METHODS AND COMPOSITIONS FOR IDENTIFYING INHIBITORS OF PAPILLOMA VIRUS REPLICATION

Abstract: Compositions and methods for identifying inhibitors of papilloma virus replication are described consisting of soluble cellular extracts supplemented with purified viral E1 and E2 proteins.

Description: METHODS AND COMPOSITIONS FOR IDENTIFYING INHIBITORS OF PAPILLOMA VIRUS REPLICATION Description This invention is in the field of molecular biology with emphasis on the identification of medicaments that can be used to treat papilloma virus diseases, particularly warts and cancers. It has been known for some time that papilloma viruses are responsible for inducing diseases in many higher vertebrates, including humans. Papilloma viruses are small DNA viruses, nonenveloped, that replicate in the nucleus of squamous epithelial cells. They are spread widely throughout nature and are causative of epithelial proliferative lesions particularly, benign fibropapillomas, or as they are more commonly known warts. Papilloma viruses are also implicated in a number of cancers. To date there have been identified about 58 distinct human papilloma viruses, based on the extent and degree of relatedness of their genomes. The clinical importance of warts varies considerably and determinative factors are the infecting viral type, the location of the wart, and factors unique to the host. For example, a wart located on the skin is often clinically insignificant, being self limiting. However, warts on the vocal cords may be life threatening as a result of respiratory obstruction. The vast majority of skin warts spontaneously regress within a few years after their initial appearance, but may persist for longer times. The exception is a rare life threatening papilloma virus disease termed epidermodyspasia verruciformis. In this disease, the infected individual does not experience spontaneous regression, but rather the infection may progress to a malignant stage. Orth, G. epitermodyspasia verruciformis, in: Salzman, N.P. Howley, P.M. Eds. the papovaviridae, vol. 2, N.Y.: Plenum Press 1987:199-243. The disease is present world-wide, but is rare and is often found among family members. Thus, genetic factors are thought to be involved in the etiology of the disease. Papilloma viruses are also involved in producing sexually transmitted warts of the genital tract. There is reported to be well over a million cases in the United States alone. Beckter, T.M, Stone, K.M, Alexander, E.R., Genital Human Papilloma virus Infection: A Growing Concern Obstet Gynecol Clin North am 1987: 14:389-396. As mentioned above, papilloma virus is thought to be responsible for several different types of cancer, including cervical cancer, of which there are about 500,000 new cases diagnosed yearly. Pto, R., Introduction: Geographic Patterns and Trends, in: Peto R., zur Hausen H. Eds. Virol Etiology of Cervical Cancer. BanBury Report 21. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1986;3-15. In addition to cervical cancer, papilloma virus has been implicated as a causative agent in nasal tumors, and various oral cancers. Warts generally regress spontaneously, and thus patients that seek treatment do so for the relief of temporary pain or discomfort or for cosmetic reasons. Treatments for warts generally consist of the application of cryotherapy, or the use of one or more DNA synthesis inhibitors, or simply removing the warts surgically. Further treatments have consisted, of the application of various inteiferons particularly against refractory genital warts. This approach has been partially successful with cure rates in the range of about 36% compared to spontaneous remissions of 3%. Part of the explanation for the lack of unified treatment strategies for controlling or curing patients of papilloma virus infections is the inability to grow the virus in vitro, and thus develop a convenient and reliable assay to identify efficacious drugs. For the most part, the study of papilloma virus has come from the development of in vitro transformation assays that has facilitated the identification of viral functions involved in the induction of cellular proliferation. The prototype papilloma virus used in these studies has been bovine papilloma virus type I (BPV-1). Papilloma viruses consist of double-stranded DNA of about 8,000 base pairs. Sequencing studies based on six animal and nine human papilloma viruses have revealed that the genomic organization of papilloma viruses is remarkably conserved A key shared feature is that all of the viruses have open reading frames located on one strand of the viral DNA. There are approximately ten open reading frames that have been classified based on their position in the viral genome. Different pathways are likely to regulate the initiation of DNA replication. For example, the post-translation modification of the proteins required for synthesis are known to be pivotal in intricate ways (see Mohr et al., EMBO J. (1987) 6_:153-160; McVey et al., Nature (1989) 241:503-507; D'Urso et al., Science (1990) 2_ϊ__:786-791; Din et al., Genes &Dev. (1990) 4:968-977; and Virshup et al., EMBO J. (1989) £.3891-3818). Interestingly, proteins that bind to origins of replication also function in the control of transcription (Depamphilis, M.L., Cell (1988) 52:635 and Brand et al., Cell (1987) 51:709.. The roles of transcription factors in regulating chromosomal replication are ambiguous. However, numerous experiments have shown that tissue- specific gene expression is correlated with early replication of the active gene and its flanking DNA, while the same gene when inactive in another tissue replicates late in the cell cycle (Hatton et al., Cancer Cell (1988) &335-340). This implies a link between transcription control and replication control. Bovine Papilloma Virus type 1 (BPV-1) provides a framework for exploring the roles of transcription factors in eukaryotic DNA replication. In transformed cells, the viral chromosome is maintained as a stable nuclear plasmid replicating in synchrony with the host DNA. Two viral proteins, El and E2, are both necessary and sufficient for replication (Ustav & Stenlund, EMBO J. (1991) lQ:449-457). El is a 68 kD protein and viral DNA with mutations in this ATP binding protein cannot be maintained as nuclear plasmids (Sun et al., J. Virol. (1990) 6.4:5093-5105). Three related site- specific DNA binding proteins are encoded by the E2 ORF (Howley, P.M. in Virology (eds. Fields & Knipe) 1625-1650 (Raven Press, N.Y. 1990), a 48 kD transactivator and two repressors lacking the activation domain, E2C and E8/E2. The 48 kD transactivator binds to DNA as a dimer, and in combination with cellular factors including SP-1 (Li et al., Cell (1991) 6^:380-400) activates transcription from a number of viral promoters. The relative concentrations of the E2 family of proteins thus intricately regulate the transcriptional program of the viral plasmids. Studies from our laboratory showed that the 48 kD E2 protein could form a tight complex with the El protein (Mohr et al., Science (1990) 25Q: 1694-1699). Partially purified El displayed a weak specific DNA binding activity, and this activity was markedly stimulated by E2. To facilitate mechanistic studies and to ascertain if E2 plays a direct role in DNA replication, we developed a cell-free replication system. Disclosure Of The Invention A first object of the invention is a description of a method of identifying compounds that inhibit papilloma virus DNA replication, consisting of isolating a cell free extract that supports papilloma virus DNA replication in the presence of papilloma virus proteins El and E2; forming a mixture consisting of the cell free extract, El and E2, assay reagents that support and permit the ctetermination of papilloma virus DNA replication, and the compounds; and measuring the amount of DNA replication that occurs in the presence of said compounds compared to the amount that occurs in their absence. A second object of the invention is a description of a composition for replicating papilloma virus DNA, comprising papilloma virus proteins El and E2, and a cell free extract that supports papilloma virus DNA replication. These and other objects of the invention will become apparent upon a full consideration of the following disclosure. Brief Description Of The Figures Figure 1 A shows the purification of BPV El and E2. The BPV El and E2 coding sequences were cloned into a baculovirus expression system and the proteins were purified by immunoaffinity chromatography as described by Mohr et al., Science (1990) 25Q: 1694-1699. El was tagged at its amino-terminus with a 9 amino acid peptide (EE epitope) and purified with a monoclonal antibody specific to the tag peptide (the antibody EE (Grussenmeyer et al., PNAS (USA (1985) £2:7952-7954) was crosslinked to protein G Sepharose, Pharmacia LKB). After washing the loaded column with LiCl, the protein was eluted with 20 mM triethylamine and concentrated (where necessary) by dialysis against solid polyethylene glycol (M.W. 8000), followed by extensive dialysis against 20 mM potassium phosphate (pH 7.5), 100 mM potassium glutamate, 1 mM EDTA, 1 mM DTT and 10% glycerol. In each lane, 200 ng of the protein preparation was fractionated by SDS-PAGE and the proteins were stained with silver. The E2 and E1-E2 complex (designated E1/E2) were similarly purified with the monoclonal antibody specific to E2 (B202)i3. MK lanes contain molecular weight markers. Figure IB shows E1/E2 and BPV origin sequence-dependent in vitro DNA replication. In lanes labelled E1/E2+, 400 ng of the purified E1/E2 complex were added. Three template DNAs are shown here: p SO contains BPV sequences from 7805-100; ρ3M contains the BPV-1 restriction fragment Hindm (6958)-Mlu (7351); and pKS is the vector plasmid (Stratagene). I and II indicate form I and form II DNA; R.I. indicates replicating intermediates. Both El with EE epitope at its N-terminus and the wild-type El showed identical activities in ATPase and replication assays. However, El with EE epitope at its C-terminus was inactive in both assays (data not shown). Figure 1C shows that replicated form I DNA is resistant to Dpnl digestion. The replication products and 200 ng of marker pKSO were mixed and separated by electrophoresis through a 1% SeaPlaque gel in 20 mM Tris-acetate (pH 8.0) buffer. The form I band was excised from the gel and hydrolyzed with Dpnl. Ethidium bromide staining showed that all detectable form ϊ DNA was cleaved by the enzyme. The MK lane uses the pKSO replication products as a marker. Figure 2A shows the time course of BPV DNA replication in vitro . The replication assay was scaled up to 200 μl. The reaction contained 640 ng of pKSO, 2.2 μg of El and 0.6 μg of E2. At each indicated time point, 25 μl of reaction sample was taken and stopped. The top portion of the figure is an autoradiograph of the time course samples after electrophoresis. The bottom portion of the figure shows total incorporation into DNA of dNMP at each time point Figure 2B shows evidence for bidirectional replication of BPV DNA. DNA samples from different time points in the replication assay were digested with Dral and BstXI and the resulting DNA fragments (A-F) were separated in a 5% polyacrylamide gel. The autoradiogram of the gel is shown on the right side. The intensity of each band was quantitated by the use of a Phosphor Imager (Molecular Dynamics). Incorporation per nucleotide was calculated for each fragment at each time point and the relative amounts of radioactivity were plotted on the left side. The open circle points represent 20 minutes; the filled circles 40 minutes; and the open squares 120 minutes. A diagram of the plasmid pKSO is shown at the bottom. The open arrow heads are Dral sites; and the filled arrow heads are BstXI sites. Figure 3 shows deletion analysis used to identify the cis elements required for replication. The physical map at the top shows the BPV upstream regulatory region (URR) which contains 12 E2 binding sites depicted as black boxes. DNA fragments spanning different regions of the viral genome were tested for their ability to function as an origin of DNA replication in the in vitro system. All reactions were carried out as described for Figure 1 with 50 ng of DNA for each reaction. The quantitation was achieved by two protocols: direct counting of the incorporated label; and by scanning and integrating each gel lane for each sample. Six picomoles of net synthesis were obtained with pKSO, and this number was set as 100%. No discrete bands were detected for templates p3H through p3M (for example Figure IB), and these templates are judged to be completely negative for in vitro DNA replication. pKSOM spans nucleotides 7805-22 (59 bps) and is the smallest fragment tested to date which shows replication activity. Where coordinate numbers are given, PCR was used to create the BPV insert placed into the plasmidpolylinker. All other fragments were inserted into the polylinker of the vector by restriction site fusions. For both pKSO and pKSOM, a BamHI site was generated at the 5' end and an EcoRI site at the 3' end by PCR with primers. Figure 4 shows the structure of the in vitro origin of BPV DNA replication. The top line of Figure 4A shows the BPV sequence from E2 binding site 11 to E2 binding site 12. An 18 bp inverted repeat centered at the Hpal site is indicated by two tail-to-tail arrows. An extensive homology between this inverted repeat and sequences present in the regulatory regions of the deer, elk, and human papilloma viruses is noted. Figure 4B shows the linker insertion at the center of the inverted repeat abolishing replication in vitro. An Ncol linker (SEQ ID NO: 1) was inserted at the Hpal site of pKSO and pKSOM. Standard replication assays were run on wild-type and mutant templates. The lanes labelled "E1/E2+ " contained 400 ng of the E1/E2 complex. All reactions were incubated at 37°C for 2 hours. Figure 5 shows E2 stimulating El dependent replication. Figure 5 A shows pKSO which contains both E2 binding site 11 and 12 was used as the template DNA in the replication assay. Standard replication assays were performed either in the presence or absence of El and E2 proteins at 37°C for 2 hours. The top figure is an autoradiogram of the replication products after fractionation by gel electrophoresis. The amounts of the viral proteins in each reaction is: El: 280 ng (lanes 1 and 4); 140 ng (lanes 2 and 5); 70 ng (lanes 3 and 6). E2: 100 ng (Janes 4-7). Lane 8 is a no added protein control. The bottom figure shows a set of El and E2 titration experiments that were carried out and the [ 3 2P] incorporation quantitated and plotted as shown. Figure 5B shows pKSOM (which contains no E2 binding site) as the template DNA in the replication assay. The top figure is an autoradiogram of the replication products. El: 280 ng (lanes 11 and 14); 140 ng (lanes 12 and 15); 70 ng (lanes 13 and 16). E2: 100 ng (lanes 14-17). No added protein control: lane 18. The bottom figure shows the titration of El and E2 protein concentrations and replication for the pKSOM template. Symbols for this figure: open circles, no E2; filled circles, 100 ng of E2; open square, 30 ng of E2. E2 concentration above 100 ng gave no further stimulation (data not shown). Figure 6 A and B show the binding of El to the origin of BPV replication stimulated by E2. DNA fragments containing BPV sequence 7805-100 were labelled with 32p at the 5' end of each strand (A, top strand with labelling at BamHI site; B, bottom strand with labelling at EcoRI site). The DNase I footprint analysis was performed as described before (Li et al., Cell (1991) 65:380-400) with the following modifications. The Z buffer was replaced with a new buffer containing 20 mM potassium phosphate (pH 7.5), 100 mM potassium glutamate, 1 mM EDTA, 0.5 mM DTT, and 10% glycerol. The binding reaction was carried out at 37°C for 15 minutes, followed by standard DNase I digestion. El concentration: lanes 2 and 7, 900 ng; lanes 3 and 8, 300 ng; lanes 4 and 9, 100 ng; lanes 5 and 10, 33 ng. E2 concentration: lanes 6-10, 100 ng. BS 11 and BS 12 indicate E2 binding sites 11 and 12. Lane M contains the AG sequence size marker. Figure 6C shows that El is required for the interaction of E2 with DNA in the absence of E2 binding sites. The interaction of El and E2 with DNA were probed by UV-CTOssHn ng of the proteins to a p2p]-labelled, bromodeoxyuridine-substituted DNA cont_-tning the minimal replication origin (i.e., no E2 binding sites). The DNA was subsequently digested and the proteins were analyzed by acrylamide gel electrophoresis. For El protein concentration: lanes A and C, 280 ng; lanes B and D, 90 ng. For E2 concentration: lanes C-E, 100 ng. Modes For Carrying Out The Invention The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference. The following methods were utilized to realize the use of the invention. Additional materials and methods are described in co-owned PCT Application No. WO 92/11290 published July 9, 1992. "E 1 and E2" refer to those papilloma virus proteins encoded by the E 1 and E2 open reading frames that have molecular mass of about 68 kD and 48 kD, respectively, and that form a complex that binds to papilloma virus DNA, and consequently are involved in initiating viral DNA synthesis. Since a key aspect of the invention described herein is the discovery that El and E2 are essential for viral DNA replication, it is intended within this definition to encompass similar papilloma virus proteins that may have different molecular mass, but that behave in a functionally related manner. It will further be appreciated that active fragments of El and E2 are intended to come within the definition. "Control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences which are suitable for procaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood, sequences. Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. "Expression system" refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. In order to effect transformation, the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome. As used herein "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus "transformants" or "transformed cells" includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny which have the same functionality as screened for in the originally transformed cell, are included. Where indistinct designations are intended, it will be clear from the context. As used herein the term " papilloma virus disease" refers to any kind of disease caused by the virus, including cancers and warts. Production Of Papilloma Virus El And E2 Proteins The invention described herein shows that the papilloma virus 68 kD El replication protein and 48 kD E2 trans-activator protein affect viral DNA synthesis. Hereinafter, reference to El and E2 will be understood to denote these proteins or proteins with similar molecular mass and function. The key functional characteristic being the capacity of the proteins to support viral DNA synthesis. Inhibitors of E1/E2 induced viral replication are appropriately used as medicaments for the treatment of papilloma virus diseases. Thus, to assay for medicaments by their capacity to inhibit E1/E2 induced viral replication, appropriate sources of the El and E2 proteins are required to carry out the assay. El and E2 are preferably produced recombinantly and isolated using various known biochemical purification protocols or modifications thereof. In general terms, the production of a recombinant E 1 or E2 typically involves the following: First, a DNA is obtained that encodes the proteins and the expression of the proteins is obtainable in an appropriate expression system capable of processing them. This sequence should be in excisable and recoverable form. The excised or recovered coding sequence is then preferably placed in operable linkage with suitable control sequences in areplicable expression vector. The vector is used to transform a suitable host and the transformed host cultured under favorable conditions to effect the production of the recombinant protein. Each of the foregoing steps can be done in a variety of ways. The constructions for expression vectors operable in a variety of hosts are made using appropriate replicons and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene. Generally, procaryotic, yeast, insert, or mammalian cells are presently useful as hosL Although procaryotic hosts are in general the most efficient and convenient for the production of recombinant proteins, eucaryotic cells, and, in particular, mammalian cells or insect cells are preferred for their processing capacity. Procaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example, Bacillus subtilis. various species ofPseudomonas. or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, £. £θJi is typically transformed using derivatives of pBR322, a plasmid derived from an £. £__11 species by Bolivar e &., 1977, Gene 2:95. pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers which can be either retained or destroyed in constructing the desired vector. Commonly used procaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang ei al., 1977, Nature 198: 1056), the tryptophan (trp) promoter system (Goeddel ei al., 1980, Nucleic Acids Res. &4057) and the lambda derived P promoter (Shimatake el al, 1981 , Nature 292: 128), and N-gene ribosome binding site, which has been made useful as a portable control cassette, U.S. Patent No. 4,711,845, issued December 8, 1987 and incorporated herein by reference in its entirety, which comprises a first DNA sequence that is the PL promoter operably linked to a second DNA sequence corresponding to the NRB S upstream of a third DNA sequence having at least one restriction site that permits cleavage within 6 bp 3 ' of the NRB S sequence. U.S. Patent No. 4,666,848, issued May 19, 1987 and incorporated herein by reference in its entirety discloses additional vectors with enhanced expression capabilities. Also useful is the phosphatase A (phoA) system described by Chang et aJ , in European Patent Publication No. 196,864, published October 8, 1986, incorporated herein by reference. However, any available promoter system compatible with procaryotes can be used. The El and E2 nucleic acid sequences may be cloned into a vector by using primers to amplify the sequence which contains restriction sites on their non- complementary ends according to the general methods as disclosed in U.S. Patent Nos. 4,683,195 issued July 28, 1987, 4,683,202 issued July 28, 1987 and 4,800,159 issued January 24, 1989 the latter of which is incorporated herein by reference in its entirety. A modification of this procedure involving the use of the heat stable Thermus aquaticus (Taq) DNA polymerase has been described and characterized in European Patent Publication No. 258,017, published March 2, 1988 incorporated herein by reference in its entirety. Also useful is the Thermal Cycler instrument (Perkin-Elmer- Cetus) which has been described in European Patent Publication No. 236,069, published September 9, 1987 also incorporated herein by reference in its entirety. Generally, the nucleic acid sequence to be cloned is treated with one oligonucleotide primer for each strand and an extension product of each primer is synthesized which is complementary to each nucleic acid strand An alternative to the use of plasmid DNAs encoding the El and E2 proteins as template for PCR is the use of RNA from any cell producing these proteins as template for PCR as described in U.S. Patent No. 4,800,159. If RNA is the available starting material, the extension product synthesized from one primer when separated from its complement can serve as template for synthesized of the extension product of the other primer. As previously mentioned, each primer contains a restriction site on its 5' end which is the same as or different from the restriction site on the other primer. After sufficient amplification has occ urred the amplification products are treated with the appropriate restriction enzyme(s) to obtain cleaved products in a restriction digest. The desired fragment to be cloned is then isolated and ligated into the appropriate cloning vector. For portions of vectors derived from cDNA or genomic DNA which require sequence modifications, site-specific primer directed mutagenesis is used. This technique is now standard in the art, and is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Construction of suitable vectors containing the desired El and E2 coding sequence employs standard ligation and restriction techniques which are well understood in the art Isolated vectors, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, andreligated in the form desired. Site specific DNA cleavage is performed by treating with suitable restriction enzyme(s) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μl of buffer solution. In the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about 1-2 hours at about 37°C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered form aqueous fractions by precipitation with ethanol followed by chromatography using a Sephadex G-50 spin column. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology. 1980, 65_:499-560. Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. cc_{i DNA polymerase I, that is, the Klenow fragment, in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15-25 minutes at 20 to 25°C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 10 mM dNTPs. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with SI nuclease results in hydrolysis of single-stranded portions. Ligations are performed in 15-30 μl volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 μg/ml BS A, 10 mM-50 mM NaCl, and 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C for "sticky end" ligation, or for "blunt end" ligations 1 mM ATP was used, and 0.3-0.6 (Weiss) units T4 ligase. Intermolecular "sticky end" ligations are usually performed at 33-100 μg/ml total DNA concentration. In blunt end ligations, the total DNA concentration of the ends is about 1 μM. In vector construction employing "vector fragments," the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent religation of the vector. BAP digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na+ and Mg+2 using about 1 unit of BAP per μg of vector at 60°C for about 1 hour. Nucleic acid fragments are recovered by extracting the preparation with phenol/chloroform, followed by ethanol precipitation. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments. In the constructions set forth below, correct ligations are confirmed by first transforming the appropriate R coli strain with the ligation mixture. Successful transformants are selected by resistance to ampicillin, tetracycline or other antibiotics, or using other markers depending on the mode of plasmid construction, as is understood in the art. Miniprep DNA can be prepared from the transformants by the method of D. Ish-Howowicz et al., 1981, Nucleic Acids Res. 9:2989 and analyzed by restriction and/or sequenced by the dideoxy method of F. S anger et al-» 1977 PNAS ________-! 74:5463 as further described by Messing et al., 1981 Nucleic Acids Res.. 9:309, or by the method of Maxam et al., 1980 Methods in Enzymology. 65:499. Host strains used in cloning in M13 consists of E. coH strains susceptible to phage infection, such as E. coH K12 strain DG98 are employed. The DG98 strain has been deposited with ATCC July 13, 1984 and has accession number 1965. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing chloride, as described by S. N. Cohen, 1972 PNAS (USA) 69:2110, or the RbCl 2 method described in Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, p. 254 was used for procaryotes. As mentioned above, numerous recombinant systems are available for the cloning and expression of El and E2. However, the preferred system is baculovirus. Thus to exemplify their cloning and expression, El and E2 were produced using bovine papilloma virus constructs expressed in baculovirus. (See PCT Application No. WO 92/11290.) As a source of bovine papilloma virus El ORF, plasmid pMTElDM was used to produce the transfer vector pAcEl. This construct was derived from plasmid pMTEl, which is described in detail by Shaw Sun ej , 1990, J. of Virology. f54:509. pMTElDM is essentially identical to pMTEl with the exception that pMTElDM has a G to A substitution at nucleotide 1236. This substitution prevents production of the spliced M protein. Thus, the 68 kD El protein produced by pMTElDM maintains bovine papilloma virus DNA as an extrachromosomal element Next, the transfer vector pAcEl was produced by removing the El encoding Xbal fragment from pMTElDM and inserting the fragment into the Xbal site of the transfer vector pAcC13. Note that the El encoding Xbal fragment from pMTElDM is downstream of the baculovirus polyhedrin promoter. To generate recombinant virus, 2 μg of transfer vector was co-transfected with 1 μg of wild type DNA into Sf9 cells as described by Summers and Smith in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures", Texas A & M Press: 1986. Recombinant virus (occlusion-negative) was isolated from the transfection supernatant by plaque purification as described by Smith el al-. 1983, Mol. Cell. Biol..3_:2156-2165. Protein production was monitored by Western analysis. The preferred electrophoresis procedure is Western blot gel analysis as described by Burnette, 1981, Anal. Bio. Chem..112:195. The Western blots are blocked, washed, and probed preferably in 10 mM sodium phosphate buffer containing 150 mM sodium chloride (pH 7.4), with 0.1% bovine serum albumin (w/v), and 0.1% ovalbumin (w/v). In addition, a detergent is preferably employed such as Tween 20 at a concentration of about 0.1%. Sodium azide may also be included in the solution at a concentration of 0.02%. The blots are washed, and subjected to autoradiography using X-ray film. Baculovirus expressing E2 was prepared similarly. The details of the E2 expression vector constructs and the urification of the E2 protein by specific oligonucleotide affinity chromatography are described by Knight and Botchan, 1991, PNAS (USA.. To produce recombinant El, Sf9 cells were infected with 5-10 PFU of recombinant virus per cell. Methods for infecting and growing Sf9 cells are well known in the art, and detailed procedures can be found in M. Summers and G. Smith in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures", Texas Agricultural Experiment Station, Bulletin No. 1555 (May, 1987) or in EPO 127,839 to G.E. Smith and M. D.Summers. Preferred media and culturing conditions can be found in co-pending, commonly owned PCT Application Nos. WO89/01027, WO89/01028 and WO89/01028, all were published February 9, 1989. These publications are hereby incorporated by reference. Sf9 cells were infected at 1-15 x 10 6 cells per ml and were harvested at 48 hours post infection. Infected cell pellets were frozen in liquid nitrogen and stored at - 70°C. Cells were lysed by thawing and immediately suspended in 5 pellet volumes of hypo buffer (10 mM Hepes pH 7.45 mM KC1, 1 mM MgCl 2 , 1 mM DTT, 10 mg/ml Leupeptin, 1 mM PMSF). A nuclear fraction was prepared with 18 strokes of a dounce homogenizer (B-pestie), and the mixture was then clarified by centrifugation at 5,000 x g for 10 minutes (HB4 rotor). The pellet was washed twice with Nucleic Wash buffer (20 mM Tris-HCL PH 8.0, 1 MM DTT, 1 mM EDTA, 1 mM PMSF, 10 μg/ml Leupeptin, 10% Sucrose), and suspended in 5 pellet volumes of Hypo buffer. NaCl was slowly added to a final concentration of 0.3 M and the mixture was slowly agitated at 4°C for 30 minutes. Following centrifugation at 10,000 rpm for 10 minutes, in a HB4 rotor, the supernatant was applied to a DEAE Sepharose fast flow column (Pharmacia) equilibrated in buffer A (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 5% glycerol, 0.05% Triton X-100) containing 0.3 M NaCl (A/300). The column was rinsed with 3 volumes of A 300 and this was fraction along with the DEAE flow- through fraction were adjusted to 0.2 M NaCl with buffer A. The individual fractions were then sequentially applied to a Phosphocellulose P-l 1 matrix (Whatmann) equilibrated in A/200. The column was washed with A 200 and eluted stepwise with buffer A containing 0.4 M followed by 1 M NaCl. These individual fractions were then dialyzed against A/200 (approximately 4 changes of 500 ml for 25 minutes each) and adjusted to 0.2 M NaCl with buffer A if necessary. Each pool was individually applied to a Mono Q 5/5 column (Pharmacia) equilibrated in 25 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100. The column was rinsed with this buffer. This was followed by a step elution with 1 M NaCl in the same buffer. Fractions containing El were identified by Western blotting, pooled, and the indicated amounts used to illustrate the invention. The conditions for BPV replication in vitro were derived from Li and Kelly, PNAS (USA) (1984) 81:6973-6977 with some modifications. Extracts from the mouse FM3A cell line were prepared as follows: Cells were grown in a 2-liter suspension culture containing RPMI 1640 media (supplemented with 25 mM HEPES, pH 7.2 and 5% calf serum). The cells were harvested at a density of 7 x 10 5 cells/ml. The cell pellet was washed with 30 ml of cold PBS and then 10 ml of hypotonic buffer (20 mM Hepes (pH 7.5), 5 mM KC1, 1 mM EDTA, and 0.5 mM DTT). The cells were then resuspended in hypotonic buffer to a final volume of 10 ml and incubated on ice for 15 minutes. After 20 strokes in a Dounce homogenizer, 500 μl of 5 M NaCl was added and the extraction mixture was incubated on ice for 30 to 60 minutes. This mixture was centrifuged in an SW41 rotor at 20 K rpm for 30 minutes and the supernatant was dialyzed twice against 1 liter of D buffer (20 mM Hepes (pH 7.5), 10 mM NaCl, 1 mM EDTA, and 0.5 mM DTT). The extract was then centrifuged in a HB4 rotor at 8 K rpm for 8 minutes and the supernatant was frozen as droplets into liquid nitrogen. The protein concentration of the extract was typically 15-20 mg/ml and the frozen extracts kept at -70°C. A standard replication assay (25 μl) contained: 10 μl extract, 40-80 ng of pure form I template DNA, 30 mM Hepes (pH 7.5), 7 mM MgCl 2 , 20 mM potassium glutamate, 4 mM ATP, 100 μM each of CTP, UTP and GTP, 26 μM each of dATP, dTTP, dGTP and dCTP, 2.5 μCi each of the [32p]-dNTP's, 40 mM phosphocreatine and 100 μg ml creatine phosphokinase and viral proteins as indicated. The reaction was incubated at 37°C for 2 hours, stopped by the addition of 25 μl of 20 mM Tris (pH 7.7), 20 mM EDTA, 2% SDS, and 50 μg ml proteinase K and incubated for another 30 minutes. The DNA was precipitated with 25 μl of 7.5 M ammonium acetate and 175 μl of 95% ethanol. The precipitation was repeated twice and the DNA was resuspended in 50 μl TE. The DNA was analyzed by electrophoresis in 0.8% agarose gel. Dried gels were exposed to X-ray film. Extracts from FM3 A cells are capable of efficient repair synthesis. This activity can be measured with damaged DNA templates, is independent of viral encoded proteins, and is essentially completed by 15 minutes of incubation (data not shown). The experimental protocol for UV-crosslinking was carried out as previously described in Lin & Riggs, PNAS (USA) (1974) 71:947-951. A primer annealed to the single-stranded pKSOM was extended by Klenow DNA polymerase in the presence of dCTP, dGTP, [α-32P]dATP and 5-bromo-2'-deoxyuridine triphosphate. The double- stranded DNA was digested with restriction enzymes BamHI and EcoRI. and the DNA fragment containing the minimal replication origin was isolated and used in the crosslin ng reaction. El and E2 proteins were incubated with the labelled DNA at 37°C for 30 minutes in 30 mM Hepes (pH 7.5), 7 mM MgCl 2 , and 100 mM potassium glutamate. The reaction mixtures were then irradiated by UV for 60 minutes at room temperature. After digestion with DNase I and micrococcal nuclease, the El andE2 proteins were separated in a 12% acrylamide gel by electrophoresis and detected by autoradiography. In Vitro Replication Using The El And E2 Proteins Cell-free extracts from virally transformed cells (ID 13) do not support the in vitro replication of exogenously added BPV- 1 DNA. The El and E2 proteins, and E1/E2 complex were over expressed in a baculovirus expression system and the proteins purified by immunoaffinity chromatography as previously described in Mohr et al., Science (1990) 250:1694-1699 and as shown in Figure 1A. When the purified E1/E2 complex was added to cell-free extracts from mouse ID 13 or FM3A cells (Nakano, N., J. Exp. Med. (1966) £8:69-84), replication activity was observed. Figure IB shows that the replication products of pKSO plasmid co-migrated with supercoiled (I) and nicked (II) pKSO markers only when the FM3A extracts were supplemented with the E1/E2 complex. In addition, a broad band of replication intermediates (R.I.) and high molecular weight forms were seen. Initially, plasmids containing the upstream regulatory region (URR), previously shown to contain the origin of replication (Yang & Botchan, Mol. Cell. Biol. (1990) :5913-5911), were used as templates for replication. Pure form I DNA template was used in these reactions to minimize repair synthesis. It was observed that completely replicated form I DNA increased with smaller template targets, thus smaller templates provide favorable substrates. Neither plasmid containing the late region of BPV-1 DNA (p3M) nor the vector (pKS) was able to direct DNA replication (Figure IB). A number of experiments suggest that the heterogeneous material labelled R.I. in Figure IB contains replication intermediates. For example, when the heterogeneous material was digested with single-cut restriction enzymes, it migrated more slowly than open circle DNA. Also upon double digestion with single-cut enzymes and Dpnl (which cuts unreplicated DNA) the heterogenous products migrated faster than the full-length linear DNA but slower than the largest Dpnl fragment (data not shown). Furthermore, the time course presented in Figure 2 is consistent with a precursor product relationship between the R.I. and the forms I and II DNA. Finally, Figure 1C shows that the replicated DNA migrating with the mobility of supercoiled plasmid is completely resistant to hydrolysis by Dpnl. Table 1 summarizes some of the essential requirements and characteristics of the in vitro papilloma virus replication system. The aphidicolin inhibition suggests that one or more of the cellular DNA polymerases α, δ or ε (Syvaoja et al., PNAS (USA) (1990) £2:6664-6668) are involved in BPV-1 replication. Furthermore, the α-amanitin resistance implies that transcription per se mediated by the E2 protein and RNA polymerase H are irrelevant. The block to in vitro replication by topoisomerase (types I and II) inhibitors suggests that the reaction requires unwinding of the DNA duplex. Table 1 Requirement for BPV DNA Replication In Vitro* *The "complete" system is the standard reaction mixture described in Figure 1 and set as 100% for relative replication comparisons. The p2p] incorporation was quantitated by scintillation counting in EcoLite (ICN Biochemicals). The actual counts incorporated for the complete reaction was 40,000 cpm (9.4 pmol). The counts for the reaction containing no template DNA was 1,000 (0.2 pmol) and set to 0%. DNA topoisomerase I inhibitor camptothecin and topoisomerase π inhibitor V-M-26 were gifts from Professor L_F. Liu (John Hopkins School of Medicine). The kinetics of incorporation shown in Figure 2A are consistent with a multicomponent or multistep reaction. After a lag period of approximately 15-20 minutes, the rate of synthesis increases for about 1 hour before reaching a plateau. At the plateau about 7 picomoles of dNTP's were synthesized into DNA in a 25 μl reaction. Similar reaction kinetics have been reported for the SV40 in vitro replication system (see Virshup et aL, EMBO J. (1989) 8:3891-3818; Stillman & Gluzman, M____ Cell Biol. (1985) 5:2051-2060; and Wobbe et al., PNAS (USA) (1986) 83:4612- 4646). To cteteπrjine the initiation site and the directionality of DNA replication, the products from various time points were analyzed after digestion with Dral and BsjXI. If the replication initiates from the BPV-1 sequences, fragment D should be labelled first. Subsequently, if replication proceeds bidirectionally, other fragments should become labelled in proportion to their molecular weight and position with respect to a unique start site. As shown in Figure 2B, a symmetrical curve peaking at fragment D is observed. The curves do not completely flatten out with time, as replication intermediates predominant in the reaction, even after 2 hours of incubation (Figure 2A). The Minimal Origin Of Replication For BPV DNA The series of plasmids used to localize the genetic elements necessary for BPV in vitro DNA replication are shown in Figure 3. We were surprised to find that plasmid PC100ΔRE replicated with the same efficiency as did the intact URR (Figure 3), as this plasmid does not contain the highest concentrations the complex bound to other regions of the URR. In the absence of E2, El displayed a weak affinity for DNA proximal to but outside of these high affinity sites (see Figure 3 and Mohr et al., Science (1990) 250:1694-1699). Consistent with our data, in vivo studies of BPV-1 replication supported the notion that these high affinity sites are unnecessary in cis as genetic elements for replication (Ustav & Stenlund, EMBO J. (1991) 1&449-457). Of the plasmids that replicated in vitro , pKSOM contains the least amounts of viral DNA. This plasmid contains a part of E2 binding site 12 (Li et al., C_e!l (1991) 61:380-400), an A T rich region and an 18 base pair paUndromic sequence, as shown in Figures 3 and 4. This palindromic sequence motif is conserved in a number of animal and human papilloma viruses. To examine the genetic significance of this palindromic sequence, mutants were created by inserting a synthetic linker into the Hpal site, as shown in Figure 4A. Neither pKSO-Nco, nor pKSOM-Nco were capable of supporting in vitro replication, as seen in Figure 4B. These results suggest that the spacing between palindromic half sites are important for replication in vitro. Stimulation Of DNA Replication Using The E2 Protein The in vitro replication system for BPV-1 was employed to examine the role E2 plays in DNA synthesis. Reactions receiving only E2 protein failed to replicate BPV- 1 templates, as shown in Figure 5, lanes 7 and 17. Reactions supplemented with only purified El protein directed a small amount of replication only at the highest levels of El, as seen in Figure 5, lanes 1 and 11). When the purified E2 protein was added to extracts along with the purified E 1 protein, a marked stimulation of replication was observed (Figure 5, top and bottom panels). Similar incorporation was detected utilizing two different templates pKSO or pKSOM. The stimulation was due specifically to the E2 protein, as the E2C protein^, purified in a manner identical to E2, did not activate El. As shown in Figure 5, the extent of E2 stimulation was dependent upon the concentration of both E2 and El. Significantly, at low El concentrations, replication was absolutely dependent upon E2. The absolute levels of the El protein in vivo during S phase are not known, but we suspect that it is lower than that of the E2 protein which we estimate to be about a few thousand molecules per cell. The absolute requirement for the E2 protein in vivo may thus reflect at least in part the low levels of the El protein in vivo. To determine if interactions between El and E2 might mediate cooperative DNA binding, DNase footprinting studies were initiated. Figure 6 shows a DNase footprint analysis of purified El protein in the presence and absence of purified E2. El alone clearly protects DNA sequences centered over the 18 b.p. palindrome (labelled Ori) of pKSO. The linker insertion mutation pKSO-Nco dramatically diminishes this protection (data not shown). The E2 protein does indeed enhance the DNA binding ability of the El protein. Protection of the El binding site in the presence of El and E2 occurs at 10-fold lower El concentrations than those which generate equivalent amounts of protection in the absence of E2. Surprisingly, cooperativity was also seen with templates pKSOM lacking intact E2 binding sites. The UV crosslinking experiment shown in Figure 6C extends this point E2 could not be crosslinked to the DNA in the absence of El , because no E2 sites exist in this target. However, in the presence of the El protein, the E2 protein can be crosslinked to the DNA (Figure 6C, lanes C and D). Together with the footprint analysis provided above, it is clear that the El and E2 proteins help stabilize the formation of a complex cont∑ώiing both proteins over the replication origin. Identification Of Papilloma Virus DNA Replication Inhibitors Papilloma virus DNA replication inhibitors can be identified by their ability to prevent E1 E2 induced papilloma virus DNA replication. Thus, those assays presented above that reveal DNA replication may be employed to identify such inhibitors by incorporating them in the assay mixture and monitoring their effect on the selected event. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: BOTCHAN, MICHAEL R. YANG, LIU LI, RONG MOHR, IAN J. CLARK, ROBIN (ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR IDENTIFYING INHIBITORS OF PAPILLOMA VIRUS REPLICATION (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: CETUS CORPORATION (B) STREET: 1400 FIFTY-THIRD STREET (C) CITY: EMERYVILLE (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 94608 (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: US 07/775,273 (B) FILING DATE: ll-OCT-1991 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: McGARRIGLE JR., PHILIP L. (B) REGISTRATION NUMBER: 31,395 (C) REFERENCE/DOCKET NUMBER: 2618 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (510) 420-3217 (B) TELEFAX: (510) 658-5239 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lι CATGCCATGG CATG (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACCGAAACCG GTAAGTAAAG ACTATGTATT TTTTCCCAGT GAATANBBRB BRBBNNCNNB 60 NNBCACACCA TCACCGTT 78 The present invention has been described with reference to specific embodiments. However, this application is intended to cover those changes and substitutions which may be made by those skilled in the art without departing from the spirit and the scope of the appended claims.

Claims: WE CLAIM: 1. A method of identifying a compound that inhibits papiUoma virus DNA repUcation, comprising the steps of: a) isolating a ceU free extract that supports papiUoma virus DNA repUcation in the presence of papiUoma virus proteins El and E2; b) fomώig a mixture comprising said ceU free extract, El and E2, assay reagents that support and permit the determination of papiUoma virus DNA repUcation, and said compound; and c) measuring the amount of DNA repUcation that occurs in the presence of said compound compared to the amount that occurs in its absence. 2. A method as described in claim 1, wherein said ceU free extract supports papiUoma virus DNA repUcation in the absence of E2 and in the presence of an elevated concentration of El. 3. A method as described in claim 2, wherein said mixture lacks E2 and comprises an elevated concentration of El. 4. A method as described in claim 1 , wherein said ceU free extract is replaced with ceUular DNA polymerases and topoisomerases. 5. A composition for repUcating papiUoma virus DNA, comprising papiUoma virus proteins El and E2, and a ceU free extract that supports papUloma virus DNA repUcation. 6. A composition as described in claim 5, wherein E2 is omitted and the concentration of El is elevated such that papiUoma virus DNA repUcation occurs in the absence of E2. 7. A composition for repUcating papiUoma virus DNA, comprising papiUoma virus proteins El and E2, ceUular DNA polymerases, and topoisomerases.

Inventors: BOTCHAN MICHAEL R, CLARK ROBIN, LI RONG, MOHR IAN J, YANG LIU, BOTCHAN, MICHAEL, R., CLARK, ROBIN, LI, RONG, MOHR, IAN, J., YANG, LIU
Applicants: CETUS ONCOLOGY CORP, REAGENTS OF THE UNIVERSITY OF, CETUS ONCOLOGY CORPORATION, UNIV CALIFORNIA
Application year: 1992
Main classification: C12Q1
IPC codes: C12Q1, G01N33