protein and Its Expression and Recovery (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Nixon, Hargrave, Devans & Doyle (B) STREET: Clinton Square, P.O. Box 1051 (C) CITY: Rochester (D) STATE: New York (E) COUNTRY: USA (F) ZIP: 14603 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Goldman Esq., Michael L. (B) REGISTRATION NUMBER: 30,727 (C) REFERENCE/DOCKET NUMBER: 19603/271 (D-1530) (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (716) 263-1304 (B) TELEFAX: (716) 263-1600 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1212 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGACAGGAC CTGGAAATGG CCTAGGAGAG AAGGGAGACA CATCTGGACC AGAAGGCTCC 60 GGCGGCAGTG GACCTCAAAG AAGAGGGGGT GATAACCATG GACGAGGACG GGGAAGAGGA 120 CGAGGACGAG GAGGCGGAAG ACCAGGAGCC CCGGGCGGCT CAGGATCAGG GCCAAGACAT 180 AGAGATGGTG TCCGGAGACC CCAAAAACGT CCAAGTTGCA TTGGCTGCAA AGGGACCCAC 240 GGTGGAACAG GAGCAGGAGC AGGAGCGGGA GGGGCAGGAG CAGGAGGTGG AGGCCGGGGT 300 CGAGGAGGTA GTGGAGGCCG GGGTCGAGGA GGTAGTGGAG GCCGCCGGGG TAGAGGACGT 360 GAAAGAGCCA GGGGGGGAAG TCGTGAAAGA GCCAGGGGGA GAGGTCGTGG ACGTGGAGAA 420 AAGAGGCCCA GGAGTCCCAG TAGTCAGTCA TCATCATCCG GGTCTCCACC GCGCAGGCCC 480 CCTCCAGGTA GAAGGCCATT TTTCCACCCT GTAGGGGAAG CCGATTATTT TGAATACCAC 540 CAAGAAGGTG GCCCAGATGG TGAGCCTGAC GTGCCCCCGG GAGCGATAGA GCAGGGCCCC 600 GCAGATCACC CAGGAGAAGG CCCAAGCACT GGACCCCGGG GTCAGGGTGA TGGAGGCAGG 660 CGCAAAAAAG GAGGGTGGTT TGGAAAGCAT CGTGGTCAAG GAGGTTCCAA CCCGAAATTT 720 GAGAACATTG CAGAAGGTTT AAGAGCTCTC CTGGCTAGGA GTCACGTAGA AAGGACTACC 780 GACGAAGGAA CTTGGGTCGC CGGTGTGTTC GTATATGGAG GTAGTAAGAC CTCCCTTTAC 840 AACCTAAGGC GAGGAACTGC CCTTGCTATT CCACAATGTC GTCTTACACC ATTGAGTCGT 900 CTCCCCTTTG GAATGGCCCC TGGACCCGGC CCACAACCTG GCCCGCTAAG GGAGTCCATT 960 GTCTGTTATT TCATGGTCTT TTTACAAACT CATATATTTG CTGAGGTTTT GAAGGATGCG 1020 ATTAAGGACC TTGTTATGAC AAAGCCCGCT CCTACCTGCA ATATCAGGGT GACTGTGTGC 1080 AGCTTTGACG ATGGAGTAGA TTTGCCTCCC TGGTTTCCAC CTATGGTGGA AGGGGCTGCC 1140 GCGGAGGGTG ATGACGGAGA TGACGGAGAT GAAGGAGGTG ATGGAGATGA GGGTGAGGAA 1200 GGGCAGGAGT GA 1212 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 404 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 Met Thr Gly Pro Gly Asn Gly Leu Gly Glu Lys Gly Asp Thr Ser Gly 1 5 10 15 Pro Glu Gly Ser Gly Gly Ser Gly Pro Gin Arg Arg Gly Gly Asp Asn 20 25 30 Kis Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly Arg Pro 35 40 45 Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro Arg His Arg Asp Gly Val 50 55 60 Arg Arg Pro Gin Lys Arg Pro Ser Cys lie Gly Cys Lys Gly Thr His 65 70 75 80 Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly Gly Ala Xaa Ala Gly Gly 85 90 95 .ly Gly Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser 100 105 110 Gly Gly Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg 115 120 125 Glu Arg Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg 130 135 140 Ser Pro Ser Ser Gin Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro 145 150 155 160 Pro Pro Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr 165 170 175 ?he Glu Tyr His Gin Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro 180 185 190 Pro Gly Ala lie Glu Gin Gly Pro Ala Asp His Pro Gly Glu Gly Pro 195 200 205 Ser Thr Gly Pro Arg Gly Gin Gly Asp Gly Gly Arg Arg Lys Lys Gly 210 215 220 Gly Trp Phe Gly Lys His Arg Gly Gin Gly Gly Ser Asn Pro Lys Phe 225 230 235 240 Glu Asn lie Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val 245 250 255 Glu Arg Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr 260 265 270 Gly Gly Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu 275 280 285 Ala lie Pro Gin Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly 290 295 300 Met Ala Pro Gly Pro Gly Pro Gin Pro Gly Pro Leu Arg Glu Ser lie 305 310 315 320 Val Cys Tyr Phe Met Val Phe Leu Gin Thr His lie Phe Ala Glu Val 325 330 335 Leu Lys Asp Ala He Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr 340 345 350 Cys Asn He Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu 355 360 365 Pro Pro Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp 370 375 380 Asp Gly Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu 385 390 395 400 Gly Gin Glu Xaa ( 2 ) INFORMATION FOR SEQ ID NO : 3 ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 624 base pairs ( B ) TYPE : nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown Iii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : ATGGGAGAAG GCCCAAGCAC TGGACCCCGG GGTCAGGGTG ATGGAGGCAG GCGCAAAAAA 60 GGAGGGTGGT TTGGAAAGCA TCGTGGTCAA GGAGGTTCCA ACCCGAAATT TGAGAACATT 120 GCAGAAGGTT TAAGAGCTCT CCTGGCTAGG AGTCACGTAG AAAGGACTAC CGACGAAGGA 180 ACTTGGGTCG CCGGTGTGTT CGTATATGGA GGTAGTAAGA CCTCCCTTTA CAACCTAAGG 240 CGAGGAACTG CCCTTGCTAT TCCACAATGT CGTCTTACAC CATTGAGTCG TCTCCCCTTT 300 GGAATGGCCC CTGGACCCGG CCCACAACCT GGCCCGCTAA GGGAGTCCAT TGTCTGTTAT 360 TTCATGGTCT TTTTACAAAC TCATATATTT GCTGAGGTTT TGAAGGATGC GATTAAGGAC 420 CTTGTTATGA CAAAGCCCGC TCCTACCTGC AATATCAGGG TGACTGTGTG CAGCTTTGAC 480 GATGGAGTAG ATTTGCCTCC CTGGTTTCCA CCTATGGTGG AAGGGGCTGC CGCGGAGGGT 540 GATGACGGAG ATGACGGAGA TGAAGGAGGT GATGGAGATG AGGGTGAGGA AGGGCAGGAG 600 CTGCGTCGTG CTTCTGTTGG TTAA 624 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 208 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : Met Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gin Gly Asp Gly Gly 1 5 10 15 Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gin Gly Gly 20 25 30 Ser Asn Pro Lys Phe Glu Asn He Ala Glu Gly Leu Arg Ala Leu Leu 35 40 45 Ala Arg Ser His Val Glu Arg Thr Thr Asp Glu Gly Thr Trp Val Ala 50 55 60 Gly Val Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu Tyr Asn Leu Arg 65 70 75 80 Arg Gly Thr Ala Leu Ala He Pro Gin Cys Arg Leu Thr Pro Leu Ser 85 90 95 Arg Leu Pro Phe Gly Met Ala Pro Gly Pro Gly Pro Gin Pro Gly Pro 100 105 110 Leu Arg Glu Ser He Val Cys Tyr Phe Met Val Phe Leu Gin Thr His 115 120 125 He Phe Ala Glu Val Leu Lys Asp Ala He Lys Asp Leu Val Met Thr 130 135 140 Lys Pro Ala Pro Thr Cys Asn He Arg Val Thr Val Cys Ser Phe Asp 145 150 155 160 Asp Gly Val Asp Leu Pro Pro Trp Phe Pro Pro Met Val Glu Gly Ala 165 170 175 Ala Ala Glu Gly Asp Asp Gly Asp Asp Gly Asp Glu Gly Gly Asp Gly 180 185 190 Asp Glu Gly Glu Glu Gly Gin Glu Leu Arg Arg Ala Ser Val Gly Xaa 195 200 205 INFORMATION FOR SEQ ID NO : 5 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 18 base pairs (B ) TYPE : nucleic acid ( C) STRANDEDNESS : single (D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : DNA (genomic ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 GGGAAGCATA TGCTACCC 18 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 GGGTAGCATA TGCATATGCT TCCC 24 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 AAAAACGTCC AAGTTGCATT G 21 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : GATCGGCATA TGGGAGAAGG CCCAAGCACT GGA 33 (2) INFORMATION FOR SEQ ID NO:9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : CTGGTGGATC CTTAACCAAC AGAAGCACGA CGCAGCTCCT GCCCTTCCTC AC 52

Claims: WHAT IS CLAIMED IS: 1. A process for recovering isolated EBNAl protein or polypeptide comprising: providing cells having a nucleus containing expressed EBNAl protein or polypeptide; recovering the nucleus containing expressed EBNAl protein or polypeptide from the cells; separating the nucleus containing expressed EBNAl protein or polypeptide into a liquid fraction containing the expressed EBNAl protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus; separating the liquid fraction from the solid fraction; and recovering EBNAl protein or polypeptide from the liquid fraction. 2. A process according to Claim 1, wherein said separating the nucleus is by centrifugation where the liquid fraction is a supernatant and the solid fraction is a pellet . 3. A process according to Claim 1, wherein said recovering EBNAl protein or polypeptide from the liquid fraction comprises : subjecting the liquid fraction to a first ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing contaminant proteins and a liquid phase containing EBNAl protein or polypeptide; subjecting the liquid phase containing EBNAl protein or polypeptide to a second ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing EBNAl protein or polypeptide and a liquid phase containing contaminant proteins; and separating the solid phase containing EBNAl protein or polypeptide and the liquid phase containing contaminant proteins. 4. A process according to Claim 3 , wherein the first ammonium sulfate treatment is at a >0 to 30% ammonium sulfate concentration. 5. A process according to Claim 3, wherein the second ammonium sulfate treatment is at a 30 to 45% ammonium sulfate concentration. 6. A process according to Claim 3, wherein said recovering EBNAl protein or polypeptide further comprises: purifying the solid phase containing EBNAl protein or polypeptide, after said separating, by affinity column chromatography. 7. A process according to Claim 6, wherein the affinity column chromatography is agarose-heparin affinity column chromatography. 8. A process according to Claim 6, wherein the affinity column chromatography is oligonucleotide affinity column chromatography. 9. A process according to Claim 1, wherein said cells are insect cells. 10. A process according to Claim 9, wherein said insect cells are Sf-9 insect cells. 11. A process according to Claim 10, wherein said EBNAl protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 2. 12. A process according to Claim 10, wherein said EBNAl protein or polypeptide is encoded by a nucleotide sequence corresponding to SEQ. ID. No. 1. 13. A process according to Claim 2, wherein the supernatant contains less than 5% of DNA. 14. A process according to Claim 1, wherein said EBNAl protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No 2. 15. A process according to Claim 1, wherein said EBNAl protein or polypeptide is encoded by a nucleotide sequence corresponding to SEQ. ID. No. 1. 16. An isolated EBNAl protein or polypeptide produced by the process of Claim 1. 17. An isolated EBNAl protein or polypeptide produced by the process of Claim 2. 18. An isolated EBNAl protein or polypeptide produced by the process of Claim 3. 19. An isolated EBNAl protein or polypeptide formulation having substantially no components which generate false positive readings when used to detect Epstein-Barr virus in human serum. 20. An isolated EBNAl protein or polypeptide according to Claim 19, wherein naturally-occurring EBNAl protein or polypeptide spans a Gly-Ala repeat amino acid sequence and said isolated EBNAl protein or polypeptide includes no more than 90% of the Gly-Ala repeat amino acid sequence. 21. An isolated EBNAl protein or polypeptide according to Claim 19, wherein the EBNAl protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 2. 22. An isolated EBNAl protein or polypeptide according to Claim 19, wherein the EBNAl protein or polypeptide is encoded by a nucleotide sequence corresponding to SEQ. ID. No. 1. 23. An isolated EBNAl protein or polypeptide according to Claim 19, wherein the EBNAl protein or polypeptide is recombinant. 24. An isolated EBNAl protein or polypeptide according to Claim 19, wherein the EBNAl protein or polypeptide is purified. 25. An isolated DNA molecule encoding EBNAl protein or polypeptide containing substantially no components which generate false positive readings when used to detect Epstein-Barr virus in human serum. 26. An isolated DNA molecule according to Claim 25, wherein said DNA molecule is isolated from any other DNA molecule which expresses protein that generates false positive readings when the EBNAl protein or polypeptide is used to detect Epstein-Barr virus in human serum. 27. An isolated DNA molecule according to Claim 25, wherein said DNA molecule encodes a protein having an amino acid sequence corresponding to SEQ. ID. No.2. 28. An isolated DNA molecule according to Claim 25, wherein said DNA molecule contains a nucleotide sequence corresponding to SEQ. ID. No. 1. 29. A recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA molecule encoding EBNAl protein or polypeptide containing substantially no components which generate false positive readings when used to detect Epstein-Barr virus in human serum. 30. A recombinant DNA expression system according to Claim 29, wherein said DNA molecule encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 2. 31. A host cell incorporating a heterologous DNA molecule encoding EBNAl protein or polypeptide containing substantially no components which generate false positive readings when used to detect Epstein-Barr virus in human serum. 32. A host cell according to Claim 31, wherein said DNA molecule encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 2. 33. A host cell according to Claim 31, wherein said host cell is an insect cell. 34. A host cell according to Claim 33, wherein said insect cell is an Sf-9 insect cell. 35. A method for detection of Epstein-Barr virus in a sample of human tissue or body fluids comprising: providing an isolated EBNAl protein or polypeptide formulation according to Claim 20 as an antigen; contacting the sample with the antigen; and detecting any reaction which indicates that Epstein-Barr virus is present in the sample using an assay system. 36. A method according to Claim 35, wherein said assay system is selected from the group consisting of an enzyme-linked immunosorbant assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay. 37. A method according to Claim 36, wherein said isolated EBNAl protein or polypeptide is produced according to Claim 1. 38. A method of expressing an EBNAl protein coding sequence in a cell, wherein said method comprises the steps of : cloning an EBNAl protein coding sequence into a baculovirus transfer vector; co-transfecting insect cells with said baculovirus transfer vector and Autographica californica nuclear polyhedrosis genomic DNA; recovering recombinant baculoviruses; and infecting cells with said recombinant baculovirus under conditions facilitating expression of isolated EBNAl protein or polypeptide in the cell, wherein naturally-occurring EBNAl protein coding sequence spans a Gly-Ala repeat amino acid sequence and said EBNAl protein coding sequence includes no less than 90% of the Gly-Ala repeat amino acid sequence. 39. A method according to Claim 38, wherein said insect cells are Sf-9 insect cells. 40. A method according to Claim 38, wherein said EBNAl protein coding sequence has an amino acid sequence corresponding to SEQ. ID. No. 2.

Inventors: O'DONNELL MICHAEL E, O'DONNELL, MICHAEL, E.
Applicants: CORNELL RES FOUNDATION INC, CORNELL RESEARCH FOUNDATION, INC.
Application year: 1995
Main classification: C07K1
IPC codes: C07K14