WO2005115458A2 - Methods and compositions related to regulation of nucleo-cytoplasmic localization of e1 dna helicase in papillomaviruses - Google Patents

Abstract

This invention relates generally to methods and compositions for regulating nucleocytoplasmic localization of E1 DNA helicase.

Description

METHODS AND COMPOSITIONS RELATED TO REGULATION OF NUCLEOCYTOPLASMIC LOCALIZATION OF El DNA HELICASE IN PAPILLOMAVIRUSES BACKGROUND OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Application

60/560,471, filed April 7, 2004, which application is incorporated herein by this reference in its entirety. This invention was funded by the National Institutes of Health, grant numbers CA83679, CA107338-02, CA36200-17, and CA36200. Therefore, the United States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions for regulating nucleocytoplasmic localization of papillomavirus El DNA helicase. The invention has broad applicability in protection against viral diseases or processes involving modulating the nuclear localization of El DNA helicase. GENERAL BACKGROUND Papillomaviruses are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been identified in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, sheep, horses, and dogs. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, of both the cutaneous and mucosal epithelia and most lesions in animals are cutaneous. In the human, more than 100 types of papillomavirus have been identified, and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral-respiratory and anogenital mucosa) (de Villiers et al. (2004) Virology 324: 17-27.) The cutaneous-related diseases include common warts, flat warts, and plantar warts. The mucosal-related diseases include laryngeal papillomas and anogenital condylomas. Some infection may progress to cervical, penile and anal carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott— Raven Pub., Philadelphia, N.Y.). Current methods of treatment for genital warts include physical removal such as cryotherapy, CO2 and other laser, electrosurgery, and surgical excision. Cytotoxic agents such as trichloroacetic acid (TCA), podophyllin or podofilox are also used. Immunotherapy is also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all infected cells, and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for papillomavirus infection since recurrence of lesions is are common with all current therapies (Beutner & Ferenczy, 1997, Amer. J. Med., 102(5 A), 28-37). What is needed in the art are compounds capable of interfering with viral DNA replication.

SUMMARY OF THE INVENTION In accordance with the purposes of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to methods of inhibiting papillomavirus replication in a subject. In another aspect, the invention relates to methods of screening for an agent that inhibits nuclear localization of El. In yet another aspect, the invention relates to nucleic acids, polypeptides, vectors, cells, and cell lines relating to El. In yet another aspect, the invention relates to a composition used in the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention. Figure 1 shows in vivo phosphorylation and subcellular localization of GFP-E1 and mutations. Figure 1 A shows functional domains and critical sequences regulating HPV-11 El nucleo-cytoplasmic localization. LRR, Localization Regulatory Region defined in this work. DBD, DNA binding domain and helicase domain were defined previously. Stars, candidate CDK MAP kinase phosphorylation sites (serine or threonine followed by a proline residue). Cross hatch, a leucine-rich nuclear export sequence (NES). Underlined, the consensus cyclin binding motif (RRL). Filled boxes, bipartite nuclear localization sequence P T/^'l3SO^tS./;ll^" H-Q (NLS). Figure IB shows in vivo phosphorylation of EE-E1 (an El protein tagged with an Glu-rich epitope at the amino terminus). EE-E1 protein purified from different sources with (+) or without (-) prior treatment with protein phosphatase 1 (PP1) was blotted with MPM-2 antibody which recognizes phospho-S/T-P. The membrane was stripped and reprobed with an antibody which recognizes the EE epitope tag of the El protein. Figure IC shows subcellular localization of GFP-El and phosphorylation site mutations in transfected COS7 cells (upper panels). Lower panels show the DAPI stained nuclei. Figure 2 shows in vivo phosphorylation and subcellular localization of GFP-El and mutations in the presence of inhibitors of CDKs or CRMl. All images were taken from transfected COS7 cells except in Figure 2C. Figure 2C shows that GFP-El phosphorylation is blocked by the mutation in the cyclin binding motif (RRL to RRA, KRA or ARA, SEQ ID NOS: 1 and 11-13). GFP-El wild type or mutated proteins were immunoprecipitated using polyclonal GFP antibody blotted with MPM-2, and reprobed with monoclonal anti- GFP after stripping. Figure 2B shows GFP 1 -El was localized to the cytoplasm (left panel) after CDK activity was inhibited by roscovatine (right panels). Immunoprecipitation and Western blotting were performed as described above. Figure 2C shows GFP-El was localized to the cytoplasm after CDK2 activity was inhibited upon p21cipl induction by IPTG in the cell line p21-9 (which was stably transduced with an inducible p21cipl gene). Figure 2D shows that inhibition of CRMl successfully restores nuclear localization of GFP - El phosphorylation mutations. In this experiment, 20 ng/ml leptomycin B (LMB), a specific inhibitor for CRMl -mediated protein nuclear export, were added to culture medium for 6 hours. Figure 3 shows identification of the leucine-rich NES necessary and sufficient for El export. All images shown were taken from transfected COS7 cells. Figure 3A shows that an El Localization Regulatory Region (LRR), with the key motifs conserved among human papillomaviruses, contains the CDK/MAP kinase phosphorylation sites, the cyclin binding motif (RRL), a putative bipartite NLS (dashed underlines), and a leucine-rich NES. Underlined, the 10-aa (residue 106-115, SEQ ID NO: 14) core of the leucine-rich NES. The minimal functional nuclear export signal sequences were determined in this study. The consensus NES sequence is shown at the top. NEm indicates the mutations in NES.

Mutations on the cyclin binding motif are illustrated on the right. Alignment with other HPVs is listed at the bottom (and is expanded in the attached sequence alignment tables). o ii'- i „i„""„^■. " ϋ ,.^• ut <:.;.» ii,,ϋ 15 ^•„„,» ,, .,.ϋ it. i'¹ if' iui Figure 3B shows subcellular localization of GFP-El NES mutations (NEm). Figure 3C shows subcellular localization of EE-E1 and mutations as revealed by indirect immunofluorescence (FITC) with anti-EE antibody. Figure 3 D shows delineation of the functional leucine-rich NES. For purposes of testing subcellular localization, El peptides were fused downstream of GFP. The positions of the appended El peptides are indicated in parentheses. Figure 4 shows activities of GFP-El mutations in transient replication assays. Transient replication assay was performed as described in Example 1. 293 cells were co- transfected with 0.5 μg of El plasmid, 5 μg of pMT2-E2 plasmid and 0.5 μg of a plasmid containing the HPV origin of replication. Low molecular weight plasmids were isolated 48 hours post-transfection, digested with restriction enzyme(s) and detected by Southern blotting using HPV origin DNA plasmid as probe. Linearized ori (arrow) in restriction enzyme Dpnl + lanes indicates the newly replicated origin DNA. Figure 4A shows ori replication by the wild type GFP-El and by phosphorylation site mutations, and by the corresponding NES mutations (NEm). Figure 4B shows signals of replicated origin DNA quantified and normalized to the wild type El to obtain the relative replication activities. Figure 5 shows subcellular localization of GFP-El (Fig. 5A) and GFP-El S89,93,107A (Fig. 5B) in the absence and presence of the origin recognition protein E2. COS7 cells were cotransfected with 5 μg each of expression plasmids of GFP-El and native E2. Cells were fixed 24 hrs post-transfection. Immunostaining of E2 was performed using anti-E2 polyclonal antibody and anti-rabbit IgG-Texas Red conjugated secondary antibody. Top and bottom panels are typical patterns exhibited by the majority and minority of cotransfected cells, respectively. Figure 6 shows that phosphorylation determines El localization by regulating its nuclear export signal. Figure 7 shows that the El protein forms first a hexamer, then a dihexamer, on the origin. Figure 8 shows immunogold-labeling of heat shock/chaperone proteins in El: DNA complexes. On the left is Hsp70, and on the right is Hsp40. Figure 9 shows helicase unwinding the origin DNA. Unwinding intermediates with double loops coated with single-stranded DNA binding protein are shown. DNA is pumped inward while being unwound to enable bi-directional replication. Figure iO diagrammatically shows assembly and activation of papillomavirus pre- replication complexes. Figure 11 diagrammatically shows HPV-11 El and E2 genes, proteins, and regulatory sequences. Figure 12 shows a eukaryotic cell cycle, and the role of cyclin expression and replication. Figure 13 shows El localization regulatory sequences. Figure 14 shows a putative MAPK docking domain alignment of papillomavirus El helicases. Figure 15 shows the sequence of HPV- 18 and E1/E2 interactions. Figure 16 shows the relationship between the MAP kinases. Figure 17 shows that MAP kinases phosphorylate HPV-11 El in vitro. Figure 18 shows the mutations of the putative El MAPK docking domains. Figure 19 shows that multiple mutations in S89, S93, or SI 07 abolish El nuclear localization. Figure 20 shows that phosphorylation determines El localization by regulating its NES. Figure 21 shows phosphorylation by MAPK and cyclin/CDK regulates HPV El protein nucleo-cytoplasmic localization and retention and therefore controls viral DNA replication. Figure 22 shows MAPK phosphorylation on El can also affect El -mediated viral DNA replication. Figure 23 shows tetracycline dosage curves in GFP-1 IE ldm cells compared to the levels of the house-keeping protein actin as a sample loading standard. The El helicase is maximally induced by adding as little as 62.5 nanograms/ml of tetracycline to the cell culture media. Lower concentrations may also be used. Figure 24 shows tetracycline dosage curves in GFP-1 lEldm cells compared to actin levels as a function of time. The induction can be sustained for at least 22 hours of continual tet-treatment. Figure 25 shows GFP-1 IE ldm wild type Tet-on 293 cells, where the gene has an inactivating mutation of the RNA splice donor site, thereby increasing the amount of El messenger RNA. p c T x Fiugu sre a 26s sh, ow ,1s, t .1h,at H_T P-pV- ,1 _Λ 1 _^ El M „A _A P „_τK, d _Joc ,ki .ng d _,omai .n mutations cannot support HPV DNA replication. Figure 27 shows that El is phosphorylated at one or more candidate cyclin/CDK sites in vivo. Figure 28 shows that single as well as combined mutations in S89, S93, and SI 07 significantly reduce El nuclear localization. Figure 29 shows that the inability to bind cyclins and be phosphorylated by CDKs leads to cytoplasmic El. Figure 30 shows that inhibition of cyclin/CDK 1 or 2 with roscovitine leads to cytoplasmic El. Figure 31 shows that inhibition of cyclin E or A/CDK2 with p21cipl leads to cytoplasmic El. Figure 32 shows that LMB, an inhibitor of CRMl -mediated nuclear export, largely restores nuclear localization of El phosphorylation mutations. Figure 33 shows that a putative nuclear export signal is conserved among HPV El genotypes. Figure 34 shows that leucine-rich nuclear export signal is necessary for efficient El export. Figure 35 shows the regulation of nucleo-cytoplasmic of GFP-El is verified by testing key mutant forms of the El protein, each tagged with the EE epitope. Detection of EE-E1 is conducted by using antibody to the EE epitope. Figure 36 shows HPV-11 El has a bipartite nuclear localization sequence. Figure 37 shows Argl24, 125 in the cyclin binding motif (RxL) are part of the nuclear localization sequence. Figure 38 shows single mutations in 3 CDK/MAP kinase phosphorylation sites significantly reduce El nuclear localization.

DETAILED DESCRIPTION OF THE INVENTION The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description. p Dcefiτnitioynss;os/ . <MtO As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a small molecule" includes mixtures of one or more small molecules, and the like. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both iri relation to the other endpoint, and independently of the other endpoint. The terms "higher," "increases," "elevates," or "elevation" refer to increases above control levels. The terms "low," "lower," "inhibits," "inhibition," "reduces," or "reduction" refer to decreases below control levels. The term "mediate" or "mediation" and "modulate" or "modulation" means to regulate, or control, in particular to increase, enhance, elevate, or alternatively to lower, inhibit, or reduce. The terms "mediate" and "modulate" are used interchangeably throughout. As used throughout, by al"subject" is meant an individual. Thus, the "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals, and birds. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. The terms "control levels" or "control cells" are defined as the standard by which a change is measured, for example, the controls are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post- treatment levels.

General Description Human papillomavirus (HPV) is a family of small DNA virus specific for mucosal or cutaneous epithelia. It causes hyperproliferation ranging from benign warts to malignant cervical cancers (zur Hausen, H., and E.-M. de Villiers Annu. Rev. Microbiol. 48:427-447 (1994)). Infections can be latent but the viral DNA persists in the long-living basal cells and can' be ή^'cάviϊeα duVi^'n 'ep s'Odes 'oi temporary or long-term immunosuppression. In the basal and parabasal cells that maintain the ability to divide, viral DNA exists as extrachromosmal plasmids in low copy numbers. Viral DNA amplification takes place only in a subset of differentiated cells in the upper strata. For initiation of viral DNA replication, HPV encodes its own replicative helicase El ( Hughes, F. J., and M. A. Romanos Nucleic Acids Res. 21 :5817-5823 (1993), Lin et al. Mol. Cell. Biol. 22:6592-6604 (2002), Seo et al. Proc. Natl. Acad. Sci. USA 90:702-706 (1993b), Yang et al. Proc. Natl. Acad. Sci: USA 90:5086-5090 (1993)) and origin-recognition protein E2 ( Chiang et al. J. Virol. 66:5224- 5231 (1992a), Mohr et al. Science 250:1694-1699 (1990), Seo et al. Proc. Natl. Acad. Sci. USA 90:2865-2869 (1993)). All other proteins necessary for replication come from host cells, including DNA polymerase α / primase, DNA polymerase δ, RPA, PCNA, and topoisomerases ( Kuo et al. J. Biol. Chem. 269:24058-24065 (1994), Yang et al. Nature 353:628-632 (1991)). The virus expresses two oncoproteins, E6 and E7, the expression of which is elevated upon squamous differentiation. Their function is to reestablish a S phase environment capable of supporting viral DNA amplification ( Chow, L. T., and T. R.

Broker, p. 267-301. In N. Nathanson (ed.), Viral Pathogenesis. Lippincott-Raven Publishers, Philadelphia (1997)). For replication, the viral E2 protein of 40 kDa recruits the El protein of 70 kDa to the origin of replication (ori), on which the El protein assembles into a dihexameric, bidirectional DNA helicase (Fouts et al. J. Biol. Chem. 274:4447-4458 (1999), Lin et al. Mol. Cell. Biol. 22:6592-6604 (2002), Liu et al. J. Biol. Chem. (1998), Sedman, J., and A. Stenlund. J. Virol. 72:6893-6897 (1998)). Assembly is facilitated by heat shock chaperone proteins. In the presence of RPA and topoisomerase I, ATP and an ATP regenerating system, the El protein then unwinds the double-stranded ori DNA (Lin et al. Mol. Cell. Bio. 22:6592-6604 (2002) ) and recruits the host DNA polymerase α / primase to the replication fork to initiate replication. Thus, E2 is only required for the assembly of the pre-RC, whereas El is essential throughout initiation and elongation ( Liu et al. 1995. J. Biol. Chem. 270:27283-27291). The HPV El protein also interacts with multiple cyclins and is phosphorylated in vitro by cyclin/CDK complexes. Eukaryotic DNA replication is strictly controlled by mechanisms that regulate cell cycle to ensure both genetic inheritance and stability. DNA replication is initiated at a precise time when the cellular replication machinery is ready, and then the genome is 'rep^icated^ϊWirigle foϊffld n S^' h cell cycle. Cell cycle entry and progression is regulated by cyclin/ CDKs that phosphorylate key regulatory proteins (Bell, S. P. et al. Annu. Rev. Biochem. 71:333-374 (2002); Pines, J. Nat. Cell Biol. l :E73-79 (1999); Sherr, C. J. et al. Genes Dev. 13:1501-1512 (1999); Weis, K. Cell 112:441-451 (2003)). At Gl/S phase transition, cyclin/CDK complexes phosphorylate the some of the components of the DΝA pre-replication complex (Pre-RC), while others including ORC, Cdc6, Cdtl, and MCM2-7 could be phosphorylated by another kinase, leading to the activation of replication initiation and at the same time preventing re-initiation in the same cell cycle (Bell et al. (2002), Blow et al. Trends Cell Biol. 12:72-78 (2002), Νishitani et al. Cells 7:523-534 (2002)). El phosphorylation by cyclin/CDK is necessary for efficient viral DΝA replication (Ma et al., 1999). El is phosphorylated by CDKs in vivo, and phosphorylation regulates its nucleocytoplasmic localization (Deng et al., 2004). Precise subcellular localization of proteins is essential for their biological functions, and reciprocally, the control of protein localization provides the cells with a convenient way to regulate their functions. Post-translational modification of proteins plays critical roles in these aspects. For instance, three of the most important outcomes after protein phosphorylation are the changes in protein localization, stability, or activity (Laney et al. Cell 97:427-430 (1999)). Proteins that shuttle between the cytoplasm and nucleus often possess short peptide sequences that are recognized by transporting factors. Proteins with nuclear localization sequence (ΝLS) can be transported into the nucleus by a family of importin α/β heterodimers, while those with nuclear export sequence (ΝES) can form a complex with export receptors (exportins) and RanGTP to be transport out of the nucleus ( Jans et al.. Bioessays 22:532-544 (2000), Komeili et al. Annu. Rev. Genet. 35:341-364 (2001), Νakielny et al. Cell Biol. 9:420-429 (1997), Ullman et al. Cell 90:967-970 (1997)). The activities of ΝLS and ΝES are often regulated by post-translational modifications, including phosphorylation, that can control the affinity for and accessibility by the protein transporters ( Jans et al. (2000), Jans, D. A., and S. Hubner Physiol. Rev. 76:651-685 (1996)). A conserved localization regulatory region (Example 7) was identified which contains a dominant leucine-rich nuclear export sequence (ΝES), the cyclin binding motif, three CDK substrate serine residues, and a putative bipartite nuclear localization sequence (ΝLS). El is exported from the nucleus by a CRMl -dependent mechanism unless the ΝES Is

It has been demonstrated that CDK phosphorylation controls El nuclear localization to support viral DNA amplification. Thus, papillomviruses adopt and adapt the cellular regulatory mechanism to regulate the El protein and to complete their reproductive cycle. The HPV DNA helicase El contains a potent leucine-rich NES, which promotes El nuclear export in a CRMl -dependent manner. The El protein is shuttled out of the nucleus unless the NES is inactivated by CDK phosphorylation on three (3) substrate sites. Thus, one mechanism by which CDK controls the timing and extent of viral DNA replication is through the regulation on El subcellular localization. These results reveal that HPV has adapted the cellular regulatory mechanism to support its own DNA amplification. Compositions Disclosed herein are El polypeptides, fragments, mutants, and nucleic acids that can be used with the methods disclosed throughout the application. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, specifically contemplated is each and every combination and permutation of the polypeptide and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

The El protein is 70 kDa (SEQ ID NO: 1). It assembles at the origin of replication (ori) into a dihexameric, bidirectional DNA helicase. A conserved region of 44 amino acids (residues 83-126, SEQ ID NO: 2) at the HPV El amino-terminal portion, a localization regulatory region (LRR) (Fig. 3A), is described herein. LRR contains not only the NES (residues 96-116, SEQ ID NO: 5), it also spans the previously identified consensus cyclin binding motif RxL (residues 124-126, SEQ ID NO: 6) ( Ma et al. (1999)), the three CDK/MAP kinase phosphorylation sites (S89, S93, SI 07), and a bipartite NLS (residues 83- 85, KRK (SEQ ID NO: 7) and residues 120-125, KKVKRR, (SEQ ID NO: 8)). These sequence elements are highly conserved among El proteins of many and very likely almost all HPV genotypes (Fig. 3 A), showing that a ubiquitous regulatory mechanism among El proteins of papillomaviruses can exist. The cell localization regulatory functions of this region are consistent with the report that the N-terminal 166 residues of HPV-11 El is dispensable for cell-free replication (Amin et al. 2000). Also disclosed are mutants of El (SEQ ID NO: 1), such as SEQ ID NO: 9 (wherein leucine at position 110 is replaced with alanine); and SEQ ID NO: 10 (same as SEQ ID NO: 1, wherein the isoleucine at 113 is replaced with alanine). The serine at amino acid 89 can be mutated, the serine at amino acid 93 can be mutated, or the serine at amino acid 107 can be mutated. In one example, alanine is substituted for serine in the mutations discussed above. Also disclosed is a non-naturally occurring polypeptide comprising SEQ ID NO: 1, wherein the consensus cyclin binding motif RxL has been mutated to RRA (SEQ ID NO: 11), KRA (SEQ ID NO: 12), or ARA (SEQ ID NO: 13). Homology/identity It is understood that one way to define any known variants and derivatives or those that might arise from the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. For example SEQ ID NO: 1 sets forth a particular sequence of full-length El, and SEQ ID NO: 2 sets forth a particular sequence of a functional fragment of El. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the [r» ii "ir ,." if is C' ndrn lo'gy"c

the two sequences so that the homology is at its highest level. Another way of calculating homology can be performed by published algorithms.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444

(1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. Peptides Protein variants As discussed herein there are numerous variants of the El protein that are known and herein contemplated. In addition to the known functional El strain variants, there are derivatives of the El proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant DNA technology transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter ^ilexpτe5sing'^!thfeⁱDNΑ^'mτe"(,ombmaht cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1 and are referred to as conservative substitutions.

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide !|'"|! ]l""' '"il"' ." II II C' |!'"ls ||™' ." "II "II " II II j!'"!ι

DactborieM nllrfe^'of'the^ubSMtion, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations. As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 1 is set forth in SEQ ID NO: 3. Another nucleic acid sequence that encodes the same protein sequence set forth in SEQ ID NO: 2 is set forth in SEQ ID NO: 4. It is also understood that ,_I( ,,.,„, ",,,". ," _I, ,j U'.", ,,..',, ||_;;." ,' „[, ,,„ ""',, ,| „ ,,.,[,

'while rib ϊn 'l d^' sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular sequence from which that protein arises is also known and herein disclosed and described. It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 1. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke,

Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs). Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH₂NH~, -CH₂S~, ~CH₂~CH₂ --, ~CH=CH- (cis and trans), ~COCH₂ --, ~ CH(OH)CH₂~, and ~CHH₂SO — (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (--CH₂NH--, CH₂CH₂»); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H₂~S); Harm J. Chem. Soc Perkin Trans. I 307-314 (1982) (~CH~CH~, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH₂-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH₂-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-- CH(OH)CH₂~); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH₂»); and Hruby Life Sci 31 :189-199 (1982) (~CH₂-S~); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is — CH₂NH~. It is understood that fl'"jι ![""^■ "'II'" ," II II C' !P"I| in" ^»|l "II -JII' il B iπi eptide ari'aiόgs'-'can 'haVe "more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like. Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference). Nucleic acids There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, El, as well as any other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that, for example, when a vector is expressed in a cell, the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. Nucleotides and related molecules A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil- 1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'- AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen base-pairing manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. It is also possible to link other types of molecules (conjugates) to nucleotides or I nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 6553-6556). A Watson-Crick interaction is at least one hydrogen-bonding interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson- Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides. Sequences There are a variety of amino acid and nucleic acid sequences related to, for example, El, as well as any other protein or nucleic acid disclosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. ^r' "'"" 'A vtlrfety O '§eq^lhe^,hces^"'-lr '^lprovided herein and these and others can be found in Genbank, at www.pubmed. gov. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art. Primers and probes Disclosed are compositions including primers and probes, which are capable of interacting with the genes disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or complement of a region of the nucleic acid. Functional Nucleic Acids Disclosed herein are functional nucleic acids that can be used with the methods disclosed herein to regulate El nuclear localization. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules can act as effectors, inhibitors,

activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules. Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of El or the genomic DNA of El, or they can interact with the polypeptide El . Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k_d) less than or equal to 10^"6, 10^"8, 10^"10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.

such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with kas from the target molecule of less than 10^"12 M. It is preferred that the aptamers bind the target molecule with a k_d less than 10^"6, 10^" , 10^" , or 10^" . Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k_d with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k_d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of aptamers, the background protein could be El . Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non- limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that Ψ'v, !l'"^f' '"'I " .'' I! |{ ϊl""* [!"'!( {!""' '' "II "II "'"!' II !| ||'"!j

"arernσt f b nσ'm natora systems ^', out which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of United States patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k_d less than 10^"6, 10^"8, 10^"10, or 10^"12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)). Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, BMBδ' ^ is f s ft^^faHKarrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in the following non-limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162. Hybridization/selective hybridization Also provided herein are sequences that selectively hydridize with nucleic acids that encode SEQ ID NO: 1, 2, or 5-8 under stringent conditions. The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or derivatives or two polynucleotides or derivatives in a sequence specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the sequences in a nucleic acid polymer dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm. The effects of temperature and the concentrations of salts and of chaotropic solutes are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The 'coπditiόnsT- Oe"^'rϊsέd"4'S"αe^*'scr^'iD d above to achieve stringency, or as is known in the art. (Chow, L.T., and T.R. Broker. 1981. Electron Microscopy in Biology, Vol. 1, (Ed. J.D. Griffith). John Wiley and Sons, N.Y. pp. 139-188; Chow, L.T. and T.R. Broker. 1989. Meth. Enzymol. 180: 239-261. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k , or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k_d. Another way to define selective hybridization is by looking at the percentage of primer that is enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. In some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation. For example, if the enzymatic manipulation is DNA extension, then selective

StøύlαWwhen at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation. Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly, it is a composition or method that is disclosed herein.

Methods of Treatment Disclosed herein are methods of inhibiting papillomavirus replication in a subject comprising the steps of identifying a subject with, or at risk of contracting, papillomavirus;' and contacting the subject with a substance that reduces nuclear localization of El, thereby inhibiting papillomavirus replication. The point of contact with the subject can be at or near an infected area or an area susceptible to infection. Alternatively, the point of contact can be systematic. Nuclear localization of El can be inhibited by inhibiting cyclin/cdk2 maintenance of phosphorylation. Cyclin-dependent kinase inhibitors include p21cipl and p27kipl . When either of these become elevated, the cyclin/cdk2 complexes are inhibited. For example, the key tumor suppressor protein p53 normally coordinates cellular defense responses against "unscheduled DNA synthesis" or aberrant DNA structures such as stalled replication forks, unresolved recombination structures, or broken chromosomes. p53, among its many properties, is an inducer of p21cipl transcription in cycling cells, thus inhibiting cyclin E/cdk2 and cyclin A/cdk2 activites. In non-cycling, differentiated cells, p21cipl mRNA transcription is p53 -independent and the protein is steadily translated. If there is no perceived evidence of !!"'!f U""' '"II"' ." || \\ >$"'; !l"'!e W'"' ^•' "If "i ' '"" It If '!'"'•

DNA ^,dataage"Or'^luhⁱsCheduled^' repMeation, p21cipl is ubiquitinated and transported to the cytoplasm to be degraded by the 26 S proteosomes. It has been shown that treatment of differentiated keratinocytes in squamous epithelia with the drug lactacystin, a potent inhibitor of the 26S proteasome, results in the rapid accumulation of p21cipl and the inhibition of viral and cellular replication. An example of another CDK inhibitor is roscovitine. The cellular replicative helicase is composed of six subunits (mem proteins 2,3,4,5,6,7 termed the mcm2-7 complex). The cellular helicase is allowed only one round of activity and then the phosphorylated subunits are inactivated or exported from the nucleus to the cytoplasm where they cannot function and may be degraded by the proteosomes. Thus papillomaviruses have adopted and adapted the cyclin E/cdk2 post-translational modification of helicases, with the unique feature that the phosphorylation of the El protein enables it to remain nuclear and able to reassemble on new templates to carry out multiple rounds of viral DNA replication and amplification, leading to production of large numbers of viruses from each infected and productive cell. There exists a stochastic distribution between infected cells that begin and permit the breakthrough of viral replication and another subset of cells in which p27kipl and p21cipl gain the upper hand and successfully inhibit viral replication. Thus, natural lesions (benign warts, papillomas and condylomas) are a mosaic of neighboring producer and non-producer cells. Nuclear importation of El can also be inhibited by reducing the interaction of El with importin-α or importin-β. Studies in vertebrates indicate that the importin proteins are the nuclear localization signal (NLS) receptors that bind to NLS-containing proteins in the cytoplasm and transport them into the nucleus. Once inside the nucleus, the importins are released from the NLS-containing proteins and exported to the cytoplasm for another cycle of import. Therefore, by disrupting the interaction of importin-α or importin-β with the El protein, nuclear importation can be reduced or prevented, and viral replication can be reduced. Nuclear localization of El can also be reduced by stimulating CRMl. CRMl is a member of the importin-β family of transport receptors. CRMl interacts with several effector proteins modulating its affinity to different cargo molecules, as well as the master regulator protein of nuclear transport, the small GTPase Ran. El contains a nuclear export seqeuence which is recognized by the nuclear exportin CRMl. Serine 107 of El is in the middle of the CRMl binding site, and ser 89 and ser 93 are just upstream in the primary sequence and very |)'"li Ii""' " " ." II jl IC' !!"'!! Ij'ϊ; , ^■' »!! "II ""II¹ I! II tf"'jj fcldϊfe ϊn"the'Tσ d^, d"El structu eT^' nosphorylation on any of these sites and especially on ser 107 blocks the binding of CRMl (Example 6). Accordingly, the El helicase remains nuclear when phosphorylated. To increase the exportation of El from the nucleus, the amount of CRMl present in the cell can be increased, thereby increasing the amount of El exported from the cell. By "papillomavirus" is meant any of the papillomavirus types. Sequence alignment data shows comparable sequences of many genotypes of papillomavirus (Table 2). The mechanisms of El regulation is universal amongst papillomaviruses, so one of skill in the art can readily identify and apply the methods and compositions disclosed herein to any of papillomavirus.

!l"^|! l 1 1,1 iι. II J !::::n ./ J II. ^■'^■H.h TABLE 2 PV Sequence Alignments

HPV54

MADNQGTEE EGTGCNGWFFVEAIVERKTGD.IISDDEPEDVE.DS.GLDMVDFID

NSVSQVEGQE.N.PQAL 67 HPV32

MADDTGTE EGLGCSGWFS VEAIVERTTEN.TISDDEDENVE.DS .GLDLVDFVD

DSRIIP.TNQLK.AQAL 66 HPV42

MADDTGTE EGLGCSGWFC VEAIVDKTTEN. AISDDEDENVD.DS .GLDLVDFVD

NSTV..IHTKQVHAQAL 66 CPV1

MADDTGTDN EGTGCSGWFLVEAIVDKTTGE.QVSDDEDETVE.DS.GLDMVDFI

DDRPI..THNSLE.AQAL 66 HPV11R

MADDSGTEN EGSGCTGWFMVEAIVEHTTGT.QISEDEEEEVE.DS .GYDMVDFI

DDRHI..TQNSVE.AQAL 66 HPV 13

MAEDTGTNN EGTGCSGWFLVEAVVERTTGQ.QISDDEDETVE.DS .GLDMVDFI

DDRPI..THNSVE.AQAL 66 HPV44

MADNTGTE GTGCSGWFLVEAIVENTTGQ.QISEDEDEAVE.DS .GLDMVDFIDD

RPI..THNSME.AQAL 64 HPV55

MADNTGTE GTGCSGWFLVEAIVEKTTGQ.QISEDEDEAVD.DS.GLDMVDFID

DRPI..THNSME.AQAL 64 HPV6bR

MADDSGTEN EGSGCTGWFMVEAIVQHPTGT.QISDDEDEEVE.DS.GYDMVDFI

DDSNI..THNSLE.AQAL 66 HPV34

MADS.G.NW EG.RCSGWFNVEAIVERKTGD.AIPADENYDGD.DTEDSEMGDFID

NAHISNIYSQQEIAQAL 67 HPV73

MADS . G.NW EG.RCTGWFNVEAIVERKTGD .PIPEDENYDGG.DTDESEMGDFID

NAHIPNIYAQQEIAQAL 67 RHPV1R

M.DPEGTPG EGVGCTGWFNVEAIVERKTGD. VVSEDEDDT.E.DT.GIDLVDFIDD

TCGSV.QTGDEAPGAL 66

HPV 10

MDDNTGTEGGA..CSESERAGGWFIVEAIVDRRTGD.PISSDDDEEED.EA.GEDFVD

FIDDTRSLGDGQEV.AQEL 71

HPV28

MDDTSGTEGDE..CSELERAGGWFMVEAIVDRRTGD.KPSSDEDEDEDADE.GEDF

VDFIDDRPVGD.GQEV.AQEL 71 l

HPV29

MADNSGTEGEEEDCSEAERAGGWFMVEAIVDRRTGD.TISSDEDEE...DE.GEDMV

DFIDDRPIGD.GQEV.AQEL 70

HPV3

MDDTSGTEGE...CSELERAGGWFMVEAIVDRRTGD.TVSSDEDEE.E.DG.GEDLVDF inui'" ii"¹ .^■■' LI C_ «''"' •" "ii "ii "".v n n jrif r

^,lDT3R!PV©D^;re E!V.A^lQEL^{,r " J}

HPV77

MADNSGTEGEEEDCSEAERAGGWFIVEAIVDKRTGD.TISSDEDEEGE....GEDMVD

FIDDRPIGD.GQEV.AQEL 70

HPV61

MADSEGTESGD.GTEAAERAGGWFLVEAVVDRTTGY.QVSSDEEDNSI.DT.GEDLV

DFIDTRRPGD.GQEV.PLAL 71

HPV2a

MEDSEGTDGT...EEDGCRAGGWFHVEAII..THGQRQVSSDEDEDET.ET.GEDL.DFI

DNRVPGD.GQE.I.PLQL 67

HPV27

MEDSEGTDGT...EEDGCRAGGWFHVEAII..THGQRQVSSDEDEDCT.ET.GEDV.DFI

DNRVPGD.GQE.I.PLQL 67

HPV57

MEDSEGTDGT...DEDGCRAGGWFHVEAII..THGQSQVSSDEDEDET.ET.REDL.DFI

DNRVPGD.GQEV.PLQL 67

HPV26

M.DCEGTNE EGRGCTGWFSVEAIVEKHTGD.TISDDETDNSS.DT.GSDLIGFIDD

SSISD.YAEQEVTQAL 67

HPV51

M.DCEGTED EGAGCNGWFFVEAIVEKKTGD.NVSDDEDENAD.DT.GSDLINFID

SETSICSQAEQETARAL 68

HPV30

MASPEGTDD EGGGCTGWFHVE AVVKKRTGD.IISEDETEEDE. GT. ASDLDGFL

DNSNVITTQADRETAQQL 69

HPV53

MASPEGTDD EG.GCRGWFHVEAIVKKRTGDVISEDETDE.E.ST.ESDLDGFIDN

SNIISTQAERETAQQL 67

HPV56

MASPEGTDG EGKGCCGWFEVEAIVEKKTGD.KISDDESDEED.EI.DTDLDGFID

DSYIQNIQADAETGQQL 69

HPV66

MASPEGTDG EGMGCCGWFQVEAIVERKTGD .TISDDESEEEN.ET.DTD VDGFID

NTLINNTQEDRETAQQL 69

HPV18R

MADPEGTDG EGTGCNGWF YVQ AIVDKKTGD . VISDDEDENAT.DT.GSDMVDF

IDTQGTFCEQAELETAQAL 69

HPV39

MANREGTDG DGSGCNGWFLVQAIVDKQTGD.TVSEDEDENAT.DT.GSDLADFI

DDSTDICVQAERETAQVL 69

HPV45

MADPEGTDG EGTGCNGWFFVETIVEKKTGD . VISDDEDETAT.DT.GSDM VDFI

DTQLSICEQAEQETAQAL 69

HPV59

MADSEGTDG EGTGCNGWFFVQAIVDKKTGD.KISDDEDENAT.DT.GSDLVDFI

DDTTTICVQAERETAQAL 69

HPV70

MANCEGTDG DGSGCNGWFLVQAIVDKQTGD.TVSEDEDENAT.DT.GSDLADFI

DDTTDICVQAERETAQVL 69 27 M;in/ i S O 5 /^' 11.7 ^ϋ'l" O

MADSPGTED GGAGCSGWFVVEAVVDKQTGD.AVSEDEDEEDIEDS.GFDMIDF

IDNSVVAEEHVELSNAQAL 70 HPV7

MADDSGTED VGSGCSGWFLVEAVVDKQTGDVVSEDEDEDAIEDS.GYDMVD

FINDTVVSE.HEELSNAQAL 69 HPV16R

MADPAGTNG EEGTGCNGWFYVEAVVEKKTGD.AISDDENENDS.DT.GEDLVD

FIVNDNDYLTQAETETAHAL 70 HPV31

MADP AGTDG EGTGCNGWF YVEAVIDRQTGD .NISEDENEDS S .DT.GEDMVDFI

DNCNVYNNQAEAETAQAL 69 HPV33

MADPEGTNG AGMGCTGWFEVEA VIERRTGD .NISEDEDETAD .DS .GTDLLEFID

DSMENSIQADTEAARAL 69 HPV35h

MADPAGTDE GEGTGCNGWFFVEAVVSRRTGD.PVSEDENEDDC.DR.GEDMVD

FINDTDILNIQAETETAQAL 70 HPV52

MEDPEGTEG EREGCTGWFEVEAIIEKQTGD.NISEDEDENAY.DS .GTDLIDFIDD

SNINNEQAEHEAARAL 69 HPV58

MDDPEGTNG VGAGCTGWFEVEAVIERRTGD.NISDDEDETAD.DS .GTDLIEFID

DSVQSTTQAEAEAARAL 69 HPV67

MEDPEGTDG EQGGCTGWFS VE AIIEKRTGD.KVSEDEDED AS .DT. GSDLIGFID

DTHIENIQADTQAARAL 69

HPV54

LHAQQLQADVEAVQQLKRKYIG.SPY.VSPVANSEPCVEKDLSPRLGAISLGRRSAK

AKRRLFD..KAQPPPNGHTD. 140

HPV32

LNRQQAHADKEAVQALKRKLLG.SPY.ESPASDLQESINKELSPRLGGLQLCRGSQG

AKRRLFQSLENRDSGYGYSE. 141

HPV42 '

LNKQQAHADQEAVQALKRKLLG.SPY.ESPVSDSQHSIDNELSPRLGGLTLCRGSQG

AKRRLFQSLENRDSGYGYSE. 141

CPV1

LNEQEADAHYAAVQDLKRKYLG.SPY.VSPLGHIEQSVDCDISPRLDAIQLSRKPKK

VKRRLFQSREITDSGYGYSE. 141

HPV11R

FNRQEADAHYATVQDLKRKYLG.SPYVSPISNVANAVESEISPRLDAIKLTTQPKK

VKRRLFETRELTDSGYGYSE. 141

HPV 13

LNEQEADAHYAAVQDLKRKYLG.SPYVSPLGHVEQSVDCDISPRLDAIKLSRNSK

KVKRRLFQSREITDSGYGYSE. 141

HPV44

LNEQEADAHYAAVQDLKRKYLG.SPY.VSPLSNIEQAVECDISPRLDAITLSRQPKK

VKRRLFDRPELTDSGYGNTE. 139 28 Bauκ.x. u s o / 1, ,17 *#-0

LNEQEADAHYAAVQDLKRKYLG.SPYVSPLSNIKEAVECDISPRLDAIKLSRQPKK

VKRRLFERPELTDSGYGNID. 139

HPV6bR

FNRQEADTHYATVQDLKRKYLG.SPYVSPINTIAEAVESEISPRLDAIKLTRQPKKV

KRRLFQTRELTDSGYGYSE. 141

HPV34

YHSQQVNADNEAIRVLKRKFAG.SAG.SSPDSKRHELKHKQRSPHILTIRDTNTT..ST

HLLC...EEQDSGYGNTE. 137

HPV73

YQSQQANADNEAIRVLKRKFTG.SPG.GSPDMKRDEFIDKQLSPQINVLSISSGRSTS

KRRLF...EEQDSGYGNTE. 139

RHPV1R

LHAQETQAHAEAVQVLKRKFVG.SPAVSPLGNYNPCVDRDLSPRLNEISLNQGSG

QAKRRLF....LPDSGYGNTE. 137

HPV 10

FQQQTAADDDVAVQTVKRKFAP.SPY.FSPVCE.QASIEHELSPRLDAIKLGRQSAK

AKRRLF...ELPDSGYGQTQ. 142

HPV28

LLQQAAADDDVAVQAVKRKFAP.SPY.FSPVCM.QPSIENELSPRLDAIKLGRQSGT

AKRRLF...QLPDSGYGQTQ. 142

HPV29

LLQQAAADDDEAVHTVKRKFAP.SPY.FSPVCV..PSIEHELSPRLDAIKLGRQSSKAK

RRLF...QLPDSGYGQTQ. 140

HPV3

LLQQAAADDDVEVQTVKRKFAP.SPY.FSPVCV.HPSIENELSPRLDAIKLGRQTSKA

KRRLF...ELPDSGYGQTQ. 139

HPV77

LLQQAAADDDVAVQAVKRKFTH.SPY.FSPLCV.DPSIEHELSPRLDAIKLGRESAKA

KRRLF...QLPDSGYGQTQ. 141

HPV61

FVQQNAQDDAATVQALKRKYTC.SPA.SSTCV...SLVDSELSPRLDAIRIHRGQDRA

RRRLF...EQ.DSGYGHTQ. 139

HPV2a

YAQQTAQDDEATVQALKRKFVA.SPL.SACSC IENDLSPRLDAISLNRKSEKAKR

RLFE.TEPPDSGYGNTQM 137 HPV27

YTQQIAQDDEATVQALKRKFVA.SPL.SACSC lENDLSPRLDAISLNRKSEKAKRR

LFE.TEPPDSGYGNTQM 137 HPV57

YAQQIAQDDEATVQALKRKFVA.SPL.SACSC IENDLSPRLDAISLNRKSEKAKR

RLFE.TEPPDSGYGNTQM 137

HPV26

FQAQQKQANTKAVRNLKRKLLG.SQN..SPLQD.ITNQHRQQSDSQQNTHQVNNS.Q

AKRRAVD..SVPDSGYGYTE. 137

HPV51

FQAQELQANKEAVHQLKRKFLV.SPR.SSPLGD..ITNQNN....THSHSQ ANES .Q VKRR

LLD..SYPDSGYGNTQ. 134

HPV30 29

ADSGYGNTLE 127 HPV53

LHAQNTHADTQTLQKLKRKYLG.SPL.GDISNQ..QTVCRE AVKRRLID.TE

VPDSGYGNTL. 124 HPV56

LQVQTAHADKQTLQKLKRKYIA.SPL.RDISNQ..QTVCRE GVKRRLIL.SDL

QDSGYGNTL. 126 HPV66

LQVQTAHADAQTLQKLKRKYIG.SPL.SDISNQ..QTVYRE EVKRRLI...LSED

SGYGNTL. 124

HPV18R

FHAQEVHNDAQVLHVLKRKFAGGSTE.NSPLGE.RLEVDTELSPRLQEISLNSGQKK

AKRRLF...TISDSGYGCSE. 141

HPV39

LHMQEAQRDAQAVRALKRKYTD.SSGDTRPYG...KKVGRNTRGTLQEISLNVSSTQ

ATQTVY...SVPDSGYGNME. 139

HPV45

FHAQEVQNDAQVLHLLKRKFAGGSKE.NSPLGE.QLSVDTDLSPRLQEISLNSGHKK

AKRRLF...TISDSGYGCSE. 141

HPV59

FNVQEAQRDAREMHVLKRKFGC.SIE.NSSEKA..AAGKKAKSP.LQEISVNVNHPKV

KRRLI...TVPDSGYGYSE. 138

HPV70

YNMQEAQRDAQSVRALKRKYGG.SNLNKSPCAK.PPGVHREQRVTLQELPVNICN

KQARTNVY...SVPDSGYGNME. 141

HPV40

LHVQQTCADAADLCELKRKYI..SPYVSPIQYSEPSIDGDLSPRLHAIRLGGGQ.KAK

RRLFQRVEQRDSGYGYSE. 143

HPV7

LHAQQTCADAVELCELKRKYI..SPYVSPIQCSEPSVDGDLSPRLHAIKLGGGK.KA

KRRLFERLEQRDSGYGYSQ. 142

HPV16R

FTAQEAKQHRDAVQVLKRKYLG.SPL.SDISG....CVDNNISPRLKAICIEKQSRAAKR

RLF...ESEDSGYGNTE. 138

HPV31

FHAQEAEEHAEAVQVLKRKYVG.SPL.SDISS....CVDYNISPRLKAICIENNSKTAKR

RLF...ELPDSGYGNTE. 137

HPV33

FNIQEGEDDLNAVCALKRKFAA.CSQ.SAAED...VVDRAANPCRTSINKNKECTYR

KRKID...ELEDSGYGNTE. 137

HPV35h

FHAQEEQTHKEAVQVLKRKYAS.SPL.SSVSL....CVNNNISPRLKAICIENKNTAAKR

RLF...ELPDSGYGNSE. 138

HPV52

FNAQEGEDDLHAVSAVKRKFTS.SPE.SAGQD....GVEKHGSPRAKHICVNTECVLPK

RKPC...HVEDSGYGNSE. 137

HPV58

FNVQEGVDDINAVCALKRKFAA.CSE.SAVED....CVDRAANVCVSWKYKNKECTH 30

HPV67

FNLQEEEDDLNAVSALKRKFTG.IQA...CGGN.SNGIENQ ACTAAKRRAY...DI

EDSGYGNTE. 126

HPV54 VEA...A.VEVNT EGTD ETETDQ 159

HPV32 VEI..RQ..EQVEN GHG APDGSM..G NGGG M

166 HPV42

VEV..QQ..TQVEH GHG AVHGTM.GN GGAV G 167

CPV1 VET...A..TQVER YGEP ENGCGG.GG DGRE....KE

168 HPV11R

VEA...A..TQVEK HGDP ENGGDG.QE RDTGR..DIE 170

HPV 13

VEA...E..TQVER NGEP ENDCGG.GG HGRD....KE 168

HPV44 VEA...E..TQVER NGEP EDCGGG..G QGRD....TE

165

HPV55 VEA...E..TQVER NGEP EDCGGG..G QGRD....TE

165 HPV6bR

VEA..GTG.TQVEK HGVP ENGGDG.QE KDTG....RD 170

HPV34

VET..YE..RQVPG PGGC LQSTSS.SN NGSQMA.SPG 168

HPV73

VET..YE..TEVPG LGAGVG.CLQNVNEE.GN QIVS....PR 171

RHPV1R VET..SL..LQVAG GGG QDVQAG 156

HPV10

VDT..ESGPKQVQGSSETQDGRQDDDEGSVVQSTLDTGNQNGRQNND EGSGRN

VGE HGSQ....EE 202

HPV28

VDT..ESGPLQVQDICETG TQDGRQDAD EGSGRNVGG NGGQ....E

E 183 HPV29

VDT..DTGPSQVQDGCETG DQNGRQQYK EGSGTKDGE NGSQ....

EE 181 HPV3

VDT..ESGPKQVQDICKTS QQDGCQGAD EGRGRNVGG NGSQ....E

E 180 HPV77

VDTDSGP..SQVQDICEAG GQDGRQTNT EGSGTEQGE NVSR....EE 182 HPV61

VEI..GASESQVPG DAQH EGGGES.V QEAE..,..E 167

HPV2a

VVG..TP..EEVTGD EESQG GRPVED.QE EERQ G 166

HPV27

VVG..TP..EEVTGD ENSEG GRPVED.KE EERQ G 166

HPV57 31 Hi jf"'' '"II"' ^■" ii i! ϊ'l" '"'"'f ill «' " "[I "II "ψ II |l |i"'|s G'Tt.ΕEVTGE...± .^...„H...DNSQG GRPVDV.RE EERQ G 166

HPV26 VET.LTP.VQVDK QYE ENGGLP..S VCSQ G

163

HPV51 VET.VEATLQVDG QHG GSQNS VCSS G

159 HPV30

VEA..TQ...QVQD NTYG....SGKQQDG.GS QTSVC...SR 156

HPV53

.ET.VEATQQVQE QYVR EASGGKEQN GGSQHSVCSR

158 HPV56

.ET..LETPEQVDE EVQG RGCGNT.QN GGSQN..STY 157

HPV66

.ET..LETSQQVEY EKG NGCGSS.QN GGSQ....NS 152

HPV18R

VEA..TQ..IQVTT NGEH GGNVCS.GGSTEAIDNGGTE G 175

HPV39

VET..AEV.EEVTV ATNT NGDAEG.EH GGSV....RE 168

HPV45 VEA..AE..TQVTV NTNA ENGGSV HSTQ S

166 HPV59

VEM..LE..TQVTV ENTG NGDSNG..S VCSD....SQ 165

HPV70

VET..AE.VEVTVVN NTNGEE EGENGG.EN GGSI....RE 173

HPV40

VET..TE..RQVET EHGGP EDTVGGSGR VTTD....EA 173

HPV7

VET..TE..TQVEE EHGEP EGIEGGSGR AATV....ET 172

HPV16R

VET..QQM.LQVEG RHETETPCSQYSGGS.GG GCSQY..SSG

174

HPV31 VET..QQMVQVEE QQTT LSCNGS 158

HPV33

VET..QQMVQQVES QNGD TNLNDL..E SSGV G 165

HPV35h VEI..QQI.QQVEG HDTV EQCSMG S 160

HPV52

VEA..QQMADQVDG . QNGD WQSNSS.QS SGVG A 166

HPV58

VET..EQMAHQVES QNGD ADLNDS..E SSGV G 165

HPV67

VET..QETMVQVEG QNGD MQCSSQ.CS TGASS...TG 157

HPV54

..VQTV.SGET...TTD....SL..GRQQITELIHNTN.IRVALFGMFKDLYGLSFMDLARPFK

SDKTVCTDWVIAAF 224

HPV32

GSVHGV.QENQ...EIGT...NT..PTTRVVELLKCKN.LQATLLGKFKELFGLSFGDLVRQ

FKSDKSSCTDWVVAAF 234

HPV42 32

FKSDKSSCTDWVIAAF 235

CPV1

GEGQVH.TEVHTESEIEQ...HT..GTTRVLELLKCKD. VRATLHGKFKECYGLSFKDLT

REFKSDKTTCGDWVVAGF 239

HPV11R

GEGVEH.REAEAVDDSTR.EHA..DTSGILELLKCKD.IRSTLHGKFKDCFGLSFVDLI

RPFKSDRTTCADWVVAGF 242

HPV 13

GEGQVH.TEVHTGSQIEE...HT..GTTRVLELLKCKD'. VRATL YGKFKDC YGLSFTDLI

RPFKSDKTTCGDWVVAAF 239

HPV44

GVEQVE.TEVQTHSNTQQ...HT..GTTRVLELLKCKN.IRATLLGKFKDCYGLSYTDLI

RQFKSDKTTCGDWVIAAF 236

HPV55

EGVEQV.ETEVQ..THSDTQLHT..ETTRVVELLKCKN.IRATLLGKFKDC YGLS YTDL

IRQFKSDKTTCGDWVIAAF 237

HPV6bR

IEGEEH.TEAEA.PTNSVR.EHA..GTAGILELLKCKD.LRAALLGKFKECFGLSFIDLIR

PFKSDKTTCLDWVVAGF 242

HPV34

ETNSGS . S SISN.MDIDM..EST..PITDITNILKS SN VKATLL AKFKEV YGLS YMELVRP

YKSDKTQCQDWVCAVF 239

HPV73

ESSSGS.SSISN.MDIET..EST..PITDITNLLQRNN.AKAALLAKFKEVYGLSYMELVRP

YKSDKTHCQDWVCAVF 242

RHPV1R

....GK.ENTR...PDD GGGDATQLLRCSN.LKATLLSKFKSVYGVSFSELVRSFKSD

RTTCADWVVGAA 217

HPV 10

ERAGGD.GEESD..LQST...STGKGAGGVVEILRASN.KKATLLGKFKEQFGLGYNELI

RHFKSDRTSCADWVVCVF 273

HPV28

ERAGGD.GEESQ..TQGV ..QTDKAACGVLAILRASN.QKATLLGKFKEQFGLGFNEL

IRHFKSSKTVCLDWVVCVF 254

HPV29

ERAGGD.GEESQ..PLST...ETEKGACGVLSILKASN.QKATLLGKFKEQFGLGYNELV

RHFKSSRTACVDWVVCVF 252

HPV3

ERAGGD.GEESQ..TESV...QTDTTACGVLAILKASN.HKATLLGKFKEQFGLGFNELI

RHFKSNKTVCSDWVVCVF 251

HPV77

ERAGGD.GEESQ..PLGA...ETERGACGVLSILKASN.QRATLLGKFKEQFGLGYNEL

VRHFKSNRTACADWVVCVF 253

HPV61

ERGGGD.GEAEATGNQET...QAQEQAADILEVFKVSN.LKAKLLYKFKDLFGLAFGE

LVRNFKSDKSICGDWVICAF 240

HPV2a

GDGEAD.LTVHT..PQS ....GT.DAAGSVLTLLRSSN.LKATLLSKFKDLFGVGFYELVR 33

HPV27

GDGEAD.LTVQT..PQS....GT.DAAGSVLTLLRSSN.LKATLLSKFKELFGVGYYELVR

QFKSSKTACADWVVCAF 235

HPV57

GDGE AD.LTVHT..PQS .... GT.D AAGS VLTLLKS SN.LKATLLSKFKEL YGVGYYELV

RQFKSSRTACADWVVCAF 235

HPV26

GSNASV.EDID..VDT....HV.NSVTQICELLKCSNVKAALLSKFKTVYGVSFAELVRV

FKSDKTCCSDWVCAAF 231

HPV51

GGSVMDVET....TESC...ANVELNSICEVLKSSN.AKATLMAKFKELYGISYNELVR

VFKSDKTCCIDWVCALF 227

HPV30

ENSIEA.DSDM...DIG.... AT..PPQQIQELLKS SN. VQ AKLC YKFKELFGIPFSELVRTFK

SDSTCCHDWICAMF 223

HPV53

DGSIGS.GSDMD.VDRQ...DI.MPLQQIQDILKCSNVQAKLYCKFKDIFGIPFSELVRT

FKSDSTCCHDWICAIF 228

HPV56

SNNSED.SVIHM..DIDRN.NET..PTQQLQDLFKSSN.LQGKLYYKFKEVYGIPFSELVR

TFKSDSTCCNDWICAIF 228

HPV66

NCSEHS.VSNM...DIDTN.MET..PTHQLQELFKSSN.VQGRLHFKFKEVYGVPYTELV

RTFKSDSTCCNDWICAIF 222

HPV18R

NNSSVD.GTSDN.SNIENVNPQC.TIAQLKDLLKVNN.KQGAMLAVFKDTYGLSFTD

LVRNFKSDKTTCTDWVTAIF 248 j

HPV39

ECSSVD.SAIDS.ENQDP...KS..PTAQIKLLLQSNN.KKAAMLTQFKETYGLSFTDLVR

TFKSDKTTCTDWVAAIF 238

HPV45

SGGDSS.DNAE...NVDP...HC..SITELKELLQASN.KKAAMLAVFKDIYGLSFTDLVRN_l

FKSDKTTCTDWVMAIF 234

HPV59

IDCSDS.SNMDV.ENIV....PT.SPTNQLLQLLHSKN.KKAAMYAKFKELYGLSFQDLV

RTFKSDRTTCSDWVTAIF 235

HPV70

ECSSVD.SAIDS.ENQDP...QS..PTAQLKTVLQANN.QKAILLSQFKHTYGLAFNDLVR

TFKSDKTICTDWVAAIC 243

HPV40

EAVEVV.EDGSH.VID....HC.SPRTQLIELFKCKD.LNAKLYGKFKELYGVGFGDLVR

QFKSDKSTCTDWVYAVF 242

HPV7

EAVEVL.EESSD.VIQ....QL.SPRTQVVELFKCKD.LNAKLCGKFKELFGVGFHDLVR

QFKSDKSTCTDWVYAVF 241

HPV16R

SGGEGV.SERH...TIC....QT..PLTNILNVLKTSN.AKAAMLAKFKELYGVSFSELVRPF

KSNKSTCCDWCIAAF 241 34 ..DGTH.SERE...NET PTRNILQVLKTSN.GKAAMLGKFKELYGVSFMELIRPFQS

NKSTCTDWCVAAF 221

HPV33

DDSEVS.CETN..VDSC...ENVTLQEISNVLHSSN.TKANILYKFKEAYGISFMELVRP

FKSDKTSCTDWCITGY 234

HPV35h

GDSITS.SSDE...RHD....ET..PTRDIIQILKCSN.ANAAMLAKFKELFGISFTELIRPFKSD

KSTCTDWCVAAF 227

HPV52

SNSDVSCTSIED..NEEN...SN.RTLKSIQNI.MCENSIKTTVLFKFKETYGVSFMELVRP

FKSNRSSCTDWCIIGM 237

HPV58

ASSDVS.SETD..VDSC...NTVPLQNISNILHNSN.TKATLLYKFKEAYGVSFMELVRP

FKSDKTSCTDWCITGY 234

HPV67

NSVDMQ.SESNS..SQEQ...SM..PLQTVENIMHVNN.IKATLMHKFKEAYGVTFTQLIR

PFKSDRTSCTDWCVTAF 226

HPV54

GIYHGITDGFKTLLEPHCLYGHIQWLTCRW..GMVLLLLTRFKCGKNRLTVSKCLG

MLLNIPETQMLIDPPKLRTPAA 300

HPV32

GVHHSIAEGFNTLIKAEALYTHIQWLTCTW..GMVLLMLIRFKCGKNRTTVSKGMC

KLLNIPANQLLIEPPRLQS VAA 310

HPV42

GVNHSIAEGFNTLIKADSLYTHIQWLTCTW..GMVLLMLIRFKCGKNRTTVSKGLSK

LLNIPTNQLLIEPPRLQSVAA 311

CPV1

GVHHSVSEAFQKLIQPLSTYSHIQWLTNYKCMGMVLLVLLRFKVNKNRCTVARTL

ATLLNIPEDHMLIEPPKIQSSVA 317

HPV11R

GIHHSIADAFQKLIEPLSLYAHIQWLTNAW..GMVLLVLIRFKVNKSRCTVARTLGT

LLNIPENHMLIEPPKIQSGVA 318

HPV 13

GIHHSVSEAFEKLMQPLTTYMHIQWLTNAW..GMVLLVLIRFKVNKSRCTVARTLA

TFLNIPEDHMLIEPPKIQSSVA 315

HPV44

GVHHSVSEAFQNLIQPVTTYSHIQWLTNAW..GMVLLALVRFKVNKNRCTVARM

MATRLNIPEDHMLIEPPKIQSGVA 312

HPV55

GVHHSVSEAFQNLIQPVTTYSHIQWLTNAW..GMVLLALLRFKVNKNRCTVARMM

ATRLNIPEDHMLIEPPKIQSGVA 313

HPV6bR

GIHHSISEAFQKLIEPLSLYAHIQWLTNAW..GMVLLVLLRFKVNKSRSTVARTLATL

LNIPENQMLIEPPKIQSGVA 318

HPV34

GVAPSLAESLKSLLTQYCLYIHLQCLTCSW..GIIVLLLARFKCNKNRLTVQKLLHGL

LNVTQEYMLIEPPRLRSTPC 315 35

GVIPSLAESLKSLLTQYCMYIHLQCLTCTW..GIIVLVLVRFKCNKNRLTVQKLLSSL

LNVTQERMLIEPPRLRSTPC 318

RHPV1R

GVHHSVAEGLKQLIQPFCSYAHIQCLTCDW..GVYLLLLARFKCGKNRLTVSKCMS

TLLNVQETHMLIEPPKLRSAAA 293

HPV 10

GVFCTVAEGIKTLIQPLCDYAHIQVLPCQW..GMTVLMLVRYKRAKNRETVAKGLS

TLLNVPESQMLIEPPKLRSGPA 349

HPV28

GVYCTLAEGIKTLIQPQCDYAHIQVLSCQW..GMTVLMLVRYKRAKNRETVAKGL STLLNVPESHMLIEPPKLRSGPA 330

HPV29

GVYCTVAEGIKQLIQPLCEYAHIQVLPCQW..GMTVLMLVRYKRAKNRETVAKGL

STLLNVPESHMLIEPPKLRSSPA 328

HPV3

GVYCTLAESFKTLIQPQCEYAHIQVLSCQW..GMTVLTLVRFKRAKNRETVAKGFS

TLLNVPENHMLIEPPKLRSAPA 327

HPV77

GVYCTVAEGIKQLIQPLCDYAHIQVLPCQW..GMTVLMLLRYKRAKNRETVAKGL

STLLNVPESHMLIEPPKLRSGPA 329

HPV61

GVYHAVAEAVKTLIQPICVYAHIQIQTCQW..GMVILMLVRYKCGKSRETVAHSMG

KLLNIPERQMLIEPPKIRSAPC 316

HPV2a

GVYYAVAEGLKKLIQPHTQYAHIQVQTSSW..GMVVFMLLRYNCAKNRDSVSKN

MSMLLNIPEKHMLIEPPKLRSTPA 311

HPV27

GVYYAVAEGIKQLIQPHTQYAHIQVLTCSW..GMVVFMLLRYNCAKNRDTVSKNM

SMLLNIPEKHMLIEPPKLRSTPA 311

HPV57

RVYYAVAEGIKQLIQPHTQYAHIQIQTSSW..GMVVFMLLRYNCAKNRDTVSKNM

SMLLNIPEKHMLIEPPKLRSTPA 311

HPV26

GVAGSVAESIKSLIQQYCLYYHIQCLTCNW..GVIVLMLVRFTCAKNRTTIKNCLCM

LLNVPETQLLIEPPKLRSTAV 307

HPV51

GVSPMVAENLKTLIKPFCMYYHIQCLSCDW..GTIVLMLIRFSCAKNRTTIAKCLSTL

VNIPQSQMFIEPPKLRSTPV 303

HPV30

GVNETLAEALKTIIKSQCMYYHIQCLTCTW..GVVILMLIRYTCGKNRKTIIKSLSSIV

NVPSEQMLVQPPKIRSPAV 299

HPV53

GVNETLAEALKTIIKTQCIYYHMQCLTCTW..GVVILLLIRYTCGKNRKTIVKSLASI

LNVPTEQMLVQPPKIRSPAV 304

HPV56

GVNETLAEALKTIIKPHCMYYHMQCLTCTW..GVIVMMLIRYTCGKNRKTIAKALS

SILNVPQEQMLIQPPKIRSPAV 304

HPV66 36

ILNVPQEQMLIQPPKLRSPAV 298 HPV18R GVNPTIAEGFKTLIQPFILYAHIQCLDCKW..GVLILALLRYKCGKSRLTVAKGLSTL LHVPETCMLIQPPKLRSSVA 324 HPV39 GVHPTIAEGFKTLINKYALYTHIQSLDTKQ..GVLILMLIRYTCGKNRVTVGKGLSTL LHVPESCMLLEPPKLRSPVA 314 HPV45 GVNPTVAEGFKTLIKPATLYAHIQCLDCKW..GVLILALLRYKCGKNRLTVAKGLST LLHVPETCMLIEPPKLRSSVA 310 HPV59 GVNPTVAEGFKTLIQPYVLYAHIQCLDCAW..GVVILALLRYKCGKNRITVAKGLST LLHVPDTCMLIEPPKLRSGVA 311 HPV70 GVNPTIAEGFKTLIQPYALYTHIQCLDTKY..GVYILLLIRYKCGKNRITVGKGLSKLL HVPESCMLIEPPKLRSPVA 319 HPV40 GVNPTIAEGFHTLLKRQALYLHTQWTSCKW..GMVLLALCRYKVGKNRETVVRQL SKMLNVPDNQILVQPPKLQSPPA 318 HPV7 GVNPTIAEGFHTLLKGQALYLHTQWTTCRW..GMVLLALCRYKVAKNRETVVRQL AKMLNVPDNQLMVQPPKLQSSAA 317 HPV16R GLTPSIADSIKTLLQQYCLYLHIQSLACSW..GMVVLLLVRYKCGKNRETIEKLLSKL LCVSPMCMMIEPPKLRSTAA 317 HPV31 GVTGTVAEGFKTLLQPYCLYCHLQSLACSW..GMVMLMLVRFKCAKNRITIEKLLE KLLCISTNCMLIQPPKLRSTAA 297 HPV33 GISPSVAESLKVLIKQHSLYTHLQCLTCDR..GIIILLLIRFRCSKNRLTVAKLMSNLLS IPETCMVIEPPKLRSQTC 310 HPV35h GIAPSVAESLKTLIKPYCLYIHIQCLSCSW..GMVILALLRFKCAKNRTTIEKLLSKLL CISAASMLIQPPKLRSTPA 303

HPV52 GVTPSVAEGLKVLIQPYSIYAHLQCLTCDR..GVLILLLIRFKCGKNRLTVSKLMSQL LNIPETHMVIEPPKLRSATC 313

HPV58 GISPSVAESLKVLIKQHSIYTHLQCLTCDR..GIILLLLIRFKCSKNRLTVAKLMSNLLS IPETCMIIEPPKLRSQAC 310

HPV67 GITPSVAESLKVLIKPQTLYTHLQCLTCDR..GIIILLLVRFKCAKNRLTVSKLMSNLL SIPETHMIIEPPKIRSTTC 302

HPV54

ALYWYRQGLSNASEIFGTPPEWLARQTVIEYSLADSQFDLSKMVQWAYDHNYID DSIIALEYAKLADIDENAAAFLGS 378 HPV32 37

SDIAYEYAQRADTDSNAAAFLKS 388

HPV42

AIYWFRSGISNASIVTGDTPEWIQRQTILEHCFADAQFNLTEMVQWAYDNDITEDS

DIAYEYAQRADRDSNAAAFLKS 389

CPV1

ALYWFRTSISNASIVTGDTPEWIARQTIVEHGLADNQFKLTEMVQWAYDNDYCDE

SDIAFEYAQRADFDSNAKAFLNS 395

HPV11R

ALYWFRTGISNASTVIGEAPEWITRQTVIEHSLADSQFKLTEMVQWAYDNDICEES

EIAFEYAQRGDFDSNARAFLNS 396

HPV 13

ALYWFRTGISNASTVTGETPEWIKRQTIVEHGLADNQFKLTEMVQWAYDNDFCDE

SEIAFEYAQRGDFDSNARAFLNS 393

HPV44

ALYWFRSGISNASIVTGETPEWITRQTIVEHGLADNQFKLADMVQWAYDNDFCEE

SEIAFEYAQRADIDANARAFLNS 390

HPV55

ALYWFRSSISNASIVTGETPEWITRQTIVEHGLGDNQFKLTEMVQWAYDNDFCEES

EIAFEYAQRADIDANARAFLNS 391

HPV6bR

ALYWFRTGISNASTVIGEAPEWITRQTVIEHGLADSQFKLTEMVQWAYDNDICEES

EIAFEYAQRGDFDSNARAFLNS 396

HPV34

ALYWYRTSLSNISETVGEVPEWIKRQTVVQHSLEDCQFDLSQMVQWAFDNDITND

CEIAYKYALLASEDSNAAAFLKS 393

HPV73

ALYWYRTSLSNISEIVGDTPEWIKRQTLVQHSLDDSQFDLSQMIQWAFDNDITDDC

EIAYKYALLGNVDSNAAAFLKS 396

RHPV1R

ALYWYRTGISNVSEVIGETPEWITRQTMFQHGLEDSIFDLSEMVQWAYDHDFTDD

SVIAYEYAQLAGIDSNAAAFLKS 371

HPV10

ALYWYKTSMSSCSDVYGETPEWIVRQTMVGHAMEDAQFSLSEMVQWAYDHDIT

DESTLAYEYALIADTDSNAAAFLSS 427

HPV28

ALYWYKTAMSNCSDVYGETPEWIVRQTMVGHALEEAQFSLSEMVQYAYDHDIT

DESMLAFEYALLADTDANAAAFLSS 408

HPV29

ALYWYKTSMSNISDVYGETPEWIVRQTMVGHALQEVQFSLSEMVQWAYDHDITD

EGTLAYEYALIADVDSNAAAFLAS 406

HPV3

ALYWFKTSLSNCSEVFGETPEWIVRQTVVGHALEEAQFSLSEMVQYAYDHDITDE

STLAYEYALQADTDANAAAFLAS 405

HPV77

ALYWYKTGMSNISEVYGDTPDWIVRQTIVGHALEETQFRLSDMVQWAYDHDITD

EGTLAYEYALIAEFDANAAAFLAS 407

HPV61

ALYWYRTAMGNASEVYGETPEWIVRQTVVGHAMQEAQFSLSMLVQWAYDNDIT 38

HPV2a

ALYWYKTAMGNGSEVYGETPEWIVRQTLVGHSMEDEQFRLSVMVQYAYDHDTV

EESVLAFEYAQLADVDANAAAFLNS 389

HPV27

ALYWYKTAMGNGSEVYGETPEWIVRQTLVGHSMEDEQFRLSVMVQFAYDHDIV

EESVLAFEYAQLADVDANAAAFLNS 389

HPV57

ALYWYKTSMGNGSEVYGETPEWIVRQTLIGHSMEDEQFKLSVMVQYAYDHDITD

ESALAFEYAQLADVDANAAAFLNS 389

HPV26

ALYFYKTGLSNISETYGDTPEWIVRQTQLEHSFDDATFDLSKMVQWAFDHDITDD

SEIAFKYAQLADIDSNAAAFLKS 385

HPV51

ALYFYRTGISNISNTYGETPEWITRQTQLQHSFEDSTFELSQMVQWAFDHEVLDDS

EIAFHYAQLADIDSNAAAFLKS 381

HPV30

ALYFYKTAMSNISDIYGETPEWIQRQTQIQHSFQDCQFELSKMVQWAFDNDVTDD

SDIAFYYAQLADVDSNAQAFLKS 377

HPV53

ALYFYKTSISNISDVYGSTPEWIERQTQLQHSFEDCQFELSKMVQWAFDNEVTDDS

QIAFHYAQLADVDSNAQAFLKS 382

HPV56

ALYFYKTAMSNISDVYGDTPEWIQRQTQLQHSLQDSQFELSKMVQWAFDNEVTD

DSQIAFQYAQLADVDSNAQAFLKS 382

HPV66

ALYFYKTAMSNISEVYGETPEWIQRQTQLQHSLQDNQFELSKMVQWAFDNEVTD

DSQIAFLYAQLADIDSNAQAFLKS 376

HPV18R

ALYWYRTGISNISEVMGDTPEWIQRLTIIQHGIDDSNFDLSEMVQWAFDNELTDES

DMAFEYALLADSNSNAAAFLKS 402

HPV39

ALYWYRTGISNISVVTGDTPEWIQRLTVIQHGIDDSVFDLSDMVQWAFDNEYTDE

SDIAFNYAMLADCNSNAAAFLKS 392

HPV45

ALYWYRTGISNISEVSGDTPEWIQRLTIIQHGIDDSNFDLSDMVQWAFDNDLTDES

DMAFQYAQLADCNSNAAAFLKS 388

HPV59

ALYWYRTGMSNISEVIGETPEWIQRLTIIQHGVDDSVFDLSEMIQWAFDNDLTDES

DIAYEYALIADSNSNAAAFLKS 389

HPV70

ALYWYRTGMSNISEVSGTTPEWIQRLTVIQHGIDDSVFDLSDMVQWAFDNDVTED

SDIAYGYALLADSNSNAAAFLKS 397

HPV40

ALFWFRAGMGNGSEVSGTTPEWIAKQTMLEHSFADTQFSLTDMVQWAYDNGHT

DECEIAYYYAQRADVDANAAAFLKS 396

HPV7

ALFWFRSGMGNGSEVSGTTPEWIAKQTMLEHSFAEAQFSLTQMVQWAYDNGHT

DECEIAYYYAQIADIDANAAAFLKS 395 39

IJ S O 5.._"'" :ll_" 1, ^!m-o

ALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVD

DSEIAYKYAQLADTNSNASAFLKS 395

HPV31

ALYWYRTGMSNISDVYGETPEWIERQTVLQHSFNDTTFDLSQMVQWAYDNDVM

DDSEIAYKYAQLADSDSNACAFLKS 375

HPV33

ALYWFRTAMSNISDVQGTTPEWIDRLTVLQHSFNDNIFDLSEMVQWAYDNELTD

DSDIAYYYAQLADSNSNAAAFLKS 388

HPV35h

ALYWFKTAMSNISEVDGETPEWIQRQTVLQHSFNDAIFDLSEMVQWAYDNDFIDD

SDI AYKY AQLAETNSN AC AFLKS 381

HPV52

ALYWYRTGLSNISEVYGTTPEWIEQQTVLQHSFDNSIFDFGEMVQWAYDHDITDD

SDIAYKYAQLADVNSNAAAFLKS 391

HPV58

ALYWFRTAMSNISDVQGTTPEWIDRLTVLQHSFNDDIFDLSEMIQWAYDNDITDD

SDIAYKYAQLADVNSNAAAFLRS 388

HPV67

ALYWFRTGMSNISEVSGQTPEWIERLTVLQHSFDDTIFDLGEMVQWAYDNDITDD

SEIAYQYAMLADVNSNAAAFLKS 380

HPV54

NCQAKYVKDCGTMCRHYIRAQKMQMTMSQWIKHRCDLVEEEGEWKEIVRFLRY

QHVDFISFMIALKQFLQGIPKHNCI 456

HPV32

NCQAKYVKDCGIMCRHYKKAQMKRMSMPQWIKHRSERTGDNGDWRPIVKFIRY

QGIDFLTFMSAFKKFLHNIPKKSCL 466

HPV42

NCQAKYVKDCGVMCRHYKKAQMRRMSMGAWIKHRSAKIGDSGDWKPIVKFIR

YQQIDFLAFMSAFKKFLHNIPKKSCL 467

CPV1

NCQAKYVKDCATMCKHYKNAEMKKMSIKQWIKYRSNKIDETGNWKPIVQFLRH

QGIEFISFLSKLKLWLHGTPKKNCI 473

HPV11R

NMQAKYVKDCAIMCRHYKHAEMKKMSIKQWIKYRGTKVDSVGNWKPIVQFLRH

QNIEFIPFLSKLKLWLHGTPKKNCI 474

HPV 13

NCQAKYVKJ3CATMCKHYKNAEMKKMSMKQWITYRSKKIEEAGNWKPIVQFLR

HQNIEFIPFLSKLKLWLHGTPKKNCI 471

HPV44

NCQAKYVKDCATMCKHYKTAEMKKMNMKQWIKFRSSKFEDTGNWKPIVQFLR

HQNIEFIPFLTKLKMWLHGTPKKNCI 468

HPV55

NCQAKYVKDCATMCKHYKTAEMKKMSMKQWIKFRSSKYEETGNWKPIVQFLR

YQNIEFIPFLTKLKMWLHGTPKKNCI 469

HPV6bR

NMQAKYVKDCATMCRHYKHAEMRKMSIKQWIKHRGSKIEGTGNWKPIVQFLRH

QNIEFIPFLTKFKLWLHGTPKKNCI 474 40 NAQAKYVKDCGTMCRHYKAAERKQMTMSQWITHRCDLIDDGGNWKHIVQFLR

YQQVEFVPFLIALKQFLKGIPKQNCI 471

HPV73

NAQAKYVKDCGTMCRHYKAAERKQMSMAQWIQHRCDLTNDGGNWKDIVLFLR

YQNVEFMPFLITLKQFLKGIPKQNCI 474

RHPV1R

NAQAKYVKDCATMCRHYKRAERQQMTMSQWIKQRCEKTDDGGDWRPIVQFLR

YQGVEFIAFLAALKLFLKGIPKKNCI 449

HPV 10

NCQAKYLKDACTMCRHYKRGEQARMSMSEWIWFRGDKVQGDGDWKPIVQFLR

YQDVEFIPFLCAFKTFLQGVPKKSCL 505

HPV28

NCQAKYVKDACTMCRHYKRGEQARMNMSEWIWFRGDKVQGDGDWKPIVQFLR

YHDVEFIPFLCAFKTFLQGIPKKSCL 486

HPV29

NCQAKYVKDACTMCRHYKRGEQARMSMSEWIRFRSNKVQGEGDWKPIVHFLRY

QNVEFIPFLCAFKLFLQGIPKKSCL 484

HPV3

NCQAKYVKDACTMCRHYKRGEQARMNMSEWIKFRGDKIQGDGDWKPIVQYLR

YQDVEFIPFLCALKSFLQGIPKKSCI 483

HPV77

NCQAKYVRDACTMCRHYKRGEQARMTMSEWIKFRSDKIQGDGNWKPIVQYLRY

QDVEFVPFLCALKSFLQGIPKKSCL 485

HPV61

NCQAKYIKDAITMCKHYRRAEQAKMTMAQWITHRGRKVADTGDWKAIVKYLR

YQQVEFVPFISALKLFLKGVPKKSCM 472

HPV2a

NCQAKYVKDAVTMCRHYKRAEREQMSMSQWITFRGNKVSEEGDWKPIVRFLRH

QGVEFVSFLAAFKLFLKGVPKKNCI 467

HPV27

NCQAKYVKDAVTMCRHYKRAERAQMSMSQWITFRGNKVLEEGDWKPIVKFLRH

QGVEFVSFLAAFKLFLKGVPKKNCI 467

HPV57

NCQAKYLKDAVTMCRHYKRAEREQMSMSQWITFRGSKISEEGDW^PIVKFLRHQ

GVEFVSFLAAFKSFLKGVPKKNCI 467

HPV26

NCQAKYVKDCATMTRHYKRAQKRSMCMSQWLQYRCSKIEEGGSWKEIAKFLRF

QHVNFIYFLQVLKQFLKGTPKHNCI 463

HPV51

NCQAKYVKDCGTMARHYKRAQRKSLSMSAWIRYRCDRAKDGGNWREIAKFLR

YQGVNFMSFIQMFKQFLKGTPKHNCI 459

HPV30

NMQAKYVKDCGIMCRHYKRAQQQQMNMKQWITHICSKVDEGGDWRPIVQFLR

YQGVDFISFLSYFKLFLRGTPKHNCL 455

HPV53

NMQAKYVKDCGIMCRHYKRAQQQQMNMKQWIKHVCSKVDDGGDWKPIVQFL

RYQGVEFISFLSYFKLFLQGTPKHNCL 460

HPV56 41

HPV66

NMQAKYVKDCGIMCRHYKRAQQQQMNMCQWIKHICSKVDEGGDWKPIVQFLR

YQGVDFISFLSYFKLFLQGTPKHNCL 454

HPV18R

NCQAKYLKDCATMCKHYRRAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRY

QQIEFITFLGALKSFLKGTPKKNCL 480

HPV39

NCQAKYVKDCATMCKHYKRAQKRQMSMSQWIKFRCSKCDEGGDWRPIVQFLR

YQGIEFISFLCALKEFLKGTPKKNCI 470

HPV45

NCQAKYLKDCAVMCRHYKRAQKRQMNMSQWIKYRCSKIDEGGDWRPIVQFLRY

QGVEFISFLRALKEFLKGTPKKNCI 466

HPV59

NCQAKYLKDCAVMCRHYKRAQKRQMSMSQWIKWRCDKIEEGGDWKPIVQFLR

YQGVEFITFLCALKDFLKGTPKRNCI 467

HPV70

NCQAKYVRDCATMCRHYKRAQKKQMTMAQWIRFRCDKCDDGGDWRPIVQFLR

YQGVEFITFLCAFKEFLKGTPKKNCI 475

HPV40 I

NNQAKYVRDCASMCKHYRLAEMRRMSMAEWIKHRGEKC.DEGDWKPIVKLLRY

QHIDIIVFLAALKKWLQGIPKKNCI 473

HPV7

NNQAKYVRDCAAMCKHYRLAEMRRMSMADWIKHRGEKC.DEGDWKPIVKLLR

YQHIDIIVFLAALKKWLHGIPKKNCI 472

HPV16R

NSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLR

YQGVEFMSFLTALKRFLQGIPKKNCI 473

HPV31

NSQAKIVKDCGTMCRHYKRAEKRQMSMGQWIKSRCDKVSDEGDWRDIVKFLRY

QQIEFVSFLSALKLFLKGVPKKNCI 453

HPV33

NSQAKIVKDCGIMCRHYKKAEKRKMSIGQWIQSRCEKTNDGGNWRPIVQLLRYQ

NIEFTAFLGAFKKFLKGIPKKSCM 466

HPV35h

NSQAKIVKDCATMCRHYKRAEKREMTMSQWIKRRCEKVDDDGDWRDIVRFLRY

QQVDFVAFLSALKNFLHGVPKKNCI 459

HPV52

NSQAKIVKDCATMCRHYKRAERKHMNIGQWIQYRCDRIDDGGDWRPIVRFLRYQ

DIEFTAFLDAFKKFLKGIPKKNCL 469

HPV58

NAQAKIVKDCGVMCRHYKRAEKRGMTMGQWIQSRCEKTNDGGNWRPIVQFLRY

QNIEFTAFLVAFKQFLQGVPKKSCM 466

HPV67

NSQAKIVKDCGTMCRHYKRAEKRKMTIGQWIQARCEKVNDGGDWRTIVKLLRY

QNVEFTQFLATFKKFLKGIPKKSCM 458

HPV54 42 TΪYG PW IK WNYMDIYMRNALDGNPMCFDRKH 534

HPV32

VLIGPPNTGKSQFGMSLVKFLAGTVISFVNSHSHFWLQPLDSAKIAMLDDATPPCW

TYLDTYLRNLLDGNPCSIDRKH 544

HPV42

VLIGPPNTGKSQFGMSLINFLAGTVISFVNSHSHFWLQPLDSAKIAMLDDATPPCW

TYLDIYLRNLLDGNPCSIDRKH 545

CPV1

AIVGPPDTGKSAFCMSLIKFLGGTVISYVNSSSHFWLQPLCNAKVALLDDATQSCW

GYMDTYMRNLLDGNPMSIDRKH 551

HPV11R

AIVGPPDTGKSCFCMSLIKFLGGTVISYVNSCSHFWLQPLTDAKVALLDDATQPCW

TYMDTYMRNLLDGNPMSIDRKH 552

HPV13

AIVGPPDTGKSCFCMSLIKFLGGTVISYVNSSSHFWLQPLCNAKVALLDDATQSCW

VYMDTYMRNLLDGNPMSIDRKH 549

HPV44

AIVGPPDTGKSCFCMSLIKFLGGTVISYVNSSSHFWLQPLCNAKVALLDDVTQSCW

VYMDTYMRNLLDGNPMTIDRKH 546

HPV55

AIVGPPDTGKSCFCMSLIKFLGGTVISYVNSSSHFWLQPLCNAKVALLDDVTQSCW

VYMDTYMRNLLDGNPMTIDRKH 547

HPV6bR

AIVGPPDTGKSYFCMSLISFLGGTVISHVNSSSHFWLQPLVDAKVALLDDATQPCW

IYMDTYMRNLLDGNPMSIDRKH 552

HPV34

VIYGPPDTGKSHFGMSLMQFMQGVVISYVNSNSHFWLSPLADAKMALLDDATPA

CWTYIDRYLRNALDGNPMCLDRKH 549

HPV73

VLYGPPDTGKSHFGMSLIKFIQGVVISYVNSTSHFWLSPLADAKMALLDDATPGC

WTYIDKYLRNALDGNPICLDRKH 552

RHPV1R

VLFGPPNTGKSYFGMSLIHFLQGSIISYVNSNSHFWLQPLADAKVAMLDDATPQC

WSYIDNYLRNALDGNPISVDRKH 527

HPV 10

VFYGPADTGKSYFCMSLLRFLGGAVISYANSSSHFWLQPLSEAKIGLLDDATSQC

WNYIDTYLRNALDGNQICVDRKH 583

HPV28

VFYGPADTGKSYFCMSLLRFLGGVVISYANSNSHFWLQPLADAKIGLLDDATSQC

WCYIDTYLRNALDGNQVCIDRKH 564

HPV29

VFYGPADTGKSYFCMSLLKFMGGVVISYANSHSHFWLQPLSEAKMGLLDDATSQ

CWSYVDTYLRNALDGNVMCIDRKH 562

HPV3

VFYGPADTGKSYFCMSLLKFLGGVVISYANSSSHFWLQPLAEAKIGLLDDATSQC

WCYIDTYLRNALDGNQVCIDRKH 561

HPV77

VFYGPADTGKSYFCMSLLRFMGGAVISYANSTSHFWLQPLSEAKMGLLDDATSQ 43

HPV61 VFYGPSDTGKSLFCMSLLNFLGGAVISYVNSSSHFWLSPLADTKVGLLDDATYQC WQYIDTYLRTVLDGNAISIDRKH 550 HPV2a VFYGPADTGKSYFCMSLLQFLGGAVISYANSSSHFWLQPLSDSKIGLLDDATPQC WSYIDIYLRNLLDGHPVSIDRKH 545 HPV27 VFYGPADTGKSYFCMSLLQFLGGAVISYANSSSHFWLQPLSDSKIGLLDDATPQC WSYIDTYLRNLLDGNPVSIDRKH 545 HPV57 VFYGPADTGKSYFCMSLLQFLGGAVISYANSSSHFWLQPLADSKIGLLDDATAQC WTYIDTYLRNLLDGNPFSIDRKH 545 HPV26 VIYGPPNTGKSQFAMSFIKFMQGSVISYVNSNSHFWLQPLEDAKVAVLDDATYSC WLYIDKYLRNFLDGNPCCIDRKH 541 HPV51 VIYGPPNTGKSLFAMSLMKFMQGSIISYVNSGSHFWLQPLEDAKIALLDDATYGC WTYIDQYLRNFLDGNPCSIDRKH 537 HPV30 VLYGPPNTGKSCFAMSLIQFFQGSVISYVNSHSHFWLQPLDNAKLGMLDDATDAC WRYIDEYMRNLLDGNPVSLDRKH 533 HPV53 VIYGPPNTGKSCFAMSLINFFHGSVISYVNSHSHFWLQPLDNTKLGMLDDATEAC WKYIDEYLRNLLDGNPVSLDRKH 538 HPV56 VLCGPPNTGKSCFAMSLIKFFQGSVISFVNSQSHFWLQPLDNAKLGLLDDATEICW KYIDDYLRNLVDGNPISLDRKH 538 HPV66 VLCGPPNTGKSCFAMSLINFFQGSVISFVNSQSHFWLQPLDNAKLGLLDDATDTC WRYIDDYLRNLLDGNPISLDRKH 532 HPV18R VFCGPANTGKSYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCW TYFDTYMRNALDGNPISIDRKH 558 HPV39 VIYGPANTGKSHFCMSLMHFLQGTVISYVNSTSHFWLEPLADAKLAMLDDATGTC WSYFDNYMRNALDGYAISLDRKY 548 HPV45 LLYGPANTGKSYFGMSFIHFLQGAIISFVNSNSHFWLEPLADTKVAMLDDATHTC WTYFDNYMRNALDGNPISIDRKH 544 HPV59 VLCGPANTGKSYFGMSLLHFLQGTVISHVNSNSHFWLEPLTDRKLAMLDDATDSC WTYFDTYMRNALDGNPISVDRKH 545 HPV70 VIQGPPNTGKSYFCMSLMHFLQGTVISYVNSTSHFWLEPLADAKVAMLDDATGTC WSYFDTYMRNALDGNPISLDRKH 553 HPV40 CIVGPPDTGKSCFGMSLMHFMQGTIISYVNSCSHFWLQSLADAKVAMLDDVTAA CWGYMDTHMRNLLDGNPTSIDRKH 551 44 irara xu s o 5 ,■ " i, ;i, 7 -Π CIVGPPDTGKSCFGMSLMHFLQGTIISFVNSCSHFWLQSLVDAKVAMLDDVTSAC WAYMDTHMRNLLDGNPTSIDRKH 550 HPV16R LLYGAANTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPC WNYIDDNLRNALDGNLVSMDVKH 551 HPV31 LIHGAPNTGKSYFGMSLISFLQGCIISYANSKSHFWLQPLADAKIGMLDDATTPCW HYIDNYLRNALDGNPVSIDVKH 531 HPV33 LICGPANTGKSYFGMSLIQFLKGCVISCVNSKSHFWLQPLSDAKIGMIDDVTPISWT YIDDYMRNALDGNEISIDVKH 544 HPV35h LIYGAPNTGKSLFGMSLMHFLQGAIISYVNSKSHFWLQPLYDAKIAMLDDATSPC WAYIDQYLRNALDGNPISLDVKH 537 HPV52 VLYGPANTGKSYFGMSLIRFLSGCVISYVNSKSHFWLQPLTDAKVGMIDDVTPICW TYIDDYMRNALDGNDISVDVKH 547 HPV58 LLCGPANTGKSYFGMSLIHFLKGCIISYVNSKSHFWLQPLSDAKLGMIDDVTAISW TYIDDYMRNALDGNDISIDVKH 544 HPV67 VICGPPNTGKTYFAMSLIHFLQGCVISYVNAKSHFWLQPLSDAKIGMIDDVTAICW TYIDDYLRNALDGNDISIDVKH 536 HPV54 RAMVQTKCPPLIVTSNINASTDDRWRYLHSRVKCFCFPNRFPFDSNGNPVYDLSN KNWKSFFKRSWSRLALN..DNDN 610 HPV32 KALTVVKCPPLIITSNTDIRTEDRWKYLYSRISLFEFPNPFPLDKNGNPVYVLNDEN WKSFFQRLWSSLEFQ...ESE 619 HPV42 KALTVVKCPPLLITSNTDIRTNDKWKYLYSRVSLFEFPNPFPLDTNGNPVYELNDK NWKSFFQRLWSSLEFQ...ESE 620 CPV1 KSLALIKCPPLLVTSNIDITTEERYKYLYSRVTLFKFPNPFPFDSNGNAVYELCDAN WKCFFARLSASLDI....QDS 625 HPV11R RALTLIKCPPLLVTSNIDISKEEKYKYLHSRVTTFTFPNPFPFDRNGNAVYELSDAN WKCFFERLSSSLDI....EDS 626 HPV 13 KSLALIKCPPLLVTSNVDITKDDKYKYLYSRVTTLTFPNPFPFDRNGNAVYELSDA NWKCFFTRLSASLDI....QDS 623 HPV44 KSLALIKCPPLIVTSNIDITKEEKYKYLCSRVTLFTFPNPFPFDRNGNALYDLCETNW KCFFARLSSSLDI....QTS 620 HPV55 KSLALIKCPPLIVTSNIDITKEDKYKYLCSRVTLFTFPNPFPFDRNGNALYDLCESNW KCFFARLSTSLDI....QTS 621 45

KALTLIKCPPLLVTSNIDITKEDKYKYLHTRVTTFTFPNPFPFDRNGNAVYELSNTN WKCFFERLSSSLDI....QDS 626

HPV34

KHLLQIKCPPLLITSNTNPKADDTWKYLHSRMKVFTFSNPFPFDSNGNPLYQLTNE

NWKAFFTKTWSKLDLT...EDD 624

HPV73

KNLLQVKCPPLLITSNTNPKADDTWKYLHSRIKVFTFLNPFPFDSNGNPLYQLTNE

NWKAFFTKTWSKLDLT...EDD 627

RHPV1R

KNLVQMKCPPLLITSNTNAGQDDRWMYLHSRMVVFTFEQPFPFDQNGNPVYELN

DKNWKSFFSRTWSRLDLQ...EEE 602

HPV 10

RALLQLKCPPLLITTNINPLTDERWKFLRSRLQLFTFKNPFPVTTQGEPMYTLNDQN

WKCFFRRLWARLSLT...DPE 658

HPV28

RALLQLKCPPLLITTNINPLEDDRWKYLRSRVQLFTFKNKFPLTTQGEPLYTLNDQ

NWKCFFRRLWARLSLT...DPD 639

HPV29

RSLLQLKCPPLLITTNVNPLEDDRWKYLRSRLQVFTFSNPCPLTSKGEPVYTLNDQ

NWKSFFQRLWARLSLT...DPD 637

HPV3

RALLQLKCPPLLITTNINPLGDERWKYLRSRLQVFTFNNKFPLTTQGEPLYTLNDQ

NWKSFFQRLWARLNLT...DPE 636

HPV77

RSLLQLKCPPLLITTNVNPLEDERWKYLRSRLQVFTFKNKFPVTSSGDPLYTLNDQ

NWKSFFQRLWARLRLT...DPD 638

HPV61

RNLTQLKCPPLMITTNINPLEDPTFKYLHSRIVVFQFLHKCPLNSNGDPVYTLNNEN

WKSFFRRSWARIEGSDQQEEE 628

HPV2a

KTLLQLKCPPLMITTNTNPLEEDRWKYLRSRLTVFTFKNPFPFASPGEPLYPLNNAN

WKCFFQRSWSRLDLN...SPE 620

HPV27

KTLLQLKCPPLMITTNINPLEEDRWKYLRSRLTLFTFNNPFPFASPGEPLYPINNAN

WKCFFQRSWSRLDLN...SPE 620

HPV57

KTLLQΠ CPPLMITTNΓNPLEEDRWKYLRSRVTLFKFTNPFPFASPGEPLYPINNAN WKCFFQRSWSRLDLN...SPE 620

HPV26

RSLLQVTCPPLIITSNINPQEDNSLLYLHSRVTVIPFPNTFPFDSNGNPVYALTDVNW

KSFFSTTWSRLDL....EED 615

HPV51

RSLIQLVCPPLLITSNINPQEDANLMYLHTRVTVLKFLNTFPFDNNGNAVYTLNDE

NWKNFFSTTWSRLDL....EEE 611

HPV30

KQLVQIKCPPVIITTNINPLHDAKLQYLHSRIHVVPFLNPFPIDTNGNPVYQLNNVN

WKCFFERTWSRLDLN...NDE 608

HPV53 46

NWKCFFERTWSRLDLD...NDE 613

HPV56

KQLVQIKCPPLLITTNINPMLDAKLRYLHSRMLVFQFQNPFPLDNNGNPVYELSNV

NWKCFFTRTWSRLNLD...NDE 613

HPV66

KQLVQIKCPPVIITTNVNPMQDAKLRYLHSRISVFKFENPFPLDNNGNPVYELSNVN

WKCFFERTWSRLNLD...NDE 607

HPV18R

KPLIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFPFDKNGNPVYEINDKN

WKCFFERTWSRLDLH...EEE 633

HPV39

KSLLQMKCPPLLITSNTNPVEDDRWPYLRSRLTVFKFPNAFPFDQNRNPVYTINDK

NWKCFFEKTWCRLDLQ...QDE 623

HPV45

KPLLQLKCPPILLTSNIDPAKDNKWPYLESRVTVFTFPHAFPFDKNGNPVYETNDKN

WKCFFERTWSRLDLH...EDD 619

HPV59

RHLVQIKCPPMLITSNTNPVTDNRWPYLNSRLMVFKFPNKLPFDKNRNPVYTINDR

NWKCFFERTWCRLDLN...EEE 620

HPV70

RHLIQIKCPPILITSNTNPVEENRWPYLTSRLTVFTFPNAFPFDQNRNPVYTINNKNW

KSFFQKTWCKLDLQ...QDE 628

HPV40

KPLAVIKCPPLLLTSNINITQDSKYQYLQSRVQVFEFPNPFPFDSNGNAVYELNDAN

WNSFFKRLASSLEL....QTP 625

HPV7

KSLAVIKCPPLLLTSNINIKHDCKYQYLQSRVTVFEFPNPFPFDSNGNAVYELSDAN

WNSFFKRLASSLEL....QTT 624

HPV16R

RPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNPVYELNDK

NWKSFFSRTWSRLSLH...EDE 626

HPV31

KALMQLKCPPLLITSNINAGKDDRWPYLHSRLVVFTFPNPFPFDKNGNPVYELSDK

NWKSFFSRTWCRLNLH...EEE 606

HPV33

RALVQLKCPPLLLTSNTNAGTDSRWPYLHSRLTVFEFKNPFPFDENGNPVYAINDE

NWKSFFSRTWCKLDLI...EEE 619

HPV35h

KALVQLKCPPLLITSNINAGKDDRWPYLHSRVVVFTFHNEFPFDKNGNPVYGLND

KNWKSFFSRTWCRLNLH...EEE 612

HPV52

RALVQIKCPPLILTTNTNAGTDPRWPYLHSRLVVFHFKNPFPFDENGNPIYEINNEN

WKSFFSRTWCKLDLI...QEE 622

HPV58

RALVQLKCPPLIITSNTNAGKDSRWPYLHSRLTVFEFNNPFPFDANGNPVYKINDE

NWKSFFSRTWCKLGLI...EEE 619

HPV67

KALVQLKCPPLLLTSNIDVATDSRWPFLHSRVVVFRFNNPFPFDENGNPVYNLNDE 47 »ISF Mffflfe&teiMP 611

HPV54 EEEE.NGDPSNTFRCVPGKASRPL. 633 HPV32 DEEE.NGDTGQTFRCVPGTVVRTV. 642 HPV42 DEED.YGETGQTFRCVPGTVVRTV.. 643 CPV1 EDED.DGDTSQAFRCVPGTVVRTV. 648 HPV11R EDEE.DGSNSQAFRCVPGSVVRTL.. 649 HPV 13 EDED.DGDNSQAFRCVPGTVVRTV. 646 HPV44 EDED.DGDNSQAFRCVPGTVVRTV.. 643 HPV55 EDED.DGDNSQAFRCVPGTVVRTV.. 644 HPV6bR EDEE.DGSNSQAFRCVPGTVVRTL.. 649 HPV34 DKEN.DGDTVQTFKCVSGRNPRTV.. 647 HPV73 DKEN.DGDTVQTFKCVSGRNPRTV.. 650 RHPV1R ETEN.DGSTCRAFKCVAGQNLRTV. 625 HPV 10 DEEE.HGNPSEPFRCVPGQNARTL. 681 HPV28 DEEE.NGNPSEPFRCVPGQNARTL.. 662 HPV29 DEEE.NGEPSEPFRCVPGQNTRTV.. 660 HPV3 DEED.NGNTSEPFRCVPGQNTRTV. 659 HPV77 DEEE.NGEPSEPFRCVPGQNARTL.. 661 HPV61 EEEDEDGVTSRPFRCVPGEITRPL.. 652 HPV2a EQDD.NGNTGEPFRCVPGDVARTV.. 643 HPV27 EQDD.NGNTSEPFRCVPGDVARTL.. 643 HPV57 DQED.NGNTGEPFRCVPGDVARTV. 643 HPV26 ADKE.NGEPLPAFKCVPGENTRLL.. 638 HPV51 EDKE.NGDPMPPFKCVPGENTRLL.. 634 HPV30 DKEN.HGDSMPTFRCVPGENSRLF.. 631 HPV53 DKEN.DGDAMPTFRCVPGENSRLF.. 636 HPV56 DKEN.NGDAFPTFKCVPEQNTRLF.. 636 HPV66 DKEN.NGDSIPTFRCVPEQNTRLL.. 630 HPV18R EDADTEGNPFGTFKCVAGQNHRPL.. 657 HPV39 DEGDNDENTFTTFKCVTGQNTRIL.. 647 HPV45 EDADTEGIPFGTFKCVTGQNTRPL.. 643 HPV59 EDADSDGHPFAAFKCVTGSNIRTL.. 644 HPV70 DEGDNDGNTIPTFKCVTGENTRTL.. 652 HPV40 GDE..DGESSQAPRFVPGTVVRTV.. 647 HPV7 EDE..DGETSQAPRFVPGTVVRTL.. 646 HPV16R DKEN.DGDSLPTFKCVSGQNTNTL.. 649 HPV31 DKEN.DGDSFSTFKCVSGQNIRTL.. 629 HPV33 DKEN.HGGNISTFKCSAGENTRSLRS 644 HPV35h DKEN.DGDAFPAFKCVSGQNTRTLRD 637 HPV52 DKEN.DGVDTGTFKCSAGKNTRSIRS 647 HPV58 DKEN.DGGNISTFKCSAGQNPRHIRS 644 HPV67 DKEN.HGGNCNTFKCSAGENSRCIRS 636

Human papillomaviruses (HPV) have been well documented. Mucosotropic HPV types are divided into "high risk" and "low risk" categories based on their potentials to cause cancers 48 l S a όierilM Ϊ t 'cϊf 'tiOfh ^' n les and females. HPV-6 and HPV-11, for instance, cause most cases of genital warts; they are considered "low risk" because they rarely lead to cancer. Other HPV strains, such as HPV 16, 31, 33, 35, 52 and 58 as well as HPV-18, 39, 45, and 70 are considered "high risk" because they have been linked with an increased risk for cervical, vaginal and anal cancers. Other examples of HPV strains include, but are not limited to HPV 2a, 3, 5, 6b, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 32, 34, 40, 44, 45, 51, 53, 55, 56, 57, 59, 61, 66, 68, 70, 72, 73, 74 and 83. Also contemplated herein are animal papillomaviruses.

Administration The agents, substances, compounds, and compositions disclosed herein can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing undue undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. The agents disclosed herein are of benefit to subjects who are experiencing viral infection or are at risk for infection Because the agents disclosed herein reduce the activity of El, thereby reducing the severity or duration of the infection, any subject that can benefit from a reduction in the activity of El can be admimstered the agents disclosed herein. The compositions comprising an agent disclosed herein in a pharmaceutically acceptable carrier may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although topical intranasal administration or administration by inhalant is typically preferred. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

49 ^W-^" L^{« if} -p rSnlteMHdh ini'slra'tiδδ'b'f the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein in its entirety for the methods taught. The compositions may be in solution or in suspension (for example, incorporated into microparticles, liposomes, or cells). These compositions may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to given tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Rof ler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor- mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

50

The disclosed agents can be delivered to the target cells in a variety of ways. For example, the agents can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro. Thus, the agents can comprise, for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage. The agents of the present invention can also be administered using methods of delivering exogenous nucleic acids, such as in gene therapy. See, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference herein in its entirely for the methods of delivery. Primary cells transfected with the gene for the agent of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, or cells. Administration of the agents disclosed herein can occur in conjunction with other therapeutic agents. Thus, the agents of the present invention can be administered alone or in combination with one or more therapeutic agents. For example, a subject can be treated with the disclosed agent alone, or in combination with chemotherapeutic agents, antibodies, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, chemokines, and/or growth factors. Combinations may be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term "combination" or "combined" is used

51 *fo^!'τef rio'^,e⁹itSeVⁱcOri};omitήn'-^', smMltaneous, or sequential administration of two or more agents. Pharmaceutically Acceptable Carriers Delivery of the agents disclosed herein can be used therapeutically in combination with a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be admimstered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including opthamalically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed compounds can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

52 "^'"'^{' i!}""' ^{i! ■}'Fό^lr MMiohgTo 't(5pfcaFaⁱαministration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Therapeutic Uses The dosage ranges for the administration of the agents disclosed herein are those large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross- reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be admimstered in one or more dose administrations daily, for one or several days. As described above, the agents disclosed herein can be administered together with other forms of therapy. For example, the molecules can be administered with antibodies, antibiotics, or other cancer treatment protocols as described above, or viral vectors. When the agent is in a vector, as described above, the vector containing the nucleic acid for therapeutic purposes can also contain the agent that modulates El activity. Screening Methods Disclosed herein are methods of screening for an agent that inhibits nuclear localization of El, comprising the steps of transfecting a cell with a tagged El protein, fragment, or mutant thereof; contacting the cell with the agent to be screened; inducing

53 ^!i'ei-pr^ssio'h¹'cff'the'tagge'dΕ'rprclt61'h, fragment, or mutant thereof; and detecting a cellular location of the tagged El protein, fragment, or mutant thereof wherein the absence of El protein in the nucleus or its presence in the cytoplasm indicates an agent that inhibits nuclear localization of El . The El protein can be SEQ ID NO: 1 , SEQ ID NO: 2, or any fragment or variant thereof. The method of identifying an El inhibitor can be performed in vitro in a cell assay, for example. Many reporter proteins are known to one of skill in the art. Fluorescent reporter proteins can also be used, such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP). Other examples include the green fluorescent protein from Aequorea coerelescens (AcGFP), DsRedExpress, and red coral fluorescent proteins (for example, AmCyan, ZsGreen, Zs Yellow, AsRed2, DsRed2, and HcRedl). For example, by utilizing GFP, fluorescence is observed upon exposure to light at 489 nm without the addition of a substrate. The use of a reporter protein that, like GFP, are directly detectable without requiring the addition of exogenous factors are preferred for detecting or assessing gene expression or location. Fluorescent proteins can be isolated from many different species, including but not limited to, Aequorea victoria (Chalfie, et al., 1994), Zoanthus species (Matz, et al., 1999), and Renilla reniformis (Ward and Cormier, 1979). The concept of the assay is to monitor the subcellular location of the El helicase in a high-throughput assay. In one example, a cell line containing a tetracycline-inducible GFP- El protein can be induced in all cells growing on the (bottom of the well) window of optical microtiter plates (eg. Corning Inc, Costar #3904 96 well flat bottom tissue culture-treated black plates with optically clear bottoms). The use of a fluorescent fusion protein which is directly detectable without requiring the addition of exogenous factors is preferred for detecting or assessing gene expression or location. For example, by utilizing GFP -tagged El, fluorescence is observed upon exposure to light at 489 nm without the addition of a substrate. In the absence of inhibitors, essentially all El protein will be nuclear. The nuclei can be marked with various DNA intercalating stains such as DAPI and various Hoechst dyes. If the nuclear dye (blue, in the case of DAPI and red in the case of Hoechst) is overlaid with green (from GFP-El), then there are comparable signals in both color channels when viewed by charge-cooled device (CCD) digital cameras or comparable recording instrumentation. Conversely, if the presence of the test compound results in the largely or completely

54

protein, then the nuclear dye (eg. blue or red) gives a relatively pure to pure signal, without GFP overlay. Initial lead compounds may generate a mixed pattern with El in both compartments. Further modifications of the lead compounds can result in an largely or exclusive cytoplasmic GFP-El . One skilled in the art will appreciate that it is desirable to screen test compounds using several different concentrations of the test compound. Computer-based image analysis software is known to those of skill in the art. El can also be fused to other fluorescence proteins, such as cyan fluorescent protein (CFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). Other examples include the green fluorescent protein from Aequorea coerelescens (AcGFP), DsRedExpress, and red coral fluorescent proteins (for example, AmCyan, ZsGreen, Zs Yellow, AsRed2, DsRed2, and HcRedl). For example, fluorescent proteins can be isolated from many different species, including but not limited to, Aequorea victoria (Chalfie, et al., 1994), Zoanthus species (Matz, et al., 1999), and Renilla reniformis (Ward and Cormier, 1979). High throughput screening methods can be used with the screening methods disclosed herein. Several commercial luminescence and fluorescence detectors are available that can simultaneously inject liquid into single or multiple wells such as the WALLAC VICTOR2 (single well), MICROBETA RTM JET (six wells), or AURORA VIPR (eight wells). Typically, these instruments require 12 to 96 minutes to read a 96-well plate in flash luminescence or fluorescence mode (1 min/well). An alternative method is to inject the test substance into all sample wells at the same time and measure the luminescence in the whole plate by imaging with a CCD camera, similar to the way that calcium responses are read by calcium-sensitive fluorescent dyes in the FLIPR or FLIPR-384 instruments. Other luminescence or fluorescence imaging systems include LEADSEEKER from AMERSHAM, the WALLAC VIEWLUX TM ultraHTS microplate imager, and the MOLECULAR DEVICES CLIPR imager. PE BIOSYSTEMS TROPIX produces a CCD-based luminometer, the NORTHSTAR TM HTS Workstation. This instrument is able to rapidly dispense liquid into 96-well or 384-well microtiter plates by an external 8 or 16-head dispenser and then can quickly transfer the plate to a CCD camera that images the whole plate. The total time for dispensing liquid into a plate and transferring it into the reader is about 10 seconds. Other

55 "^'system's ari'&Φerøds f rϊiigli th ughput screening are known in the art and contemplated herein. Also disclosed are agents identified by the screening methods disclosed herein. The agent can follow the guidelines of "Lipinski's Rule of Five." (Lipinski, 1997). Lipinski's Rule of Five is particularly useful when the goals of compound design are (i) to have less than 5 hydrogen donors, (ii) less than 10 hydrogen bond acceptors, (iii) molecular weight of less than 500 Daltons and (iv) the log of the partition coefficient, P (where P = the concentration of the compound in water divided by the concentration of the compound in 1 octanol) is less than 5. The Lipinski Rule of Five is a useful guideline, however, the composition is not limited to these parameters. A wide variety of small molecular weight compounds can be used in the screening methods disclosed herein. Such compounds include, but are not limited to, any compositions which are being tested for drug discovery or development. Such compounds include, but are not limited to, nucleic acids including functional nucleic acids, amino acids including peptides and proteins and fragments thereof, and various other chemical compounds. Compounds can be aqueous- or lipid-soluble. Compounds can be dissolved or suspended within solution, or affixed to a solid-support. Solid supports may include, but are not limited to, insoluble polymer beads or a polymeric matrix coated with one or a plurality of individual compounds, or with combinatorial chemistries. Dosages and volumes which are administered in the screening methods can be varied so as to optimize dosages for further studies or to rank compounds as to their toxicity and/or potency. Information resulting from variations in conditions can be used to prioritize chemicals for further study, to delineate the relative toxicities of structurally related chemicals, and/or to identify the proper dose range for subsequent toxicity studies (see e.g., Harris, et al., Fundam. Appl. Toxicol. 19:186-196). Most importantly, the known structures of lead compounds can then be varied through (prior or subsequent) combinatorial chemistry to work toward more favorable properties (eg. higher efficacy and specificity, lower toxicity, desired solubilities, optimized bioavailabilities). With respect to the generation of small molecular weight compound libraries, the combination of biochemical diversity is often synergistic with the metabolic diversity obtained from the in vivo production of "natural products". Collections of starting compounds, for example small molecule chemicals, can be administered to cultures of microorganisms. In accord, each microbial strain may potentially create numerous modified

56 ''^"small mdle' &'fcften ic ,'', "thus "generating a "metabolite library". Because each of these aforementioned cultures can contain a very complex mixture of metabolites, a highly efficacious method of screening is required (i.e., high throughput screening). An aliquot of the library is incubated with the cell lines induced for GFP-El protein expression, and the nucleo-cytopolasmic localization of the GFP-El protein will be recorded utilizing an HTS- based assay. Furthermore, natural product diversity can be screened by creating a mixture of combinatorially-tagged liposomes; wherein each liposome preferably encapsulates only one member or a simple mixture of a natural product compound library. Vectors and Cells Disclosed is an expression vector comprising SEQ ID NO: 1, or a fragment or variant thereof, operatively linked to a tag. Also disclosed is an expression vector comprising SEQ ID > NO: 2, or a fragment or variant thereof, operatively linked to a tag. Also disclosed are cells and cell lines comprising the vectors described herein, which can be used with the screening methods disclosed herein. The tag can be a fluorescent tag, such as GFP or RFP, for example, as described above. Also disclosed is a plurality of cells stably transfected with the vectors described herein, wherein the expression of a polypeptide encoded by the vector is inducible. At least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells can be induced to express the polypeptide. For example, contemplated is a green fluorescent protein chimeric tag fused to the amino-terminus of an El protein. The chimeric protein is active in transient replication assays, and the GFP moiety does not materially affect helicase location or function. Expression of the GFP-tagged helicase can be induced in all cells in a population. This was achieved in two stages: (1) creating a mutant version of El in which the dominant splice donor site (surrounding nucleotide 847) was mutated to curtail splicing of the primary transcript, and thereby to dramatically increase the translation of the full-length El helicase protein; and (2) driving the expression of GFP-tagged El helicase from a tetracyclin- inducible promoter. Cell lines were created to contain an integrated cassette consisting of a regulatable Tet-on promoter driving expression of the GFP-El. The GFP-1 lEldm open reading frame (ORF) was inserted into the pcDNA4/TO vector and linearized by Seal restriction endonuclease digestion. The linearized dsDNA was transfected by lipofectamine 2000 into the T-REx-293 or T-REx-COS 7 cell line which

57 ^ii"*cⁱbhtaihs'me"Te1 repress r^"frorh^'pcDNA6/TR. Cells were selected by the antibiotic Zeocin, and single colonies were picked up and tested for their characteristics. El helicase is maximally induced by adding as little as 62.5 nanograms/ml of tetracycline to the cell culture media (Figure 23). Lower concentrations may also be used. The induction can be sustained for at least 22 hours of continual tet-treatment (Figure 24). Pulsed, cyclical treatments can also be used. GFP-El helicase was induced in all cells, and it was nuclear, providing a highly controlled, robust means for expression of fluorescence- tagged El helicase. These tagged El proteins can also be made into vectors containing mutations, including but not limited to those in the various regulatory motifs described herein. These serve as both controls for cytoplasmic El localization in response to potential inhibitory drugs. Organotypic epithelial keratinocyte raft cultures are also contemplated (Wilson et al. Cell Growth and Differentiation 3: 471-483; Dollard et al. Genes Development 6: 1131- 1142; Chow et al. Clinics Dermatol. 15: 217-227). Complete stratification and differentiation of primary human keratinocytes has been achieved using these rafts. Preparation of organotypic raft cultures comprised of cells with induced (or also transient) expression of tagged El protein enables the development of stratified epithelial (eg. mucosal or cutaneous) skin equivalents with non-cycling cells in which the papillomaviral El helicase can be induced in the organotypic context, and that anti-El drug validation (specificity, selectivity, bioavailability, persistence, and (lack of) toxicity) can include such an organotypic testing environment. The raft cultures enables ex vivo validation of efficacy. Kits Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include El, an attenuated virus, and a suitable container. The kit can also include combinatorial libraries of small molecules. The kit can also include a screening platform, such as well plates for screening molecules. The present invention is more particularly described in the following examples, which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

58 ⁱr- ^{€,„■ ιi} .'^• Tlrr uinbuf this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

EXAMPLES Example 1: Methods For the examples, specific experiments conducted with HPV-11 and its El DNA helicase are described. The very high conservation of sequence motifs among HPV El proteins shows that all types have similar control over cytoplasmic-nuclear translocation and retention. Assays described and drugs eventually identified based on HPV-11 are fully applicable to the other HPV genotypes. Plasmids and antibodies. The HPV-11 EE-El and E2 expression plasmids pMTX- EE-E1, pMT2-E2, and origin-containing plasmid p7730-99 have been described (Chiang et al. Proc. Natl. Acad. Sci. USA 89:5799-5803 (1992), Kuo et al. (1994)). The NES mutation, the phosphorylation site mutations, and the cyclin binding motif mutations were each constructed in the context of pGFP-1 IE ldm, which encodes a GFP-El fusion protein and is mutated at the dominate splice donor site at nt 847 without affecting the encoded amino acids (18). pMTX-EE-Eldm was derived from pMTX-EE-El by incorporating the Eldm mutation just described. pMTX EE-El S107A and pMTX EE-El S 107A-NEm were

59 ^rtfeι pf ep eϊ! m'pMTX-FiE^'-El"drn. Oligonucleotide-directed mutagenesis was performed using standard PCR method. All PCR-amplified fragments were confirmed by DNA sequencing. The anti-E2 polyclonal antibody and the anti-EE monoclonal antibody has been described previously (Chiang et al. (1992), Grussenmeyer et al. Proc. Natl. Acad. Sci. U S A. 82:7952-7954 (1985), Kuo et al. (1994)). For immunoprecipitation, EE was crosslinked to CNBr-activated Sepharose 4 FastFlow beads (Amersham Biosciences Corp., Piscataway, NJ) according to manufacturer's instruction. Phospho-Serine/Threonine-Proline specific monoclonal antibody MPM-2 was purchased from Upstate (Lake Placid, NY), the anti-GFP monoclonal antibody JL-8 and polyclonal antibody from Clontech Laboratories (Palo Alto, Ca), anti-mouse IgG-FITC conjugate from Molecular Probes (Eugene, OR) and anti-rabbit IgG-Texas Red conjugate from Vector Laboratories (Burlingame, CA). Cell culture, transfection, and drug treatment. COS 7, 293 and p21-9 ( Chang et al. Proc. Natl. Acad. Sci. USA 97:4291-4296 (2000) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO₂. Cells were transfected by electroporation as described( Chiang et al. (1992), Zou et al. J. Virol. 74:3761-3770 (2000)). Cells were treated with roscovitine (Sigma Co., St. Louis, MO) at 20 μM 4 hrs post-transfection and cultured for another 20 hrs. For p21cip induction, isopropyl-β-thio-galactosidase (IPTG) (Invitrogen Life Technologies, Carlsbad, CA) was added to the culture medium to a final concentration of 50 μM 4 hrs post- transfection and maintained for 20 hrs. Leptomycin B (LMB) (Sigma) at 5 μg/ml was added to the culture medium to a final concentration of 20 ng/ml 24 hrs post-transfection. Cells were then cultured for 6 more hrs for fluorescence microscopy. Fluorescence microscopy. After electroporation, lxlO⁵ cells were grown on 2-well chamber slides (Nalge Nunc International, Naperville, IL), treated with the chemicals as indicated above. Cells were then fixed at the indicated time point with 4% paraformaldehyde. For GFP-El observation, slides were directly overlaid with coverslips using mounting media with DAPI (4',6-diamidino-2-phenylindole) (Vector Laboratories, Inc., Burlingame, CA). Immunostaining of EE-El was performed using monoclonal anti- EE antibody followed with anti-mouse IgG-FITC conjugate. Immunostaining of E2 was performed using polyclonal anti-E2 antibody followed with anti-rabbit IgG-Texas Red conjugate. These slides were mounted with DAPI-containing media. Images were

60 '^"c ϊf)tured'^!a;nd" p' dcessed^' with ah Olympus AX70 fluorescence microscope with FITC/DAPI filters using an AxioCam digital camera (Carl Zeiss, Inc., Germany). Protein purification, immunoprecipitation, and protein phosphatase treatment. Purification of E. co//-expressed EE-El and Sf9-expressed EE-El protein were described ( Kuo et al. (1994), Ma et al. (1999)). For EE-El immunoprecipitation, 5xl0⁷ COS7 cells or 1.5x10⁸293 cells transfected with pMTX-EE-Eldm plasmid were lysed in 5 ml NETN buffer (20mM Tris, PH 8.0, lOOmM NaCl, ImM EDTA, 5mM NaF, 0.5% NP-40) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% of Protease Inhibitor Cocktail (Sigma). Lysates were mixed with 200 μl Sepharose 4 beads to which anti-EE antibody was coupled at 4°C overnight, then extensively washed five times with NETN buffer. Proteins were eluted with 400 μl of 0.1 M glycine buffer (PH 3.0) and neutralized with 4 μl of 1.5 M Tris buffer (PH 9.0). The final solution was concentrated using Nanosep Centrifugal columns (Pall Co., Ann Arbor, MI). The concentrations of EE-El proteins recovered were determined by western blotting with anti-EE antibody using purified EE-El as standards. 50 ng each were used for subsequent analysis. For phosphatase 1 treatment, 2 μl of purified or immunoprecipitated EE-El (50 ng) was mixed with 1 unit of protein phosphatase 1 catalytic subunit (Sigma) in reaction buffer (50 mM Tris, pH 7.5, O.lmM EDTA, ImM MnCl₂) to a final volume of 10 μl. The mixture was incubated at 30°C for an hour. All samples were resolved by electrophoresis through SDS- 8% polyacrylamide gels, followed by immunoblotting with individual antibodies and detected by enhanced chemiluminescence (ECL) reagents (Amersham). The MPM-2 antibody was used to detect phosphorylated protein. The membranes were stripped and reprobed with monoclonal anti-EE antibody. GFP-El immunoprecipitation from transfected COS7 cells was performed similarly except a polyclonal anti-GFP antibody was used to recover the fusion protein and bound protein was eluted from protein A-Sepharose beads (Amersham) with 2 x SDS-PAGE sample buffer and subject to immunoblotting. MPM-2 was used to detect phosphorylated protein. The membranes were stripped and re-probed with monoclonal anti-GFP antibody (JL-8). Transient replication assay. Transient replication assays were performed using 293 cells as described (10, 18, 84). In each assay, 0.5 μg of GFP-El expression plasmid, 5 μg of pMT2-E2 plasmid, 0.5 μg of HPV-11 origin-containing plasmid p7730-99 were cotransfected into 5x10⁶ cells by electroporation. Cells were harvested 48 hrs post- 61 ir^hs cti H^!'iM^;tew-ffi l cular-weight DNA was isolated. One half of the recovered DNA was digested with Hind III to linearize the ori plasmid. The other half was digested together with Dpn I, which cut the unreplicated input plasmids into small fragments. Standard Southern blotting was then performed using ²P-α-dCTP labeled probes from the ori plasmid p7730-99. The results were analyzed by Phosphorlmager (Molecular Dynamics Inc., Sunnyvale, CA). Example 2: El is phosphorylated by CDKs in vivo. A predominant intragenic splice occurs within the HPV-11 El transcript, abolishing El expression. Consequently, pGFP-El, which encodes the green fluorescence protein (GFP) fused to the N-terminus of the El sequence, expresses primarily a pancellular GFP variant of slightly higher molecular mass ( Deng et al. J. Virol. 77:10213-10226 (2003)). A mutation at the donor splice site at nt 847 (Eldm), which does not alter the encoded amino acids, abrogates this splice and pGFP-Eldm encodes abundant, replication-competent GFP- El protein ( Deng et al. (2003)). Thus, this mutation not only increases the quantity of the El protein expressed, it also allows the subcellular localization of the GFP-El protein to be followed. All El expression vectors described herein contain this donor site mutation. HPV- 11 El is phosphorylated by multiple cyclin/CDK complexes in vitro, except cyclin D/CDK4 (43). To assess whether the El protein is also phosphorylated in vivo, HPV-11 El tagged at its amino terminus with glu-rich (EE) epitope was expressed in insect Sf9 cells, monkey COS7 cells and human 293 cells. The EE-El functions as a potent DNA helicase in vitro and is replication competent in transient replication and cell-free replication assays ( Kuo et al. (1994), Lin et al. (2002), Ma et al. (1999)). The protein was purified from Sf9 cells as described ( Kuo et al. (1994), Lin et al. (2002), Liu et al. (1995), Liu et al. (1998)), or immunoprecipitated from transfected mammalian cells by using , Sepharose beads conjugated to monoclonal antibody to the EE epitope. The recovered protein was then analyzed by SDS-PAGE and western blotted with an antibody specific for phospho-Ser/Thr-Pro resulting from phosphorylation by CDK kinases. Of several such antibodies tested, only MPM-2 specifically recognizes EE-El . Fig. IB shows that this antibody detected EE-El protein expressed in insect and mammalian cells (lanes 3, 5, 8). However, if the EE-El was first treated with protein phosphatase 1 (PP1), a serine/threonine phosphatase, reactivity with the MPM-2 antibody was abolished (Fig. IB, lane 4, 6, 9) while reactivity with the anti-EE antibody was unchanged (Fig. IB, lanes 3-6,

62 8, 9). EE-El purified from E. coli was only detected by the anti-EE antibody, not by MPM-2 antibody with or without PP1 treatment (Fig. IB, lane 1 and 2). These results demonstrate that the HPV-11 El is indeed phosphorylated in eukaryotic cells. To substantiate this conclusion, COS7 cells expressing EE-El were treated for 20 hours with 20 μM roscovitine. Roscovitine is a purine derivative that reversibly competes for ATP binding and specifically inhibits the activity of CDK 1 and CDK2, but has no effect on CDK4 and CDK6 ( De Azevedo et al. Eur. J. Biochem. 243:518-526 (1997), Meijer et al. Eur. J. Biochem. 243:527-536 (1997)), nor on the MAP kinases ( Bain et al. Biochem. J. 371:199-204 (2003)), which have the same consensus substrate sites as CDKs. Upon treatment with roscovitine, the ability to detect EE-El with the MPM-2 antibody was abolished (Fig. IB, lane 7). Thus, the HPV El protein is phosphorylated in vivo by one or more CDK complexes on the consensus CDK substrate site(s). Mutational analyses described below showed that CDK phosphorylation substrates in HPV-11 El helicase included S89, S93, and SI 07. Example 3: Single mutations in S89, S93 or SI 07 significantly reduce nuclear localization. Mutations at four CDK phosphorylation sites abrogated El function to support viral origin DNA replication in vivo ( Ma et al. (1999)). In the majority of the transfected cells, GFP-El was restricted to the nucleus, whereas a small percentage of the cells exhibit cytoplasmic or pancellular signals ( Deng et al. (2003)). To understand the mechanism by which CDK phosphorylation regulates the El replication activity, it was determined whether phosphorylation could affect its subcellular localization. The subcellular localization of GFP-El fusion protein was tracked after the incorporation of single or multiple Ser to Ala mutations at the candidate CDK phosphorylation sites. All direct fluorescence microscopy of GFP-El or mutations was conducted in monkey kidney epithelial cell line COS7 cells and in human kidney epithelial cell line 293 cells. The results were identical. By fluorescence microscopy, the majority of cells expressing the S89A, S93A, or S107A single mutations exhibited cyctoplasmic GFP-El (Fig. IC). S107A exhibited the most severe defect in El nuclear localization. More than 80% of the cells with GFP-El S107A contained only cytoplasmic signals, whereas in S89A and S93A expressing cells, about 60-70% had exclusivly cytoplasmic signals. In the remaining cells for all three

63 mutations, there was some nuclear signal in addition to a dominant cytoplasmic signal. With the three double mutations S89,93A, S89,107A, or S93,107A, more than 90% of the transfected cells had exclusively cytoplasmic GFP-El protein (Fig. IC). When all three serine residues or all four CDK substrates were mutated, as in S 89,93, 107A and S89,93,107,T468A, all the GFP-El protein was restricted to the cytoplasm (Fig. IC). These , observations show that CDK phosphorylation on S89, S93 and SI 07 are all required for effective retention of El in the nucleus. Example 4: Lack of phosphorylation by cyclin/CDK leads to cytoplasmic El. El mutations unable to bind cyclins and are no longer able to be phosphorylated by CDK complexes should exhibit a phenotype similar to the phosphorylation site mutations. To demonstrate this, GFP-El mutated in the consensus cyclin binding motif RxL ( Ma et al. (1999)) was constructed and tested. The RRL sequence was mutated to RRA, KRA or ARA (Fig. 2A). The ARA mutation has previously been shown to disrupt the binding to cyclins (43). HPV-11 El protein has a bipartite nuclear localization sequence (NLS), which partially overlap the cyclin binding motif, and mutation of the RR residues decreased but did not abolish El nuclear localization (see Fig. 2A). All three mutations were localized to the cytoplasm (Fig. 2A, left panel). Western blots with the phospho-S/TP- specific antibody MPM-2 failed to detect the GFP-El RRA, although the protein was detected at a similar level as the wild type protein by using an anti-GFP antibody (Fig. 2A, right panel, compare lanes 2 and 3). In contrast, the wild type GFP-El, which is primarily nuclear (Fig. IC), was detected by the MPM-2 and GFP antibodies, consistent with the observation with the EE-El protein (Fig. IB). In addition, the GFP-El S89,93,107A,T468A protein, which is exclusively cytoplasmic (Fig. IC), did not produce a detectable signal when probed with the MPM-2 antibody (Fig. 2 A, right panel, lane 1). Collectively, these results show that El phosphorylation at S89, S93 and SI 07 is required for efficient nuclear localization. Example 5: Inhibition of CDK activities results in cytoplasmic El protein. To verify the above interpretation further, COS7 cells transfected with GFP-El were treated with 20 μM roscovitine for 20 hours. The results showed that specific inhibition of CDK activities resulted in a GFP-El which was no longer detectable with the MPM-2 antibody (Fig. 2B, right panel). On the cellular level, the GFP-El protein was observed exclusively in the cytoplasm in treated cultures (Fig. 2B, left panel, compared

64 with Fig. IC). Similarly, roscovitine treatment of transfected cells also resulted in the cytoplasmic relocalization of the GFP-El T468A. In another experiment, elevated p21cipl expression was used to inhibit CDK activity. p21cipl is a potent inhibitor of cyclin E/CDK2 and cyclin A/CDK2 complexes that regulate Gl/S phase transition and also plays a key role in Gl/S or G2 checkpoint after DNA damage ( Abraham, R. T. Genes Dev. 15:2177-2196 (2001), Sherr et al. (1999), Sun et . JGen Virol 79:1651-1658 (1998)). GFP-El expression vectors were transfected into the p21-9 cell line. This cell line is derived from HT1080 human fibrosarcoma cells with an inducible p21cipl gene under the control of the bacteria lac I repressor ( Chang et al. (2000)). Maximal induction of the p21cipl protein was achieved by adding isopropyl-β- thio-galactosidase (IPTG) to the medium at 50 μM for' 14 to 24 hrs ( Chang et al. (2000)). In the absence of IPTG, the GFP-El protein was primarily nuclear, as in COS7 or 293 cells. Upon induction with IPTG, GFP-El was observed in the cytoplasm in the majority of the cells (Fig. 2C). Collectively, these results show that phosphorylation of El by cyclin/CDK complexes, including CDK2, is required for El nuclear localization. Example 6: Mutations in El phosphorylation sites promote CRMl mediated nuclear export. To determine whether phosphorylation affected El nuclear import or nuclear export, cells were treated with leptomycin B (LMB). LMB is a potent antifungal antibiotic which binds to the nuclear export receptor CRMl in a highly specific manner and inhibits nuclear export ( Kudo et al. Proc. Natl. Acad. Sci. USA 96:9112-9117 (1999), Nishi et al. J. Biol. Chem. 269:6320-6324 (1994)). COS7 cells expressing wild type GFP- El proteins were treated with 20 ng/ml LMB for 6 hours (Fig. 2D). Virtually all cells were observed to have the GFP-El protein in the nucleus. In untreated cells, about 20-30% contained some cytoplasmic signal (Deng et al., 2003). In the presence of LMB, GFP-El S107A, GFP-El S89,93,107A, and GFP-El L126A (in the RxL motif) were also observed in the nucleus, in contrast to their original, primarily cytoplasmic distribution in the absence of LMB (compare Fig. 2D with Fig. IC and 2A). Prolonged treatment with LMB increased nuclear localization and all the protein was localized to the nucleus at 11 hrs.. Similarly, all other El phosphorylation site mutations were also found in the nucleus upon LMB treatment. Collectively, these results demonstrate that cytoplasmic distribution for each of these El mutations was caused by the rapid, continuous export from nucleus mediated by CRMl and that the dominant, CRMl -dependent nuclear export is inactivated

65 by phosphoryation by cyclin/CDK complexes. These observations also indicate that neither the loss of phosphorylation by CDK nor the mutation in the overlapping bipartite NLS and consensus cyclin binding motif (RRL) is able to abolish its nuclear entry completely. Example 7: A conserved, putative leucine-rich NES controls El export. CRMl binds specifically to proteins with a leucine-rich NES and, in a complex with RanGTP, and transports the cargo out of the nucleus ( Stade et al. (1997)). Leucine- rich NES is a short peptide sufficient to promote rapid protein export from the nucleus ( Fischer et al. (1995), Wen et al. (1995)). It has a core sequence of about 10 residues that are highly enriched with large hydrophobic residues such as leucine, isoleucine and valine. A consensus sequence for the core peptide has been proposed to be L-X(2-3)-L/I/V/F/M- X(2-3)-L-X-L/I (SEQ ID NO: 16). Upon inspecting the whole El sequence with special attention to the region close to the three CDK substrates, one region (residues 83-126, SEQ ID NO: 2) was identified which is rich in leucine, isoleucine or valine residues (Fig. 3A, green box), including a putative NES core consensus sequence (green underline), ISPRLDAIKL (residues 106 to 115, SEQ ID NO: 14). To test whether this region might be an NES, two hydrophobic residues (LI 10 and II 13, SEQ ID NO: 9 and 10) were mutated in this apparent core sequence to alanine substitutions (designated NEm) in the wild type GFP-El and a number of CDK phosphorylation site mutations. These GFP-El mutations were transfected into COS7 cells. The nuclear localization of S89A-NEm, S93A-NEm, and S89,93,107A-NEm were restored to different extent. In contrast, the wild type El -NEm and SA107A-NEm were both exclusively nuclear (Fig. 3B, compared with Fig. IB). The dramatic difference between S107A and S107A-NEm, compared with the other phosphorylation sites mutations and the corresponding NEm, demonstrates that the primary function of phosphorylation of SI 07 is to inactivate the NES. Example 8: CDK phosphorylation inactivates the NES of the El protein. To substantiate the hypothesis that one of the critical roles of CDK phosphorylation is to inactivate the NES, the NEm was also introduced into the GFP-El LI 26 A mutated in the cyclin binding motif. GFP-El LI 26 A was cytoplasmic (Fig. 2 A) and was not detected by the MPM-2 antibody in western blots (Fig. 2A). The introduction of the NEm did not restore CDK phosphorylation, as the protein did not react with the MPM-2 antibody (Fig. 2 A, right panel, lanes 3, 4). Nevertheless, the GFP-El RRA-NEm protein was primarily

66 located in the nucleus (Fig. 3B). Thus, this observation shows that the LI 10AX113A mutation inactivates the NES and that the NES mutation can at least partially substitute for the requirement for CDK phosphorylation for El nuclear retention. Cells transfected with the NEm mutations were also treated with roscovitine. This CDK inhibitor caused the wild type El protein to redistribute to the cytoplasm (Fig. 2B), but it had no effect on the subcellular localization of any of the NES mutations. These results show that the NES is effectively inactivated by the LI 10A 113A mutation and hence CDK phosphorylation is no longer necessary for its inactivation. Collectively, these results show that the leucine- rich peptide spanning residues 106-115 (SEQ ID NO: 14) functions as an NES and one of the functions of CDK complexes is to inactivate it by phosphorylation. The above localization experiments were conducted by direct observation of GFP- tagged El protein. This protein is fully functional in supporting transient replication ( Deng et al. (2003)). To verify that these observations are not confounded by the GFP moiety, three EE-tagged El or mutations were expressed in COS7 cells. Indirect immunofluoescent staining of EE-El, EE-El SI 07 A, EE-El S107A-NEm using antibody against the EE-epitope showed that each of the protein exhibited exactly the same subcellular localization patterns as the corresponding GFP-El counterparts (Fig. 3C, compare with Figs. IC and 3B). EE-El was observed in the nucleus in most of the cells. The phosphorylation site mutation SI 07 A was primarily in the cytoplasm. The double mutation S107A-NEm was localized entirely in the nucleus. Thus, through observations made on both GFP-tagged and EE-tagged El, it was demonstrated that El phosphorylation by CDKs determines its nuclear localization through the disruption of a leucine-rich NES. Example ?: Delineation of a bonaβde leucine-rich NES sufficient for protein export. To delineate the NES of the El protein, a series of short peptides spanning the 10 amino acid NES core (106-115, SEQ ID NO: 14) were fused to the C-terminus of the GFP protein. The region between residues 86 and 119 was purposefully limited to circumvent any complications which might result from the NLS or from phosphorylation by cyclin/CDK, the association of which depends on the consensus cyclin binding motif (residues 124-126, SEQ ID NO: 6). As shown in Fig. 3D, because of it small size, GFP alone was observed both in the nucleus and in the cytoplasm. The localization of the fusion proteins showed that each of the El peptides spanning residues 96-115 (SEQ ID NO: 15)

67 conferred an exclusive cytoplasmic localization to GFP. Each of these peptides contains the 10-amino acid long core sequence (residues 106-115, SEQ ID NO: 14) and also includes two additional valine residues (Fig. 3 A). In contrast, the 10 amino-acid core sequence was not sufficient (Fig. 3D). Thus a bonaβde leucine-rich NES in HPV-11 El protein between residues 96 and 115 was defined and determined to be necessary and sufficient for export from the cell nucleus. Example 10: El replication activity correlates with its subcellular localization. If one of the functions of El phosphorylation is to determine its nuclear localization, this would explain why the El S89,93,107,T468A mutation supported virus origin DNA replication in vivo extremely poorly, if at all ( Ma et al. (1999)). A prediction of this conclusion is that, if nuclear export could be abolished, a recovery of the replication activities of the phosphorylation site mutations should be observed. Therefore, transient replication assays were performed (Fig. 4). 293 cells were co-transfected with 0.5 μg of an El expression plasmid, 5 μg of pMT2-E2 expression plasmid and 0.5 μg of a HPV origin- containing plasmid. The extent of the viral origin DNA replication was evaluated by southern blotting of the newly synthesized origin plasmid (Dpn I + lanes) 48 hr post transfection. A subset of the GFP-El phosphorylation site mutations and their corresponding NES mutations (NEm) were tested. Fig. 4A is an autoradiogram of one of several experiments. The signals were quantified in Fig. 4B to obtain the relative replication activities. The data show that El proteins mutated at phosphorylation site(s) or cyclin binding motif had much reduced activities to support virus DNA replication (Fig. 4A, upper panel). The extent of decrease correlated largely with the percentages of cells containing primarily cytoplasmic El protein (see Fig. IC). When nuclear localization of these mutations was restored through mutations in the NES (Fig. 3B), their abilities to support replication were increased, although to different extent (Fig. 4A, lower panel). These experiments were conducted using different amounts of GFP-El plasmids and a similar tendency was observed. These results clearly demonstrate that cyclin/CDK phosphorylation inactivates El NES, promotes its nuclear retention, and consequently controls virus DNA replication. The results also show that the phosphorylation site mutations per se did not completely abrogate the ability of the El protein to function as a replicative helicase or to interact with the host proteins to initiate replication. However, mutations involving SI 07 appeared to have additional functional impairment, as its

68 replication activity was the lowest among all the mutations even when NEm was introduced (Fig. 4). Example 11: Altered subcellular localization of El S89,93,107A protein in the presence or absence of the E2 protein. To investigate the reason for the poor replication activity of E1S89,93,107, subcellular localization of the GFP-El wild type protein and GFP-El S89,93,107A was examined in the absence and in the presence of the native E2 in transfected COS cells. E2 was revealed by indirect immunofluorescence detection with E2 antibody. In a great majority of the cells, the wild type GFP-El colocalized with E2 and formed small nuclear foci above diffuse El and E2 nuclear signal (Fig. 5 A, top panels). Only in less than 5% of the cells positive for nuclear E2, cytoplasmic GFP-El was observed (Fig. 5A, lower cell in bottom panels). In the absence of E2, up to 20-30%) of the cells exhibited some cytoplasmic signals. Thus, under the condition of high levels of protein expression, E2 was able to increase the percentage of cells with nuclear wild type GFP-El protein. In contrast, in the majority of cells expressing GFP-El S 89,93, 107 A and the native E2 protein, the GFP-El protein remained cytoplasmic (Fig. 5B, top panels). These results show that the E2 fail to overcome effectively the powerful NES in El to prevent the nuclear export of this El phosphorylation mutation. In addition, this mutation might have other defects. This is inferred from the pattern of El distribution in the remaining cells in which GFP-El was observed in the nucleus or in both compartments. When GFP-El S89,93,107A was observed in the nucleus, the two proteins colocalized in dots much brighter and larger than the foci formed by wild type El and E2 proteins. In addition, there was very little diffuse GFP-El remaining in the nucleoplasm (compare Fig. 5 A top panels with Fig. 5B bottom panels). These observations indicate that in a low percentage of cells, this mutated El was able to interact with E2 and, in doing so, almost all of the GFP-El and most of the E2 protein were sequestered into these dots (Fig .5). Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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77

Claims

What is claimed is:

1. A method of inhibiting papillomavirus replication in a subject comprising the steps of: a. identifying a subject with, or at risk of contracting, papillomavirus; and b. contacting the subject with a substance that reduces nuclear localization of El, thereby inhibiting papillomavirus replication.

2. The method of claim 1, wherein the substance reduces nuclear localization by reducing the interaction of El with importin-α.

3. The method of claim 1, wherein the substance reduces nuclear localization by inhibiting cyclin/cdk2 maintenance of phosphorylation.

4. The method of claim 3, wherein the substance is roscovitine.

5. The method of claim 1 , wherein the substance reduces nuclear localization by stimulating CRMl.

6. The method of claim 1 , wherein the substance reduces nuclear localization by increasing p21cipl or p27kipl.

7. The method of claim 8, wherein the substance increases p21cipl by inhibiting 26S proteasome.

8. The method of claim 7, wherein the substance is lactacystin.

9. A method of screening for an agent that inhibits nuclear localization of El , comprising the steps of: a. transfecting a cell with a tagged El protein, fragment, or mutant thereof; b. contacting the cell with the agent to be screened; c. inducing expression of the tagged El protein, fragment, or mutant thereof; and d. detecting a cellular location of the tagged El protein, fragment, or mutant thereof wherein El's absence in the nucleus or presence in the cytoplasm indicating an agent that inhibits nuclear localization of El.

10. The method of claim 9, wherein tagged E 1 protein is SEQ ID NO : 1. ^

11. The method of claim 9, wherein the tagged El protein is SEQ ID NO: 2.

12. The method of claim 9, wherein the tag is a fluorescent tag.

13. The method of claim 12 , wherein the tag is GFP .

14. The method of claim 12, wherein the tag is RFP.

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15. The method of claim 9, wherein the method of screening comprises using a high throughput screen.

16. An agent identified by the method of claim 9.

17. An expression vector comprising SEQ ID NO: 3, or a fragment thereof, operatively linked to a tag.

18. An isolated cell comprising the expression vector of claim 17.

19. A cell line comprising the vector of claim 17.

20. The vector of claim 17, wherein the tag is a fluorescent tag.

21. The vector of claim 20, wherein the fluorescent tag is GFP.

22. The vector of claim 20, wherein the fluorescent tag is RFP.

23. An expression vector comprising SEQ ID NO: 4, or a fragment thereof, operatively linked to a tag.

24. An isolated cell comprising the expression vector of claim 23.

25. A cell line comprising the vector of claim 23.

26. The vector of claim 23, wherein the tag is a fluorescent tag.

27. The vector of claim 26, wherein the fluorescent tag is GFP.

28. The vector of claim 26, wherein the fluorescent tag is RFP.

29. A plurality of cells stably transfected with the vector of claim 25, wherein the expression of a polypeptide encoded by the vector is inducible.

30. The cells of claim 25, wherein at least about 80% of the cells are induced to express the polypeptide.

31. The cells of claims 25, wherein at least about 90% of the cells are induced to express the polypeptide.

32. The cells of claims 27, wherein at least about 95% of the cells are induced to express the polypeptide.

33. The cells of claims 25, wherein at least about 98% of the cells are induced to express the polypeptide.

34. A non-naturally occurring polypeptide comprising SEQ ID NO: 2.

35. A non-naturally occurring polypeptide comprising SEQ ID NO: 5.

36. A non-naturally occurring polypeptide comprising SEQ ID NO: 9.

37. A non-naturally occurring polypeptide comprising SEQ ID NO: 10.

38. A non-naturally occurring polypeptide comprising SEQ ID NO: 2.

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39. The polypeptide of claim 38, wherein serine at amino acid 89 is mutated.

40. The polypeptide of claim 38, wherein serine at amino acid 93 is mutated.

41. The polypeptide of claim 38, wherein serine at amino acid 107 is mutated.

42. The polypeptide of any one of claims 39-41 , wherein alanine is substituted for serine.

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