EP1297126A1 - Nucleotide and amino acid sequences of a cellular inhibitor of the dna replication in the epstein-barr herpes virus - Google Patents

Abstract

The invention relates to the identification of a cellular inhibitor of DNA replication in the Epstein-Barr herpes virus (EPV), called TB7. The invention further relates to the discovery of the nucleotide and amino acid sequences of TB7 and the use of TB7, in particular for the production of a pharmaceutical preparation for suppression of the multiplication of EBV in infected patients.

Description

 NUCLEOTIDE AND AMINO ACID SEQUENCES OF A CELLULAR REPRESSOR OF DNA REPLICATION OF HERPESVIRUS EPSTEIN-BARR

The present invention relates to the identification of a cellular repressor designated TB7, the DNA replication of the herpes virus Epstein-Barr (EBV), the elucidation of the nucleotide and amino acid sequences of TB7 and the use of TB7, in particular for the production of a pharmaceutical preparation, to suppress the proliferation of EBV in infected patients.

The Epstein-Barr virus

The Epstein-Barr virus (EBV) belongs to the family of herpesviridae, which are classified into three subfamilies (α-, ß- and γ-herpesviruses) based on their pathogenicity, the cell types they infect and their nutritional properties. The Epstein-Barr virus is classified in the subfamily of γ-herpes viruses, which are characterized by a very narrow host spectrum. A characteristic feature of all herpes viruses is their ability to persist in a latent state for the rest of their life after the primary infection in the infected host without the formation of infectious particles. adjourn. In this state, the genome is present in the cell as a histone-packed, episomal plasmid.

The primary infection with EBV usually occurs in early childhood via cells of the nasopharynx and proceeds without clinical symptoms. If contact with EBV occurs only in early adulthood or later, it is often associated with the clinical picture of infectious mononucleosis (IM). This acute phase of the infection is accompanied by a massive proliferation of the B lymphocytes, which leads to a strong T cell activation and thus to the clinical symptoms of IM. Most EBV-infected cells are usually eliminated very efficiently by the immune system, but all infected people have EBV-positive B-lymphocytes in their blood for life and some latently infected people excrete the virus via saliva. The B lymphocytes that remain in the body after infection with EBV contain the virus and have the ability to grow into immortalized cell lines in vitro. However, EBV can lead to fatal mononucleosis in patients with a weakened immune system or in connection with genetic defects. EBV is also associated with the development of tumors. For example, the EBV genome is found in at least three types of cancer in humans, Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal cancer (for an overview: Rickinson and Kieff, 1996).

The EBV genome consists of linear double-stranded DNA with a length of 172 kbp and codes for about 90 different proteins. It was the first herpesviral genome to be fully cloned and sequenced (Skare and Strominger, 1980; Baer et al, 1984). At the end of the genome there are approx. 500 bp long repetitive sequences (TR, "terminal repeats"), which occur in different numbers depending on the EBV strain. After infection of the host cell, the genome is circularized via these sequences (Kintner and Sugden, 1979).

The cycle of multiplication of the EBV, like that of all herpes viruses, runs in two phases. In the primary infection, the virus infects epithelial cells and B-lymphocytes, which are located in the mucous membranes of the nasopharynx. During this phase, the virus genome is replicated in most cells, which leads to a massive release of infectious virus particles and the death of the host cells. In contrast, in some of the infected B lymphocytes the latent phase is established, in which the viral genome as an extrachromosomal plasmid is synchronous with the Cell cycle is replicated and passed on to the daughter cells. The replication in these two phases is shown schematically in Figure 1.

The latent phase of the Epstein-Barr Nirus

After infection of B lymphocytes, the genome is circularized by fusion of the terminal sequences ("terminal repeats") and amplified via a mechanism that has not yet been clarified, so that between 2 and 250 copies per cell are present in the latent phase (Sugden et al ., 1979; Metzenberg, 1990). In this phase only a few viral genes are expressed which are required for the immortalization of the infected cells and for the stable transmission of the viral genome (Hammerschmidt and Sudgen, 1989; Tomkinson et al., 1993).

The replication of the EBN genome in the latent phase takes place at the cw-active origin of replication oriP, which is composed of two components for initiation (DS element) and termination (FR element) (Gähn and Schildkraut, 1989). The virally encoded protein EBΝA1 (EBN nuclear antigenl) binds to the FR element of oriP and, probably through interaction with chromatin, ensures the even distribution of the viral episome at the end of mitosis (Aiyar et al., 1998) (see Fig.1.1 B). No other viral factors are involved in replication in the latent phase. Like a chromosomal origin of replication, the EBV genome replicates exactly once with the cell cycle with the aid of cellular replication enzymes and is thus passed on stably to the daughter cells (Yates and Guan, 1991; Davenport and Pagano, 1999)

The lyrical phase of the Epstein-Barr Nirus

The lytic replication of the EBN takes place in vivo in epithelial cells of the pharynx and is important for the spread of the virus. The mechanisms that reactivate the lytic cycle in latently infected cells in vivo are not known. Since there is no natural cell culture system for the study of lytic replication in epithelial cells, the previous findings are limited to observations in latently infected immortalized B- Lymphocytes. However, activation of the lytic cycle is rather the exception in latent EBV-infected B lymphocytes.

Viral replication proteins

In contrast to replication in the latent phase, which is controlled by cellular replication enzymes, herpes viruses in the lytic phase provide most of the proteins required for replication themselves. The EBV genes responsible for lytic replication were identified in transient transfection experiments (Fixman et al., 1992) and are shown in Table 1.1. These six viral gene products, together with BZLF1, are sufficient for the replication of orzXyt plasmids. However, no typical initiation protein could be characterized which has helicase activity and specifically binds to the lytic origin of replication oriLyt, as described for herpes simplex virus 1 (UL9), papillomavirus (El) and SV40 (T-Ag) (Challberg, 1986; Hassel! And Brinton, 1996; Stenlund, 1996). The binding of BZLF1 to oriLyt is necessary for its activation, but BZLF1 has no intrinsic helicase activity.

Table 1.1: Lytic replication factors of the Epstein-Barr virus.

The lytic origin of replication oriLyt

In the lytic phase, the replication of the EBV genome takes place from the origin of replication oriLyt (Hammerschmidt and Sugden, 1988), which is not functional in the latent phase. OriLyt is a complex replication origin that contains both transcription and transcription elements Replication contains and its structure differs from all previously known eukaryotic origins of replication (Fig. 2). The central area of oriLyt contains two essential components which represent the minimal origin of replication and which, due to their location, are referred to as "upstream" and "downstream" components (Schepers et al., 1993). This central area is flanked by so-called replication enhancers, sequences that are important for lytic replication but not essential (Hammerschmidt and Sugden, 1988). Within the "upstream" component is the promoter of the BHLF1 gene, which is due to the binding of the viral

Transactivators BZLFl to neighboring ZRE binding sites (ZRE 1-4; ZRE: BZLFl responsive element) is activated directly. For the activation of oriLyt, the binding of BZLFl to the ZRE binding sites of the "upstream" component is absolutely necessary, the binding to ZRE 5, 6 and 7, which is located between the "downstream" component and the promoter of the BHRF1 gene , however, is unnecessary for lytic DNA replication (Schepers et al., 1996). Between the binding sites ZRE 5 and 6 there are also two binding sites for a further viral transactivator BRLF1 which, together with BZLF1, has a transactivating effect on the BHLF1 promoter (Gruffat et al., 1990; Giot et al., 1991).

The "downstream" component of oriLyt is approximately 530 bp from the "upstream" component. Mutation studies have shown that within this component a G / C-rich sequence section, the TD element, is absolutely necessary for lytic DNA replication and that point mutations already lead to a loss of replication efficiency in oriLyt (Schepers et al., 1993 ). Therefore, it is believed that this sequence is a platform for the specific binding of proteins involved in lytic replication. Binding of the viral transactivators BZLFl and BRLF1 does not take place, but several cellular protein complexes have been identified which bind to the TD element in a sequence-specific manner (Gruffat et al., 1995).

Regulation of lytic replication

The lytic cycle can be experimentally induced in some latently infected cells by treatment with chemical substances (zur Hausen et al., 1978). Already one hour after stimulation, the expression of very early ("immediate early") genes which code for the viral transactivators BZLFl and BRLF1 is observed (Takada and Ono, 1989; Sinclair et al, 1991). These very early gene products represent the first stage of cascading expression of more than 80 genes, with the transactivators of each stage activating the genes for the activators of the next stage until all genes important for virus production are finally expressed (Kenney et al., 1989; Holley et al, 1990). The lytic cycle can also be induced directly by transfection of BZLFl or BRLF1 in B lymphocytes (Countryman and Miller, 1985; Ragoczy et al., 1998) or in epithelial cells (Zalani et al, 1996).

BZLFl is a transactivator that has homologies to members of the AP-1 transcription factor family, such as c-Jun and c-Fos (Farrell et al., 1989). BZLFl binds as a homodimer to ZRE motifs in oriLyt and can also bind with high affinity to cellular or viral API or TRE (TPA responsive element) sequences (Urier et al., 1989). BRLF1 is also a sequence-specific, DNA-binding transactivator (Gruffat et al, 1990; Hardwick et al., 1992), which can synergistically activate viral promoters with BZLFl (Cox et al., 1990). However, the activity of BRLF1 alone is not sufficient for the induction of lytic DNA replication, as replication studies with a recombinant BZLFl -defective EBV genome have shown (Feederle et al., 1999).

The genes for BZLFl and BRLF1 are located in an overlapping transcription unit and the BZLFl gene can be transcribed both by its own promoter and by the neighboring promoter of the BRLFl gene (Manet et al, 1989). The regulation of the BZLFl and BRLF1 genes is subject to a complex mechanism that is not yet understood in detail. The complexity is increased by the fact that both BZLFl and BRLF1 mutually influence each other in their expression and can also have an autoregulatory effect (Flemington and Speck, 1990; Sinclair et al., 1991). The transcriptional regulation of these two gene products by cellular factors is essential for maintaining latency. Switching from the latent to the lytic phase is likely to require not only the activation of cellular transcription factors that activate the BZLFl and RR Ei transcription, but also the inactivation of repressing factors.

It could be shown that the promoters of the BZLFl and BRLFl genes are activated by binding Spl (Zalani et al., 1992; Liu et al., 1997). By binding the transcription factor YY1 (Yin-Yang 1) (Shi et al., 1991) to these promoters, their transcription is negatively regulated (Montalvo et al., 1995; Zalani et al., 1997). Other works could show that the cellular transactivators RelA (Gutsch et al., 1994) and Sμbp-2 (Zhang et al., 1999) have an inhibitory effect on BZLFl -dependent promoters and are therefore probably responsible for maintaining the latent phase. In addition to the transcriptional control, post-translational regulatory mechanisms that directly influence the activity of the BZLFl protein have also been described. An interaction of BZLFl with p53 inhibits the transactivation by BZLFl (Zhang et al., 1994) while the interaction with the basic transcription factors TFIIA and TFIID stabilizes the binding to promoters (Lieberman et al., 1994).

Recent studies show a direct interaction of BZLFl with the CREB binding protein (CBP), a transcriptional coactivator with histone acetyl transferase activity (Adamson and Keney, 1999; Zerby et al., 1999). The CBP-mediated acetylation of histones could contribute to an altered chromatin structure ("chromatin remodeling") and thus on the one hand promote the transactivation of BZLFl -regulated promoters and on the other hand facilitate the formation of an initiation complex on oriLyt (Kodadek, 1998).

At the viral regulatory level, the involvement of the alternative spliced protein RAZ (BRLF1 and BZLFl) is discussed, which is composed of the DNA binding and dimerization domain of BZLFl and the DNA transactivation domain of BRLF1 and inhibits its function by heterodimer formation with BZLFl (Furnari et al ., 1994).

The regulation of the transition from latent to lytic replication can thus be carried out by cellular control of the BZLFl and BRLF1 activity. In addition, direct activation and / or repression of the lytic replication appears to take place by binding cellular factors to oriLyt. As already mentioned, the TD element within the "downstream" component of oriLyt is absolutely essential for lytic replication. Since neither BZLFl nor BRLF1 interact with this sequence, other, as yet unidentified factors seem to regulate the transition from latent to lytic replication via binding to the TD element. Binding studies identified several protein complexes on the TD element, including the transcription factor Spl. Recent data on the function of Spl in lytic replication could show that Spl and at least one other cellular transcription factor bind to the TD element and recruit viral replication factors to the origin of replication (Baumann et al., 1999). So far, no repression of oriLyt by binding cellular factors has been demonstrated. The identification and characterization of a cellular protein that inhibits the lytic replication of EBV would not only be of scientific interest; such a protein could be used in particular for the treatment of patients suffering from EBV infections.

It is therefore an object of the present invention not only to demonstrate the existence of such a cellular repressor of lytic DNA replication, but in particular also to show its genomic DNA sequence, its cDNA sequence and amino acid sequence.

This object is achieved according to the invention by proving the existence of the TB7 protein and by describing the genomic DNA, cDNA and amino acid sequences of TB7, as set out in the appended claims. The cellular repressor TB7 is suitable for suppressing the replication of EBV and thus for the treatment of EBV infected patients, in particular for the treatment of infectious mononucleosis. Since EBV is also associated with tumor diseases, TB 7 can also be used to treat tumors whose development is associated with EBV, in particular for the treatment of Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinoma.

Possible further uses of TB7 in the pharmacological sense as an antiviral active ingredient are the very closely related human gamma herpes viruses, example HHV8, and the relatively closely related human beta herpes viruses, example human cytomegalovirus. These herpes viruses have very homologous replication origins, which due to their structural similarities can be expected to have similar effects in TB7.

Further refinements of the invention result from the subclaims, the following description and the exemplary embodiments. However, the invention is not limited to the following preferred embodiments.

The invention relates in particular to the genomic DNA sequence, the cDNA sequence and the RNA sequence of the TB7 gene and the amino acid sequence of the protein encoded thereby. The invention further relates to fragments and other derivatives and analogs of the TB7 protein. The invention furthermore encompasses nucleic acids which code for these fragments or derivatives. The invention includes in particular the TB7 gene of human origin, but also such TB7 genes from vertebrates, in particular from mammals, for example the mouse, the rat or the rabbit. The invention also encompasses processes for the production of said proteins and derivatives, for example by recombinant techniques.

The invention also encompasses fragments and derivatives of these fragments of TB7 which contain one or more functional units. The invention further relates to polyclonal or monoclonal antibodies which are directed against TB7 and its derivatives and analogs, in particular its epitopes and domains.

Some terms used in the above application are defined in more detail below.

The term "isolated" for nucleic acids means that the nucleic acid is from its naturally occurring environment, i. H. the chromosome of the cell. The nucleic acid is therefore in a cell-free solution or in a cellular environment different from the original cell. The expression does not mean that no other nucleic acid components can be present, but in particular that it has been removed from its natural, chromosomal environment. The same applies analogously to proteins or polypeptides.

The term "enriched" means that the proportion of nucleic acid or protein is higher than the proportion naturally occurring in the cell.

The term "cleaned" does not mean that absolute purity, i.e. H. a homogeneous preparation. The term "purified" means that the nucleic acid or protein is of a higher purity than that in the natural environment.

The term "hybridization" means that a DNA or RNA nucleic acid with high affinity binds to a given nucleic acid molecule, which can be DNA or RNA. Under highly stringent hybridization conditions, only highly complementary nucleic acid sequences hybridize. "Highly stringent" is preferably understood when, in the case of one or two mismatches in 20 consecutive nucleotides, hybridization no longer takes place. Stringency is caused by different salt concentrations or concentrations of denaturation _¬ centering agents. Highly stringent conditions mean, for example, the following hybridization conditions: hybridization in 50% formamide, 5 x SSC, 50 mM NaH ₃ PO ₄ , pH 6.8, 0.5% SDS, 0.1 mg / ml ultrasound-treated salmon sperm DNA, 5 x Den-hart solution at 42 ° C overnight; Washing with 2 x SSC, 0.1% SDS at 45 ° C; Wash with 0.2 x SSC, 0.1% SDS at 45 ° C. It is known to the person skilled in the art that the conditions can be varied depending on the specificity and selectivity.

CDNA means those molecules that have been reverse transcribed by the mRNA.

The term "mammals" includes organisms such as mice, rats, rabbits, goats, monkeys and preferably humans.

According to the invention, “derivatives” are understood to mean modifications or modifications or analogs of chemical compounds. Analogs according to the invention are derivatives of chemical compounds in which the spatial structure of the compounds remains essentially unchanged.

Nucleic acid derivatives include, for example, nucleic acids in which one or more nucleotides have been exchanged, modified or modified. The nucleic acids in which one or more nucleotide bases or the sugar portion have an additional alkyl, aryl and / or arylalkyl radical, which in turn can be derivatized with functional groups. The nucleic acid derivatives also include analogs in which, for example, the sugar portion of a nucleotide is replaced by another compound or the sugar portion is modified.

According to the invention, amino acid derivatives include, for example, those compounds in which the side chain is modified, exchanged and / or substituted. Suitable modifications or substituents are, for example, alkyl, aryl and / or arylalkyl radicals, which in turn can be derivatized with functional groups. According to the invention, in addition to the commonly occurring L-amino acids, the D-amino acids are also included. Preferred configurations are described below.

The invention discloses a nucleic acid in isolated, enriched or purified form which codes for a TB7 polypeptide or which contains a nucleic acid which codes for a TB7 polypeptide. This includes, for example, the genomic DNA sequence with the exon sequences of the TB7 gene, the cDNA for the TB7 gene, but also vectors which contain a nucleic acid which codes for a TB7 polypeptide.

The nucleic acid provided according to the invention comprises in particular the genomic DNA according to SEQ ID NO: 1 and the cDNA of SEQ ID NO: 3. The invention also includes those nucleic acids which are complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and those nucleic acids which hybridize with the nucleic acid of SEQ ID NO: 1 under highly stringent conditions, as defined in more detail above, and code for a functional TB7 polypeptide. The invention also encompasses those nucleic acids which code for a functional part of the polypeptide TB7.

According to the invention, the term “functional part” is understood to mean a nucleic acid which codes for a part of the TB7 polypeptide which fulfills functional features. These include, for example, epitopes and domains of TB7, in particular those domains of the polypeptide which are responsible for the recognition and / or binding to the EBV-DNA, in particular the TD element of EBV, or which have antigenic properties.

This includes in particular the DNA-binding domain of the gene product TB7 with amino acids 103 to 151, starting from the definition of the zinc finger domains.

A preferred source of the nucleic acid is a mammal, for example a mouse, rat, rabbit, guinea pig, goat, etc., furthermore preferably monkey and particularly preferably human.

The invention also includes those nucleic acids which have been replaced by another nucleic acid due to the degeneration of the genetic code. The invention also encompasses nucleic acids which code for derivatives of the TB7 polypeptide or derivatives of functional parts of the TB7 polypeptide, as are described in more detail here. The invention also encompasses RNA derived from a nucleic acid as set out above.

The invention also includes a nucleic acid probe for the detection of a nucleic acid encoding a TB 7 polypeptide. “Nucleic acid probe” is understood to mean a nucleic acid molecule which is complementary to a nucleic acid sequence and can bind to it, which is specific for the TB7 polypeptide, preferably under highly stringent hybridization conditions, as described above.

Methods using these probes include detection of the presence of TB7 RNA or DNA or quantitation of the TB7 RNA or DNA in a sample, wherein a nucleic acid probe is contacted with a sample under such conditions that hybridizes and allow detection of the binding of the probe to the TB7 RNA or DNA.

To carry out this and subsequent investigations, reference is generally made to the methods known to the person skilled in the art and to literature which describe these methods. Examples of methodology manuals are Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.

In a further embodiment, the invention also comprises vectors which contain a nucleic acid molecule with the TB7-DNA according to the invention or functional parts thereof. A vector is understood to mean single-stranded or double-stranded, usually circular nucleic acid molecules which can be transfected into cells or with which cells can be transformed and which replicate independently or together with the host cell genome. Such vectors are known to the person skilled in the art. Particularly preferred are vectors which enable expression of the polypeptide TB7 or its functional parts in a host cell. The host cell is to be understood as any prokaryotic or eukaryotic cell into which a recombinant vector containing TB7 DNA can be introduced and replicated. To the particularly preferred prokaryotic cells include, for example, the E. coli cell. To the Special _¬ DERS preferred eukaryotic cells include for example a B cell.

¹

Methods for introducing DNA, in particular vector DNA, into a prokaryotic or eukaryotic cell are known per se. The terms "transformation" and "transfection" describe such methods. The vector DNA can either replicate extrachromosomally in the cell or it can also integrate into the genome of the host cell and thus replicate together with the host cell DNA.

The TB7 DNA introduced into such a host cell is generally under the control of a promoter element which is naturally not responsible for controlling the transcription of the TB7 gene in the cell and which is preferably inducible. The promoter element comprises binding sites for transcription factors, for the polymerase and for the transcription of the vector DNA in mRNA. In addition to the promoter element, other elements known per se can control the replication and transcription of the vector DNA and facilitate their handling and can be integrated on the vector DNA. These include, for example, the origin of replication, ribosome binding sites, resistance genes, nucleotide sequences that enable secretion of the TB7 polypeptide or its functional parts, signals for purification of the polypeptide, etc.

The invention also encompasses recombinant cells which contain a recombinant TB7 DNA or functional parts thereof or their derivatives. This can be both prokaryotic and eukaryotic cells, but also cell lines. A typical example of a prokaryotic cell is E. coli, a typical example of a eukaryotic cell is a mammalian cell, preferably a human cell or cell line. The recombinant TB7 DNA or functional parts thereof or their derivatives can be present in the recombinant cell, cell line and then of course in the recombinant organism either in extra-chromosomal form or integrated into the chromosome. You can either replicate with or independently of the host genome, recombine with the host cell genome and integrate into it or not.

The present invention also describes isolated, enriched or purified TB7 polypeptide or functional parts of this polypeptide as well as derivatives of the polypeptide. The polypeptide is encoded by the nucleic acid according to SEQ ID NO: 1 or SEQ ID NO: 2 and has the in SEQ ID NO: 3 and 4 described amino acid sequence. It is preferably in a non-glycosylated form.

The invention also includes derivatives of the TB7 polypeptide, i.e. H. such polypeptides or functional parts thereof, in which one or more of the amino acids have been replaced by another amino acid, one or more amino acids have been deleted and / or one or more amino acids are additionally present, the function of TB7 as a repressor of the DNA Replication of EBV is preserved. The same applies to functional parts of the TB7 repressor.

"Functional parts" are understood to mean domains and epitopes of the TB7 polypeptide. "Domain" means a part of the TB 7 polypeptide that is responsible for the function, interaction or activity of the protein. In particular, this is understood to mean the part of the TB7 polypeptide which interacts with the TD sequence of the EBV.

The term "derivatives" encompasses those amino acid sequences of the polypeptide which differ from the native sequence by the exchange, addition or deletion of amino acids described above. These can be so-called conservative or non-conservative exchanges of amino acids. “Conservative” means the substitution of an amino acid by an amino acid with similar properties, for example in relation to the charge, hydrophobicity, structure, etc.

“Epitope” is to be understood as a sequence of amino acids which has antigenic properties and is specific for the TB7 polypeptide. Such epitopes can be used to produce antibodies.

The invention also relates to recombinant polypeptides of TB7 which have been produced by recombinant DNA techniques as described above and which comprise the polypeptides described above, functional parts thereof and their derivatives.

The invention also describes monoclonal and polyclonal antibodies directed against the TB7 polypeptides, functional parts thereof and their derivatives. Such antibodies with a specific binding to a TB7 polypeptide can be used for detection and / or Quantity determination of the TB 7 polypeptide can be used in a sample. Appropriate methods are known to the person skilled in the art and are described in the literature.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen or a functional part thereof.

Monoclonal antibodies are essentially homogeneous populations of antibodies against a specific antigen, here against an epitope of the TB7 polypeptide. Methods for producing monoclonal antibodies in cell cultures are known. An example of this is the original literature by Köhler et al., Nature 256: 495-497 (1975) and U.S. Patent No. 4,376,110. The invention also includes hybridoma cell lines, i. H. immortalized cell lines that produce monoclonal antibodies and that are suitable for recognizing and binding the TB7 polypeptide.

The invention also describes a method for inhibiting the lytic replication of EBV, wherein a TB7 polypeptide or a functional part of the TB7 polypeptide or a derivative thereof is brought into contact with an EBV-containing cell, the TB7 polypeptide or a functional part of the TB7 polypeptide is present in such an amount that lytic replication is suppressed by binding TB7 to the TD element of EBV. In an alternative embodiment, the genetic information encoding the TB7 polypeptide or a functional part thereof or a derivative is introduced into the cell to be treated or into a patient to be treated and the expression of the polypeptide is induced to prevent the replication of EBV or at least to suppress it.

In a further embodiment of the invention, a pharmaceutical composition is provided which contains the TB7 polypeptide or a functional part of the polypeptide or derivatives thereof in a pharmaceutically effective amount. The TB7 polypeptide or a functional part thereof or a derivative is present together with conventional carriers and auxiliaries. The pharmaceutical composition can be administered either systemically or locally. Suitable techniques for formulation and administration are mentioned, for example, "Remington's Pharmaceutical Sciences", 1990, 18 ^th ed., Mack Publishing Co., Easton, PA. The exact manner of formulation, administration and dose may depend on the disease to be treated, the patient to be treated, the doctor and the Pharmacists can be selected. The corresponding methods are described, and we refer, for example, to Fingl et al., In "The Pharmacological Basis of Therapeutics", Chap. 1 p. 1, 1975.

The invention also discloses a method for producing a TB7 polypeptide or a functional part of the TB7 polypeptide or a derivative thereof. Here, for example, a nucleic acid is introduced into a cell, which contains the information for the expression of the TB7 polypeptide or a functional part or derivative thereof, together with conventional control and regulatory sequences such as a promoter,

Contains enhancer elements, polyadenylation signals, etc. on a vector. Suitable methods are available to the person skilled in the art and only the method of electroporation or transfection is mentioned here, for example. Induction of the transcription results in the expression of the TB7 polypeptide or a functional part or derivative thereof. The recombinant cell or cell line is cultivated and the recombinant TB7 polypeptide or the functional recombinant part or derivative of TB7 is obtained and purified by suitable methods.

The isolation and characterization of TB7 DNA and polypeptide is described in more detail below.

An attempt to isolate DNA-binding proteins represents a modified form of the genetic selection system in yeast described for the first time by Wang and Reed, which has meanwhile entered the textbooks under the name "one hybrid screen" (Wang and Reed, 1993). This system is based on the observation that many eukaryotic transcription activators contain a DNA binding domain and an activation domain. If any cDNA which contains a DNA binding domain is fused to an activation domain, the fusion protein can regulate the activity of indicator genes (for example HIS3, LacZ) by binding to the target sequence. This selection in yeast has the advantage over biochemical methods that the proteins are present in their native fold in the cell and thereby the sensitivity of the DNA-protein interactions is increased. Isolation of TD-binding proteins using the "one hybrid screen" in Saccharomyces cerevisiae

A corresponding yeast strain had to be developed for the genetic selection system of TD-binding proteins in S. cerevisiae (Fig. 3 A). For this, two linearized integration plasmids were subsequently transformed into the yeast strain RH1533 (MATα; leu2-3, -112; ura3-52; his3-del200; trpl-del901; lys2-801; suc2-del9; MCI-). These plasmids carry multimerized TD elements and either the His3 or LacZ marker gene under the control of various minimal promoters. The plasmids were integrated into the Ey-s2 or. Ura3 gene of the yeast strain selected by histidine and uracil prototrophy. This genetically modified yeast strain was transfected with a cDNA library derived from an EBV-immortalized human B cell line (Durfee et al, 1993). The cDNAs in this library are fused to the activation domain of GAL4. After specific binding of a fusion protein to the sequence motifs of the TD element, the promoters for the histidine or LacZ gene are activated by the GAL4 activation domain. To increase the stringency of the selection, a previously tested concentration of 3-aminotriazole (3-AT) was added to the medium, which inhibits the synthesis of imidazole-glycerol-3-phosphate dehydratase, an H..s3-encoded enzyme , With leucine deficiency, selection was made for the successful transfection of the plasmids from the cDNA library which carry the leucine gene on the vector portion (FIG. 3 B).

It should be noted that TB7 and TD-BP7 are used as identical terms.

Two clones were isolated which had grown on minimal medium without leucine, uracil and histidine with 30mM 3-AT and which were able to grow again on minimal medium after preparation of the plasmid DNA and retransformation in yeast (data not shown). The DNAs from these clones were then sequenced. The cDNA sequence referred to below as TD binding protein 7 (TD-BP7) was 951 bp long. The isolated clone contained partial cDNAs with two zinc finger domains. The identified open reading frame started directly with the isolated cDNA and contained no start codon in the first position. The sequence has no homologies to known proteins. Only in the area of the zinc finger domains are there recognizable homologies to other proteins with zinc finger structures. To study the function of the protein, the full length cDNA was cloned. Isolation of the Complete cDNA: Searching a cDNA library ι In order to clone the complete cDNA, a commercially available cDNA library, which was obtained from

RNA from human HeLa cells was created (Clontech, # HL1152X), searched with the partial cDNA from the "one hybrid screen" as a probe. In this library, the cDNA is cloned into the RαmHI - ^ - bαl interface of the phage vector λpDR2 in such a way that it is flanked by two rectified loxP recognition sequences of the Pl phage. The purpose of this construction of the phage vector is that the cDNAs do not have to be elaborately subcloned, as in conventional lambda vectors, but during the infection of a Cre-expressing E.co/t strain (AMI) in vivo by site-specific recombination at the loxP -Recombine sequences. The recombination results in plasmids which contain the cloned cDNA.

To search the library, approximately 1, 2 × 10 ⁶ plaque-forming units (pfu) were first plated out on 24 bacterial plates (0 140 mm) (approx. 50,000 pf / plate) and incubated at 37 ° C. overnight. The plaques were then transferred to nitrocellulose filters and, after denaturing and fixing the DNA on the filters, the hybridization was carried out at 32p. radiolabelled probes. The partial cDNA TD-BP7 isolated from the "one hybrid screen" was used as the specific probe. In order to exclude non-specific signals, 2 filter deductions (replicas) were made per plate and the signals of both filters compared. Hybridization with the TD-BP7-specific probe showed a positive signal, which was confirmed on the replica filter. After Cre-mediated site-specific recombination and subsequent circularization, the newly formed recombinant plasmid was isolated and the DNA was purified. The cDNA was first analyzed by restriction enzyme cleavage and subsequent gel electrophoresis. The clone isolated from the "one hybrid screen" could be confirmed and the sequence added in the 5 'and 3' direction. After sequencing, the length of the TD-BP7 cDNA was 2308 bp with an open reading frame of 193 amino acids. Characterization of the genomic sequence of TD-BP7

To determine regulatory elements and the structure of the gene for TD-BP7, a human placenta library was searched with a TD-BP7-specific probe. A total of 5x10 ^ plaques were hybridized using the standard method (Sambrook et al, 1989) and the DNA was purified from three positive phage plaques. In order to isolate the sought genomic sections of the TD-BP7 gene from the phages, these were cleaved with the restriction enzyme Sali, the corresponding fragments isolated and ligated into the pACYC177 vector linearized with Jöiol. Three recombinant plasmids could be established (p2147, p2148, p2149). After transformation and preparation of the plasmid DNA, the clones were subjected to a comprehensive restriction enzyme analysis. It was found that clone p2147 was smaller than clones p2148 and p2149 and that the latter two clones were identical and only cloned into the plasmid vector in the opposite orientation. In addition, the agarose gels with the separated restriction fragments were subjected to Southern blot analysis using a TD-BP7-specific probe in order to confirm the identity of the DNA and to be able to distinguish the genomic fragments from the vector portion.

Starting from the already known TD-BP7 cDNA sequence and the pACYC vector sequence, primers were selected and the sequencing of clone 2148 was carried out. After a total of 23 sequencing reactions, the sequence of the genomic section was 14328 bp in length, which is referred to below as the TD-BP7 gene. This designation corresponds to the designation TB7. By computer analysis and comparison of the genomic sequence with the cDNA sequence, the following structure could be determined for the TD-BP7 gene (Fig. 5): The TD-BP7 gene contains a 5'-UTR, which is within a CpG island and which is preceded by a binding site for the transcription factor Spl. The second exon contains the translation initiation codon, which is embedded in a good Kozak consensus sequence (AT-CAGCatgG; (Kozak, 1996)). The open reading frame is interrupted by the second intron after 9bp. Another five exons of different lengths follow; the seventh exon ends the open reading frame with a stop codon. The mRNA ends after the 1.2 kbp 3'-UTR with a poly-A sequence, which, however, is not preceded by a clear polyadenylation signal. The zinc finger coding sequences (type C2H2) are located in exons 5 and 6. With the exception of the transitions of the 5th intron (GT ... CA), all 5 'and 3' splice points correspond to the splice consensus motifs (GT ... AG). The TD-BP7 and TD-BP17 cDNAs encode unknown proteins

/ To determine whether the cDNA of TD-BP7 codes for a protein with known functions, a comparison of the sequence was carried out with the BlastN algorithm and the GENIUSnet database (DKFZ, Heidelberg). Homologies were found for the cDNA sequence in the 5 'and 3' untranslated regions to EST cDNAs ("expressed sequence tag"). In addition, as already mentioned, there were homologies to known zinc finger proteins in the area of the zinc finger coding sequences. Figure 4 shows a graphic representation of the homology areas.

Production of monoclonal antibodies against TD-BP7

In order to be able to examine the expression of the cDNA at the protein level and to detect specific DNA interactions of TD-BP7 in binding studies, monoclonal antibodies against this protein were produced. To immunize rats, the protein antigens first had to be produced and purified. For this purpose, fusion plasmids of parts of the TD-BP7 cDNA with the glutathione-S-transferase (GST) or the maltose binding protein (MBP) were prepared and, after overexpression, purified in E. coli.

Monoclonal antisera were obtained by immunizing Lou / c rats with MBP-TD-BP7 antigens. The required fusion of isolated spleen cells from the rat with a mouse myeloma cell line and the double recloning of positive clones were carried out according to standard methods (Kohler and Milstein, 1975). The testing of 20 culture supernatants of each hybridoma cell line was carried out by Western blot analysis. To rule out that the monoclonal antibodies are directed against the MBP (TD-BP7) portion or possibly against a neo-determinant, the hybridoma supernatants were tested in each case with partially purified antigens which were present as GST (TD-BP7) fusion proteins, and that had not been used for immunization. A monoclonal antibody against TD-BP7 (10C1) was selected. The monoclonal antibody recognizes the respective antigen. However, detection of endogenously expressed TD-BP7 in HeLa protein extracts was not possible with these antibodies. After expression of pCMV-TD-BP7 in 293 cells, however, specific signals could be detected. It was thus possible to produce monoclonal antibodies against TD-BP7, the affinity of which, however, was insufficient to detect endogenous gene products from TD-BP7. The antibodies were tested in gel retardation experiments for their ability to bind to non-immobilized protein-DNA complexes.

Interaction of TD-BP7 and TD-BP17 with the downstream component of oriLyt: in vitro binding studies

The protein interaction with the TD element in the "one hybrid screen" in S. cerevisiae led to the isolation of the cDNA from TD-BP7. In the following it will be investigated whether the proteins encoded by this cDNA also bind to the TD element in vitro. Since it is known from previous studies that a total of six cellular complexes are formed on the "downstream" component (Gruffat et al., 1995), it should also be checked whether these proteins are part of the complexes described.

The recombinant proteins TD-BP7 and TD-BP17 bind in vitro to the TD element of oriLyt

To determine whether the protein TD-BP7 binds to the TD element in vitro, gel retention analyzes were first carried out with recombinant fusion proteins. The 62bp sequence of the oriLyt TD element was radioactively labeled and served as a DNA sample for incubation with recombinant MBP-TD-BP7 protein extracts. In order to be able to assess the specificity of the complexes, additional competitions were carried out with unlabelled oligonucleotides (T1-T6 and TD) from the area of the downstream component. It was found that the protein interacts with the TD element. After incubation of the TD Elements with the MBP-TD-BP7 extract formed two complexes, of which the upper complex I was almost completely competed by adding unlabeled TD element, as was shown by the competition with oligonucleotide T4 from the "downstream" component same result, the binding of the upper complex I was completely blocked with the oligonucleotides T3, T3.4 and T6. The addition of unlabeled oligonucleotides inhibited the formation of the lower complex II only a little. The specificity of the complexes was also confirmed by the formation of a super shift after the addition of monoclonal antibodies against the protein TD-BP7.

The competition of the complex formation on the TD element by the oligonucleotide T6, which lies outside the TD sequence, is due to the high sequence similarity of both sequences. These results show that the recombinantly expressed proteins TD-BP17 and TD-BP7 specifically bind to the TD element of oriLyt in vitro. The interaction takes place exactly in the area of the TD element (EBV # 53,355 - # 53,395) in which point mutations already lead to the complete blocking of the lytic DNA replication of EBV (Schepers et al., 1993).

Influence of the protein TD-BP7 on the lytic replication of EBV

The results to date have shown that the protein TD-BP7 specifically binds to the TD element of oriLyt in vitro. A key question was whether this protein had an impact on the efficiency of lytic replication in EBV. The lytic origin of replication of EBV was identified by W. Hammerschmidt with the help of a transient replication attempt (Hammerschmidt and Sugden, 1988). With this method, the replication of recombinant plasmids was investigated in EBV-positive cells that are able to support the lytic phase of EBV. For this purpose, recombinant plasmids carrying orz ^' Jyt sequences of the EBV strain B95-8 were transfected together with a BZLFl expression plasmid into EBV-positive D98 / HR1 cells. BZLFl induces the lytic cycle of endogenous EBV. Two days after transfection, the cells were harvested, lysed and the DNA isolated (Hirt, 1967). After restriction cleavage with BamHI and Dpnl, the newly replicated DNA was quantified by Southern blot hybridization. Prokaryotic sequences that specifically recognize the plasmid DNA were used as the probe. The distinction between transfected and newly replicated DNA is achieved by cleaving the DNA with Dpnl, an enzyme that cleaves only DNA that has been methylated in a dam ⁺ -E.co z ^' strain. Since the Dj-ml recognition sequence GATC occurs very frequently, the transfected DNA is severely fragmented. DNA that has replicated in eukaryotic cells is not cleaved and remains intact. The replication efficiency of the or / Eyt plasmid p968.22 was tested after cotransfection of CMV expression plasmids for the open reading frame (ORF) of TD-BP7 in a transient replication experiment. The co-transfection of pCMV-BP7 / ORF led to a significant repression of the replication efficiency compared to the vector control. In order to determine whether TD-BP7 competes with the binding of essential replication factors to the TD element or whether the observed effect is active repression, an expression plasmid of the DNA-binding domain of TD-BP7 was compared (pCMV-BP7 DBD). It was shown that the TD-BP7 DNA binding domain alone is sufficient to suppress replication. The repression of the replication of oriLyt is therefore due to a competitive binding of TD-BP7 to the TD element, which is likely to lead to the displacement of essential replication factors.

In order to rule out that during transfection the pCMV expression plasmids compete for transcription factors and therefore the induction of the lytic cycle by pCMV-BZLFl was different in the batches, the transfected cells were additionally examined in Western blot analyzes for the amount of BZLFl expression. The BZLFl expression was comparable in all approaches.

The results so far show that TD-BP7 binds specifically to the oriLyt TD element. The expression of TD-BP7 led to complete repression of the lytic replication, which could be attributed to the binding of the protein to the lytic origin of replication.

TD-BP7 represses the binding of Spl to the TD element

The contact points of TD-BP7 with the TD element lie within a sequence which has been described as a binding motif for the transcription factor Spl (Gruffat et al., 1995). Recent studies on the role of Spl in lytic replication have shown that the cellular transcription factor binds to the TD element and that viral replication factors recruit to the lytic origin of replication oriLyt (Baumann et al., 1999). Since TD-BP7 represses the lytic replication of EBV, it made sense to check whether TD-BP7 can suppress the binding of Spl to the TD element. This hypothesis was tested with DNasel protection experiments. Increasing amounts of recombinant Spl or MBP-BP7 were found incubated with a radioactively labeled fragment of the "downstream" component, the DNA was then cleaved under defined conditions with DNasel and separated in a denaturing PAA gel (Fig. 6). Even at the lowest amount of MBP-BP7 used, the binding to the "downstream" component, in accordance with the competitive gel retardation experiments, led to a clear protection of the DNA against the DNasel cleavage in the region of T3.4 and T6 (EBV # 53,403-53,424 and EBV # 53,364-53,391), which became more apparent with increasing amounts of MBP-BP7. Binding of the protein made the sequence hypersensitive to DNasel on both sides of the binding sites. Spl binding also produced protection in the area of T6 and the TD element, but was less well developed and slightly offset compared to MBP-TD7 (EBV # 53,403-53,422 and EBV # 53,359-53,379). The simultaneous incubation of the sample with a constant amount (highest concentration of the single incubation) of Spl and increasing amounts of MBP-BP7 or vice versa inhibited the formation of the Spl protection pattern almost completely in all approaches.

These results show that TD-BP7 competes with the transcription factor Spl for binding to the "downstream" component of oriLyt. The observed repression of lytic replication by binding of TD-BP7 could therefore be explained by the displacement of cellular and / or viral factors from the origin of lytic replication.

Expression of TD-BP7 inhibits EBV DNA replication

In order to investigate the influence of TD-BP7 on the lytic DNA replication of EBV, lytic replication was induced in the cell line 2098-31, which carries the recombinant EBV genome, by transfection of BZLF1. At the same time, CMN flag expression plasmids for the open reading frame or the DΝA binding domain of TD-BP7 were co-transfected. After three days, Raji cells were incubated with filtered culture supernatants and, after a further three days, examined for GFP expression in the fluorescence microscope (FIG. 8). With supernatants obtained by transfection of the control of the nector, about 40% of the Raji cells expressed GFP. Cell culture supernatants obtained after transfection with pCMN-BP7 / DBD or pCMN-BP7 / ORF contained significantly fewer infectious particles, as can be seen from the infection rate of the Raji cells. This effect was particularly pronounced after transfection of the Plasmid pCMN-BP7 / ORF, which obviously particularly strongly represses the DΝA replication of EBN. Western emblot analyzes of the transfection approaches with BZLF1 and Flag-specific antibodies were used to control the expression level of the transfected plasmids (Fig. 8). The weak expression of the pCMN-BP7 / DBD plasmid explains the reduced repression of replication in comparison to the effect after transfection of the pCMV-TD-BP7 / ORF plasmid. These results confirm the data from the transient replication experiments and binding studies. TD-BP7 strongly represses the DΝA replication of oriLyt, and this effect is due to the binding of the protein to the lytic origin of replication.

It could thus be shown in transient replication experiments that the expression of the cellular factor TB7 leads to an almost complete suppression of the DNA replication of oryLyt (Fig. 7).

The attached pictures show:

Fig. 1 The two phases of the Epstein-Barr virus. After infection of the host cell, the linear viral DNA is released and circularized via the terminal repetitive (TR) sequences. In the lytic phase (A), DNA replication takes place at the oriLyt origin of replication. DNA synthesis is probably unidirectional via the so-called "rolling circle" mechanism, as described for the replication of bacteriophage D. The product is more concatemic molecules, which are cut in a base-specific manner at the TR sequences and packaged in virus capsids. In latently infected cells, the viral genome is an extra-chromosomal episome. In this latent phase, the viral genome replicates in sync with the cell cycle using the plasmid origin of replication oriP (B). Only a few latently infected cells support spontaneous lytic replication. Treatment of the cells with chemicals or the virally coded transactivator BZLFl can, however, experimentally induce the lytic cycle in some of the cells (C).

Fig. 2 Schematic representation of the lytic origin of replication oriLyt of the Epstein-Barr virus. The origin of lytic replication is complex and contains elements of transcription and replication. In the central area (approx. Lkbp) are the two "upstream" and "downstream" components that are essential for replication. flanked opposite genes are flanked. These flanking areas are called replication enhancers because they support replication but are not essential. The previously defined elements of oriLyt are shown in the lower part of the figure. The promoter of the BHLFl gene (TATA) colocalizes with the "upstream" component and contains four binding sites for the viral transactivator BZLFl (ZRE: BZLFl responsive element). Further ZRE binding sites are located in an area between the "downstream" component and the BHRF1 promoter, where there are also two binding sites (R) for the viral transactivator BRLF1. The "downstream" component contains a 60 bp long G / C-rich sequence (TD element) to which the binding of Spl and other cellular proteins that have not yet been characterized has been demonstrated. Mutations within the TD element prevent the lytic replication of EBN (according to Gruffat et al., 1995).

Fig. 3 Scheme of the "one hybrid screen" in S. cerevisiae. (A) Preparation of the yeast strain. The yeast strain RH1533 was transformed with two plasmids that directedly integrated into the defective LYS2 or URA3 genes. These integration plasmids introduce the two marker genes HIS3 and LacZ, which are under the control of different promoters and whose activity is determined by multi-merized TD elements. After integration of the marker gene HIS3, the yeasts are able to grow on histidine-free medium due to minimal HIS 3 promoter activity. For the selection after transfection of the cDNA library, this residual activity of the promoter must be inhibited by the addition of 3-aminotriazole. The selection for integration was made by growth on a medium without histidine and uracil. (B) Transformation of the cDNA library into the yeast strain produced. The promoters of the HIS3 or LacZ gene are only activated if a fusion protein specifically binds to the DNA sequence motifs of TD and thereby brings the GAL4 activation domain close to the promoters. With leucine deficiency, selection was made for the successful transfection of the cDNA library which carries the Leu2 gene on the nector portion.

Fig. 4 TB7 codes for an unknown gene product. Graphical representation of the homology areas between EST sequences from the GENIUSnet database and the cDNA of TB7. The numbers of the arrows refer to the sequences described in Table 1. The direction of the arrow indicates whether the homologous sequences relate to the coding or non-coding strand. White bars show the location of the zinc finger domains. Fig. 5 Schematic structure of the TD-BP7 gene. The cloned sequence of the JE> RP7 gene _. is 14328bp. The coding areas with Εxons 1-7 and 3'-UTR (hatched), the introns (gray) and the genomic sequence (white) are shown. The transcription start point (+1) lies within a CpG island and a putative binding site for the transcription factor Spl is marked.

Fig. 6 TD-BP7 competes with Spl for binding to the "downstream" component.

A 242bp fragment of the "downstream" component was radioactively labeled and incubated with DNasel in the presence of recombinant Spl and / or MBP-TD-BP7. The amounts of recombinant proteins used are indicated. A G + A sequence reaction (G + A) of the fragment according to Maxam-Gilbert was used to determine the sequence positions. A DNasel reaction without prior protein incubation was used as a negative control. DNasel-sensitive areas are marked by asterisks. The areas of the "downstream" component protected from DNasel cleavage by the binding of TD-BP7 or Spl are shown schematically on the side (dotted or hatched bars), fpu: "footprinting units".

Fig. 7 Transient replication experiments with TB7 and mutants. The structure of the open reading frame of TB7 and various deletion mutants are shown schematically in the upper part of the figure. After cotransfection of these different expression plasmids and an expression plasmid for BZLFl (pCMN-BZLFl as inducer of lytic replication) in D98 / HR1 cells, the replication efficiency of an oriLyt plasmid was examined. Expression of the intact TB7 protein or a variant that is slightly shortened in the 5 'range completely suppresses the lytic DNA replication (p2065 or p2181). The deletion of the 5 'region results in a partial abolition of this repressive effect (p2608) while after the deletion of the DNA-binding zinc finger domains the lytic DNA replication is not impaired.

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SEQUENCE LISTING

<110> GSF Research Center f. Environment and Health GmbH

<120> Nucleotide and amino acid sequences of a cellular repressor of the DNA replication of the Hepesvirus Epstein-Barr

<130> P12332

<140> <141>

<160> 4

<170> Patentin Ver. 2.1

<210> 1

<211> 14646

<212> DNA

<213> Homo sapiens

<220>

<221> genes

<222> (1) .. (14646)

<223> TB7 genomic DNA

<220>

<221> misc_feature <222> (2762) .. (2973) <223> CpG island

<220>

<221> misc_feature <222> (4065) .. (5181) <223> CpG island

<220>

<221> mRNA

<222> (4422)

<223> start site primer extension

<220>

<221> stem_loop

<222> (4474) .. (4505)

<220>

<221> 5 * UTR

<222> (4530) .. (4679)

<223> 1st exon

<220>

<221> misc_feature

<222> (4530)

<223> Start site of the Hela cDNA clone

<220>

<221> intron

<222> (4680) .. (5518)

<220>

<221> DNA

<222> (5519) .. (5834) <223> 2nd exon

<220>

<221> misc_feature

<222> (5826)

<223> AUG start codon

<220>

<221> intron

<222> (5835) .. (8672)

<220>

<221> DNA

<222> (8673) .. (8822)

<223> 3rd exon

<220>

<221> intron

<222> (8823) .. (9168)

<220>

<221> DNA

<222> (9169) .. (9258)

<223> 4th exon

<220>

<221> intron

<222> (9259) .. (9735)

<220>

<221> DNA

<222> (9736) .. (9859)

<223> 5th exon

<220>

<221> intron

<222> (9860) .. (10172)

<220>

<221> DNA

<222> (10173) .. (10290)

<223> 6th exon

<220>

<221> intron

<222> (10291) .. (11280)

<220>

<221> DNA

<222> (11281) .. (11371)

<223> 7th exon

<220>

<221> 3'UTR

<222> (11372) .. (14646)

<223> 3'-UTR

<220>

<221> polyA_signal

<222> (14622) .. (14627)

<223> AAUAAA - PolyA signal <400> 1 cctgcaggtc aacggatcac ctgaggtcac ctgaggtcag gagttcgaga ccagcctgac 60 caacatggtg aaaccccgtc tctactaaaa atacaaaaat tagccgggtg tggtggtgca 120 tgcctgtaat cccagctact tgggaggctg aggcagaaga atcgcttgaa cccgggaggc 180 agaggttgca gtgagccaag atcgcaccat tgcattccag cctgggcagc aagagcaaaa 240 ttccatctca aaaaaaaaaa caagaatacc agaaggggct gggagcagtg gctcacgcct 300 gtaatcccag cactttggga ggccagggcg ggtggatcac gaggtcagga gatcgagacc 360 atcctggcta atatggtgaa accccatctc tactaaaaat acaaaaaatt agcccggcat 420 ggtggtgggc acctgcagtc ccagctactc aggaggctga ggcaggagaa cggcgagaac 480 ccgggaggcg gagcttgcag tgagccgaga tcatgccact gcactccagc ctgggtgaca 540 gagcgagact ccatctcaaa aaaaaaaaaa atatttattt attattttag agaataagtt 600 ttgctgtatc acccaggctg aagtgcagta gcacaatcat gtcttactgc agcttcgaac 660 tcttgggctc aaacaattct cccacctcag cctcctgagt agctaggact acaggcacat 720 gccagcatgc cgggttaatt ttttaatttt ttgtaaacag ggtctcacta tgttgcccag 780 gctggtctta aactcttggc ttcaagcaat cctcccacct cagcctccca aagcactggg 840 attacaggtg tg agccacct cgcctggcca aaatataatg tttttattgt gaatcagatt 900 atttattcta tggtatttta gtgttggtta tattcttata ttttatttat ataatttttg 960 tttgttaatc tgacatctgg tcatgttgct gaactccata gttttgatac attgtcaatc 1020 gtttctcttt gtaagatgaa tatatatgaa atcaaggagg ctttagggtg gccaggagct 1080 ctcctaaaat cttagtttta atagtaaaca tccctggctt gttcctagca aagacagaaa 1140 tgtttcaaat attttcccat aaagcatgac ttttgtctca aaagctgaaa gataagtgaa 1200 aaagagaaaa tattttaaat gcagacgaca aagaatagtt attatccata acacagacag 1260 gatttctacc atcaataaga aatatacaaa tgatctacct ttttttaatg ggcaaaggat 1320 atgaacagac agtttacaaa aacaattaaa atggtcaaag agcaataaaa tagtcccacc 1380 tatttggaag gctgaagtgg gaggatcact tgagctcacg agttggaaac cagcctaagg 1440 caacaaagcg tggagaccca tctatttatt tatttattta tttatttatt ttttgagaca 1500 gagtttcgct cttgttgccc aggctggagt gcaatggcac aatatgggct cactgcaacc 1560 cccgcctccc gggttcaagt gattctcctg cctcagccac ctggtaactg ggattacagg 1620 cacgtgccac caagcccagc taattttttt gtatttttag tagagayggg gtttctccat 1680 gttggcccag ctggtctcaa actcctggcc tcaggtgatc cgcccacctc ggtctcccag 1740 agtgctggga ttacaggcgt gagccaccgt gcctgtccag agacccatct cttaaaaaaa 1800 aaaaaggcaa taagagaata ttaacttcac tattaatgac atgaaaatta aaacaaatac 1860 tatttttcac ccattaaact agtaactgta taaaaaattc atagcataca ctaaacattg 1920 ccaaagttgt aggaataggg aacattcaca catggttctg agtaaacatg ggagttgtta 1980 caaactttta caattcattt tcacagaatc tattaaaatt tacatatccg gccaggcatg 2040 gtggctcaat cctgtaatcc tagcactcta ggaggccgag gccaatggat agcttgagcc 2100 taggaattcg agaccagcct ggggcaacat ggcaaaaccc tgtctctaca aaaaacctaa 2160 aaaacaaaaa aaaaactaca aaattatcca ggcacagttg cccacatctg tagtcccaac 2220 tacttgggag gctaagatgg gaggattcct tgagcccagg aggttgaggc tgcagtgtgc 2280 cctgactgca ccattgcact ccagcctggg cgacagacca agaccctatc tttaaacaga 2340 atgaaaatag ggccgggtgt ggtggctcac acctggaatc ccagcacttt gggaggccaa 2400 ggtgggtaga tgacctgagg tcaggagttc aagaccagcc tgaccaacat ggtgaaactc 2460 catctctact aaaaaataca aaaaaattag ccgggctggc agcaggcgcc agctactcag 2520 gaggctaagc caggagaatt gcttg aacct gggaggtgga ggttgcagtg agccaagatt 2580 gtgccattgc actctagcct gggtgacaga gtgagactcc atctcaaaaa aaaataaaaa 2640 taaaaataaa tttaaataaa agcacatatc ctatctataa atccttataa atgcaaaagg 2700 taactttttt ttcttttttg agacagagtt tcactcttat tgcccaggaa aggcacgact 2760 ctcgcctcac cacaactccc gcctcccggg ttcaagtgat tctcctgact cagcctctcg 2820 agtagctggg attacaggca tgtgccacca cgcccggcta attttgtatt tttagaagag 2880 acgcggtttc tccatgttgg tcaggctggt ctggaactcc cgacctcaga tgatccgccc 2940 acctcggctt cccaacgtgc tgggattaca ggtgtgagcc accctgcccg gagcaaaaag 3000 ataactattt aaggatgttc attattgtat tgtttataaa gcaaaaaaat ctgaaaccta 3060 aatgttcatc aagatgggac aaggcacaca cataaactgg aatactgtgc aacagttttt 3120 taaatgttag ctgactcaga tgtctaaaat acactgtatg gcgcattaca ggtgctcaat 3180 aaatactggt tgaaaagatt tttgtatgaa cacagaaaaa agcctaaaag catatatacc 3240 aaactggtaa gaatgattcc cttcaggagc atgggataag tagaggggga gaataaccat 3300 tttgtgtctt ttatactttt gcgttgtgtc gatttgtaaa ataagtagga gctaattttc 3360 aaaaaagatt taaaagactt attcctggtg atgtttcttt atctctctct ttctccattt 3420 tgtctctatt ttagaatgat cctaaaacac aaaaaacgta ctttcaatgg cccaaatcat 3480 ggagtggctc agggaaatga aatctgtcta caaaaaaaaa aaagttgaga atccttgtct 3540 ttttttcatc atacagtttt tttttttctt ttggtatcac cattacttct cctaaagtca 3600 cgacataata tcacctacag cagaaatgag gacttcagtg gaattgacat ctccttccat 3660 caagcgcatt tgcagtccat ctttagcagg acctctctct aatggctaag gctaggcaga 3720 agaatttgaa gacagtctgg tttccatttt cctaatgcag cctggcccaa gagccaggac 3780 tcccataaga ctagtctact gtgggagtca gggtcttttc cacgtgtgga ggctagtgtt 3840 tgatgctgga cggcctggag taaagggcga aggggagaag gtaaccaacc aggctggcct 3900 cgggattcgc agaggagggc tggggctttg acttctgcag ggaggtgagc agtgtggcca 3960 agggctactc aaggaacgcc tctgcgagaa ttcagactgt ggttgagcgc acaaagcaag 4020 agtgtgcgaa gaaaagttaa cgcctctccc agctgacccc aaaatctgct aatctgggcc 4080 tgacggaaaa aattcggaga agaaccacct cggccgagtt acgtgaaaag gctgggtacg 4140 aactactctg gctctacttg gggcgccggc tcagagcgat gggcaggacg tccgcgagga 4200 agaaagagtg acgatgattt ttcaaggagt ctgaaccagc tcacccagca aaccctcctc 4260 ccatcgagca cccagcgtac aatcatcgaa gggccgaggg tcctgccctc agtctcgatg 4320 gcggccgggg cgcgccccgg ccacggcgca cgcgccggcc tcagcgctcc ctcctccatg 4380 ccctcggcgc acggcgccgc ccactcaccc agctgcacct ctccgtgcgt ccccaagctc 4440 acccgctctg aggctgcccc cgctagtcta gctgccgagc ccggaagtgg atgcagccgc 4500 tcggcggaga ggacgagggg gagggggaag tgcttccggg gaaacggacc ggggttggtg 4560 tttgta aact tgcctcggtc ccggtggggg cagccgcggc ggtggggttg gcagggtgtg 4620 ctggggcctg gaggaggcgc cgcgcggcca gggagccagc gggaggccgc gcctggcagg 4680 taggagcaag ccccaaagac cgcagcgtcg tccgtacaga cggcagcgct tcagtagctc 4740 gcaagccgtg gggtgccgcg cgggaagggg gaggggaggg gaggaggggc gcgtggcgaa 4800 gggaggggga tctccggggg agggagggga aaggccgtgc gcgcacaccg ccccctgccc 4860 gcgcgtcccg ccgcgtcccc ggcccgggag agggctctcg gaggggacgc gggcgcgagc 4920 acgcacctcg cgcgcttctc ggcactctcc ggctctgctc ccggttggtg cagcgggtgc 4980 agaggctggc tggccggagc gaggcgggcg acacgccgcg ccgggcttcg cctgcctgtc 5040 ctccacctcc gcagcaagtt tccctacctt cgggcggacg gggagaacgc acacgtgtgg 5100 gcactgccag tcccgtttct gcccgccacc gaagcaccca gcgatggccc ttgagctttc 5160 ctccacccta gtcttgccgc ctctgtaaag tgcgagcttt tcgggccggc ccactgggca 5220 caaaagcccc aaagggtctc cgcccccttc ttccattcct gagatggcaa atccaagcaa 5280 ctttcctttt ggcagagttc ctccatagga ggccccaatt acttttgctc ctgttggctt 5340 ccactccttg cccaggagac cagttcctcc tattgaccta gaaagaaaac tcttcttgac 5400 tcatgtcctc g ttctgagct tcacagagag cgcccagcaa aaaaaaaatt catatcgcac 5460 gttgtaatgg cctgaagatg ttgcatttag cagcccccct cttcctttct ggtttcaggt 5520 ttctgaaaca gatcgtgagc ttcatcaaga gaattcaact ggaaaaccaa gactggtaga 5580 ctctcttttt cttcagacaa taggcaggag ccaggcagag tccagggatt cttggaacac 5640 ctatcttttc ttcggaggac actaagttct atttgaagac aaagttcaat atggcaacag 5700 gactgatggg acacgaagga gtcgctaccg tgatttggtg acagttcttc aaaacgacag 5760 tgtctcaagg aaaggtggac ctaggaactc ctgaactttt gggttgcctt aagtgagaaa 5820 tcac _f catggc tcaggtaaaa agctctagag tcctcctcca agataactgg gaagcctgtt 5880 tgcagtttca ctcttgaatg ctgccttagt caacatattg accaacccca gtgatccaag 5940 ctctgtatta agcaccaggg ggagatctag aggaaggaaa ggagacaggc cacagcccct 6000 gccttcaggg agcttccagt caaatgaagg aggtaggcta tactgcatca caggcactga 6060 tgggacattt gcaaagcaac gtgtactcag tttacatttg gtacattaag tgctgagggg 6120 tgtaagggag tgaatagtac ggtgaatgct tcttttgttt ttttccaaca ggaatttatt 6180 gagttcctgt tgcatgcaag gcattgtgct gggaatacaa agatgaatac gattgtcctt 6240 gtccccagtg AGCT tccagg ctagttgggg tcgggagagg gatggaaagc catttatttt 6300 tatattcaca atgccttgta tagtggctgg ctagtagtag agactctgaa tctgttgatt 6360 gaggaatgtg caataatagg ggtatgtaca taggagagaa cagtacttct gcttggatag 6420 aaaaagaaga tggaggttga aagatgcata ggattttgct aaacaagcga gaagggtgtt 6480 tggacaaaag gaacagcatg ggcaggagct tagagtcatg ggaaaatgtg atgaggttcc 6540 gagtactgca ggttgtttga tagttacgga gtaagggatt taaatgggga tagttatgga 6600 ggtagtattg acagtgatag gtgaggaagt tgttccctgc agtaagcaga gtgtaaccca 6660 aaggcctttt atgcctcgct aagttaagca gcagagtagc atcagatcag tgttatgtag 6720 aaggtcatta ttgtggaggt agaggatgga ctggagagac agaatgagca gataggctgt 6780 tgtagtcatc tgagtagaat agctatgtgc acacatctcc agagcttctc ccagcttgct 6840 tctagggaac ctccctacca gtctcaccag gatggcagtc tgattgtagt ataattggct 6900 ttcctccagc tgcttctgaa tgcacagctt cctcctttcc atatagaata ggagcctgtc 6960 agtttcttct tcctttgtgt tttgcaaccc acttctcagc attgtattca gtcttctgag 7020 aatgctgcct tccactaagg actgaacttt gtgctagaca ctgttaagcg ttttacgtct 7080 tagtttactt ttcatcaacc ctgtaaggat aagcattatg acaggcattt ttcatgtaag 7140 gaaactaagg cttgcaaagg tggaataact tgcccaagat tatagagtgc aaacagctga 7200 gctaagattt taactctgga tctttcactc caaagtccca gaaggtggag ggaggggtcc 7260 cagaacgtac tgatcatcta aaaggaattt cttcataaat ttgtgagttt gctgaccaac 7320 gatttcttaa aatttttttt ttcagtaaga aaagtatttt ctttaatgcc ggctaaccga 7380 agtcagtttg aagaatgtag cagttttctg gaaatttact taaacatacc ttacttaagt 7440 aattgtttaa attatatatg gagcaaggaa tgtgaaaaaa aatgtgatca tacaagcaca 7500 catataaatg gcctaagagg tcaagagaaa tgctgaactc caaaaatcat atcagttgca 7560 attggtaaat acttagggag tttaaaatct aaagggagaa atagcctgta gtcccaggac 7620 cttaggagac tgaggctgga ggattgttta agctcaggag ttccacacca gcctaggcaa 7680 catggtgaaa ccccgtctct ataaatacaa aaaaaaatag ccgggtgtgg tggcattctc 7740 ctgaagtccc agttactcag gaggtagagg tagaaggatc gcttaagcca gggaggtcaa 7800 gagtgcagtg agccgagatc gtgccactgc actccagcct gggcgacaga gtgacaccct 7860 gtctcaacca acaagcaaaa aatgaaacaa accgatagaa aatacagttg gcaccagcaa 7920 gatctgccaa tcaaaagtgg gctctgaagc taaggggggt gtgtgtgtgt gtgtgtgtgt 7980 gtgagtgacg gagggattct cgctctgttg cccaggctgg agtgcagtgg cgcgatttcg 8040 gctcactgca gcctccgcct cccgggttca agcaattctt ctgcctcagc ctcccgagta 8100 gctgggaccg caggtgtgtg ccaccacacc tggctaattc ttgtattttt agtatagaca 8160 aggtttcacc tattgtccag gcttgtctcg aactcctgac ttcgtgatcc gcccgcctca 8220 ttctcccaaa gtgctgggat tataggtgtg agccactgca ctcggcccag ctgagggctg 8280 ttgagggagg aaggaggaag gtcctgaagg aatctcctgt gtcagtgggt tagcaagatg gaacct 8340 cata cccactcaca gttaaggcct agaaatacct tcaaagaaat atgtagccac 8400 acactgccta gtgctgatga caaaggtgtc agtaatactt gattatttac ttgatctata 8460 tttaaatgct gaaggatgac aagataagtc gttcctggtg agatgagtta gggaggactc 8520 ttgaaggaag caaggctttg gcagtgagga gggacattcc ctggagagaa gcagtttgta 8580 gcagggtgta aaaggtagtt ctatagagga tggcagtctg tttttgccct gccctgccag 8640 gtgacctgac acctcatctc ctttcaactt aggcaagtct cctggcttgt gaaggcctag 8700 caggtgtgag tttggttccc actgcagcca gcaagaagat gatgctgagc cagattgcca 8760 gcaagcaggc cgagaatggc gagcgggcag gtagccctga tgtgctgagg tgctcgagtc 8820 aggtacagcg cttgagtcca ttgtggcacc tggggatcgg gtggcccaag ctctctgcca 8880 ggagatacca agctctgatt ccttgatgct tcctcatagg gaagtggggc aaggggatcc 8940 aactggggaa tggatgaaca ttggtatttc agaaagctaa ggaaggtctt ttgtgtgcct 9000 tcaactcttt taagaccagg tgcttggaga ggaaatagtt gtatacttcc tctaagcttt 9060 cggggcaggt ggtttactct catttatgaa ttttgtctga gagctgcatt gggtctcatc 9120 tcatgttccg agatgggcta tgagtttaga tttgatttgt cttcaaaggg tcaccgaaaa 9180 gacagtgata a gtcccggag ccgcaaagat gatgacagct tgtctgaggc ctctcattca 9240 aaaaagactg ttaaaaaggc aggtaatggg gtgatccccc aaactcatac agtttggtaa 9300 tggggtaata ttcctccttc ttccctctac catgtaacct ggagctttcc ctagatgctt 9360 tgatctctga tagccagtga gtgagtcccc cagatttcat ccagagacta ccttggtagt 9420 tgatctcctg ccttgctgga acagtaatgg tacaggttgg ggagcatgag atcccagatc 9480 ccgaggcatg ggagcaggca ggctccagct caagtcatac tcagactgct gggtttgaca 9540 ggatagacag cttacccact ccacctcctt ccccaaaggg tctcttacct ccatggagta 9600 caggagatgg cagcaccttt agacagcatc agagaggttc ctttctccaa cagctggaag 9660 tggacccaga gtagaacagt gtctccagga agagggcagt ggtggcctca tgacatgcct 9720 gattattgcc cccaggtggt ggtagtggaa caaaatggtt cttttcaagt aaagattccc 9780 aaaaattttg tttgtgaaca ctgctttgga gcctttcgga gcagttacca cctaaagagg 9840 cacatcctta ttcacactgg taagtctctt gtttatattc aaaaggggga gaatagtcta 9900 tcgcttgtat tccatgtctg tttgtggctc tagttgggtt tattttaatg ggtaagtatt 9960 agaaatgaag gaagaagata agtcaatgaa gcagagagat tgcatggctt tgtatctggt 10020 gtcactgaga ccagca cctt tggggacctc tccccattta tgatcactta ggtgtctaca 10080 cattactaag agttaaagtt tctgagaaaa ctggaatgag gtccctactg tacatacctc 10140 cccacattca cttctctgtc cacctgggcc aggtgagaag ccatttgaat gcgatatatg 10200 tgatatgcgt ttcatccaga agtaccacct ggaacgccac aagcgtgtgc acagtggtga 10260 aaagccttac cagtgtgaac ggtgtcatca gtaaggctca tccctgctac aataccccac 10320 acacactttt ccttccccaa gaatcagggc caccctccac tcctgcctga caaaagagcc 10380 accagcgtta ctcgccataa tagttaacac atcggggtga agtgcatctg tctggcctta 10440 tgcagctgat catggggagc agcaattaga acattgtaat aataataata atagcattcc 10500 atatcactta ctctatgcca ggcactgtga gagcatactt tacatagatg attttattta 10560 atcctcacat tgactcccta aggtagctgt attgtcctta tttcacgtat gggaaaattg 10620 agattcagag aagttaagtt tcccagggac accgcagatc acgaacagag ctgggattca 10680 gtccaacttc agagctaatg cactttggag gaagagatac aatctttgcc ctgggggtca 10740 gtatctgatc tgatgagaat gtaagagctg gggccaaaac aagacaagaa caagagatga 10800 gaaatgataa tagctaacac ttcctggatg cattgctagg tcccaagctt tgctctgcac 10860 tcttacact g aatcttcaga cgctagaata aaagctccat gagggcagag actttgttcc 10920 tctggttctc tactctgtat ctgccttgag atagatactc aataaatctt tgatgaataa 10980 agcagctgtg ctgtgagtag gtacttttag cctcgctttg cagttttgca gatgagtatg 11040 cagattctgg gctgttaggt aacttgccca aggtcacaca gctgctgagt gatagagcca 11100 agattcagcc ccaggcggtc tggcctgaga gcttggattc tccaccaccg tgtgctcctg 11160 ctgtctgtgc acgtgctgag gggatggaga atgtggtatc tgcctcctgt gagaggaatg 11220 agttagccaa caggtagctg cgtttgtctg ctcaggcact taaccctccc tttcttctca 11280 gtgtttttct cggacagatc gattactcag acacaaacgg atgtgccaag ggtgccagtc 11340 caagacttcc gacgggcagt tttctctata ggtgcaaggg gccccgggtg gtgggagtga 11400 tcagaagaac ctgccgaaga gcacaccccc tctggtcgat ggtcccacca ccgccccatg 11460 tgaacctgtc ccactcctgg aggagtggac ccaggagacc agaagagtgc atcaggggac 11520 agtggcccca gaacctcagc tctgtaaata atctgagact atgagtatct cggggaagtt 11580 cttacagcat tcctgggtag gggagctagt ccctggaccc ttggctgagg tcataggtgg 11640 ggactcaagg tacaggacca agactgaagt gtggctccca caaccttgga ctccagctgg 11700 cagctgaaat ggaaaagatt ggggaagggg atttgtttgt tctgttcttg tttttgttta 11760 tccaaaagca acttcagcgg gtaaactgtg gatacaggta atccagaggc ataagagtcc 11820 tggtccaaag tagggactat agctggtcca gctctcccca aaaattgggt ttttagttgc 11880 agtagctcct cagtgtttca caatcctgca cttcgcctcc tttttgagga agaataaggt 11940 caggaatggc agctgagctt actttggctt ccttatacac tgtagggaca aaaggaaaaa 12000 cggggtggag cagacaaggg ctggatccaa aaagtggatg gcacctcttc cttcctcaag 12060 ggaaaaggaa gagttgtgcc agtgggtgtt gtcatggccc atgtcacatt ggcacctga t 12120 ggcatcagct caaatgcctt taggaggaaa cttgggtgag agtggcccat gaagcccttt 12180 tcttcttttt ttggggagtc ggtcttggag atggtatgtg gctacctcgc tgccattgac 12240 tcctaaagtg agacagatgt gttgagcaag gagtcatgga agtcaaagtg ggttctctct 12300 ccttgtgccc tcttaggaac aacccaagac tggtcccaag ttggctaaca gctgctcaag 12360 aaacaaagga atctggggtc ttctgacatg cctgttgagc agtaggagct gttctccctc 12420 cccaagtcct atcactgtat tcaggtagag gaggtaggga ggggtctgtc ctttggtccc 12480 aaaaaacaag atctcggctt gcttcctgat ggccatccct acccaacaga gtgtggggga 12540 tgggggtggg ggtgctgcag agaaaagact aaagaagaaa cctaatgtag gttccattct 12600 taaccatatt ctttgctcct ctctgctcca ataccaccac cttgtccact cccaaaaaag 12660 taatcgggtg tgtgtgtgtg tgtgagagag agtgtgtgtg agatatgaat gcatgtattt 12720 gtatgcttgc aggagatggc atattattga gccaaaccct tctctggccc tcaccttcag 12780 ggaggatgag tggaaaatag cctcctcttg cccaaacatg aagggtgctt gaggtctcag 12840 caggagctga agaacacaat ttggggaaca gaagggcttt tgttttggag ggtttttttt 12900 tttaagatga attcaacagc ctggtggtta ggatgtttct a aacccaagcc gggcttgtg 12960 aacatgaatt ttctaaggtg aaataaacct tttctttctg ctaaagaaag aatccttatg 13020 ggaagttttg gggtttggtt gacccccact gtaacctgac ctgacctgtg gcgggtacgg 13080 gaactgtctt agtaatcatg gagactggct gaacctgagt gccaagctgt cagtagggtc 13140 tgagatsgga aactcctgaa agtatgattt catttcccag aaaatgtmaa gaatggatac 13200 cagcaatgat tcctggcwgt gacctcatct ggaggggtgg ggttggggag ggggttgtcc 13260 atacctctgt ggtatgagta tttcagggaa aaagaaagca ggcatggcac ccattcgatt 13320 ttccctgaca gcatctgaga tccttttggg gagacgctga ggagtgtttg ctgccatgta 13380 ctcttacagc tctatgctga cactcccatt tgatgtggtc cagaacttag acctcagtcc 13440 ttggatcagc cctgtggtcc ctgcaagcag gggcatcttt ctgcatgtga gccagccccc 13500 ttcctgttca agggttctgc tggatctggg cttttcctgt ccttcacttc tgggatgatt 13560 caccccacat cttccagtac cctgtaaacc attttaaaat atttagaaaa ctatcctccc 13620 aaaaatgctt ttgaaaatga gagccctctg tccctgccac ttacagctag tctctttggg 13680 ataggggtgt atgtggagag attcatgtaa gtctcacatg agtgacctgt gcccctatgt 13740 gtactaatgt gtgtactggg tcagaaggtg ccctgggttc CCAC agacct tggtttcctg 13800 cctgggtggg tgggagggag ggaacttcag agagtatgtg cgaacagcca tagcagagcc 13860 acccctgcca aggacccagt gagggaaact caggagcagg tggttggttc agttcccgcg 13920 ctgatgtctg gggcagtgca tgtgtgagaa gagaggcgca aagcagtgaa aggagtgatt 13980 tggcatccag agggcccggg agccccacgg ataccccagg gaagccactg actcacaccg 14040 ctgcagaggc tcctgggccc aatgcccgac ccctgctggc cagcatgggg cctagcactg 14100 gaagatgcct caggtgtgtt tggagtgggg gaaacggaag tagggcacgc tctcagagac 14160 caggatagag agtcagggaa cctgggaaag agtagtgcca gttccgggta ggctgctgca 14220 ccaggcccta ccagctcagg ctataaacag tctgctttgc cccaagtctt ttccgtccac 14280 ccccaacagc cttgtcatca cttagctact gatcacgccc atggcttgac attggagggt 14340 tacattagtg gagtccgcca cagcttcgaa ccctctcccc agatgccctt tgcctctgct 14400 gtggccctgg ggttttatca actgtcccct tcctatcctc ctctccaccc tgttttttgt 14460 tttttttttt ttttaaataa tattttgcta tgggggaggg gaatatcaaa tggtggacaa 14520 gatcactttt aaatggtagt ttttataaga tgttataaac ttgtatcttt ttaccattaa 14580 agtgcagtgt atgttccttc gtgatatatt taggata ttt aaataaaagg aaaatggggc 14640 ttttct 14646

<210> 2

<211> 4440

<212> DNA

<213> Homo sapiens <220>

<221> mRNA

<222> (1)

<223> start of transcription (primer extension)

<220>

<221> 5'UTR <222> (109) .. (258) <223> 1st exon

<220>

<221> misc_feature

<222> (109)

<223> Start site of the HeLa cDNA clone

<220>

<221> DNA

<222> (259) .. (> 565)

<223> 2nd exon

<220>

<221> mRNA

<222> (<566) .. (574)

<223> 2nd exon. [Split]

<220>

<221> mRNA

<222> (566)

<223> Translation start

<220>

<221> CDS

<222> (566) .. (1147)

<223> Open reading frame TB7

<220>

<221> mRNA <222> (575) .. (724) <223> 3rd exon

<220>

<221> mRNA <222> (725) .. (814) <223> 4th exon

<220>

<221> mRNA

<222> (815) .. (938)

<223> 5th exon

<220>

<221> misc_binding <222> (873) .. (1006) <223> Zn-finger (2x)

<220>

<221> mRNA

<222> (939) .. (1056)

<223> 6th exon

<220><221> mRNA <222> (1057) .. (1147) <223> 7th exon

<220>

<221> 3'UTR

<222> (1148) .. (4422)

<223> 3'-UTR

<220>

<221> polyA_signal

<222> (4398) .. (4403)

<223> AAUAAA poly A signal

<400> 2 tccgtgcgtc cccaagctca cccgctctga ggctgccccc gctagtctag ctgccgagcc 60 cggaagtgga tgcagccgct cggcggagag gacgaggggg agggggaagt gcttccgggg 120 aaacggaccg gggttggtgt ttgtaaactt gcctcggtcc cggtgggggc agccgcggcg 180 gtggggttgg cagggtgtgc tggggcctgg aggaggcgcc gcgcggccag ggagccagcg 240 ggaggccgcg cctggcaggt ttctgaaaca gatcgtgagc ttcatcaaga gaattcaact 300 ggaaaaccaa gactggtaga ctctcttttt cttcagacaa taggcaggag ccaggcagag 360 tccagggatt cttggaacac ctatcttttc ttcggaggac actaagttct atttgaagac 420 aaagttcaat atggcaacag gactgatggg acacgaagga gtcgctaccg tgatttggtg 480 acagttcttc aaaacgacag tgtctcaagg aaaggtggac ctaggaactc ctgaactttt 540 cgtgtgagaactt gagtcgactt gtccgccc

Met Ala Gin Ala Ser Leu Leu Ala Cys 1 5 gaa ggc cta gca ggt gtg agt ttg gtt ccc act gca gcc agc aag aag 640 Glu Gly Leu Ala Gly Val Ser Leu Val Pro Thr Ala Ala Ser Lys Lys 10 15 20 25 atg atg ctg agc cag att gcc agc aag cag gcc gag aat ggc gag egg 688 Met Met Leu Ser Gin Ile Ala Ser Lys Gin Ala Glu Asn Gly Glu Arg 30 35 40 gca ggt agc cet gat gtg ctg agg tgc teg agt cag ggt cac ega aaa 736 Ala Gly Ser Pro Asp Val Leu Arg Cys Ser Ser Gin Gly His Arg Lys 45 50 55 gac agt gat aag tec egg agc ege aaa gat gat gac agc ttg tet gag 784 Asp Ser Asp Lys Ser Arg Ser Arg Lys Asp Asp Asp Ser Ser Leu Ser Glu 60 65 70 gcc tet cat tea aaa aag act gtt aaa aag gtg gtg gta gtg gaa caa 832 Ala Ser His Ser Lys Lys Thr Val Lys Lys Val Val Val Val Glu Gin 75 80 85 aat ggt tet ttt caa gta aag att ccc aaa aat ttt gtt tgt gaa cac 880 Asn Gly Ser Phe Gin Val Lys Ile Pro Lys Asn Phe Val Cys Glu His 90 95 100 105 tgc ttt gga gcc ttt egg agc agt tac cac cta aag agg cac atc ctt 928 Cys Phe Gly Ala Phe Arg Ser Ser Tyr Hi s Leu Lys Arg His Ile Leu 110 115 120 att cac act ggt gag aag cca ttt gaa tgc gat ata tgt gat atg cgt 976 Ile His Thr Gly Glu Lys Pro Phe Glu Cys Asp Ile Cys Asp Met Arg 125 130 135 ttc atc cag aag tac cac ctg gaa cgc cac aag cgt gtg ca 'c agt ggt 1024 Phe Ile Gin Lys Tyr His Leu Glu Arg His Lys Arg Val His Ser Gly 140 145 150 gaa aag cct tac cag tgt gaa egg tgt cat cag tgt ttt tet egg aca 1072 Glu Lys Pro Tyr Gin Cys Glu Arg Cys His Gin Cys Phe Ser Arg Thr 155 160 165 gat ega tta ctc aga cac aaa egg atg tgc caa ggg tgc cag tec aag 1120 Asp Arg Leu Leu Arg His Lys Arg Met Cys Gin Gly Cys Gin Ser Lys 170 175 180 185 act tec gac ggg ttt cag tet cta day gtgcaagggg ccccgggtgg 1167 Thr Ser Asp Gly Phe Gin Ser Leu 190 tgggagtgat cagaagaacc tgeegaagag cacaccccct ctggtcgatg gtcccaccac 1227 cgccccatgt gaacctgtcc cactcctgga ggagtggacc caggagacca gaagagtgca 1287 tcaggggaca gtggccccag aacctcagct ctgtaaataa tetgagaeta tgagtatetc 1347 ggggaagttc ttacagcatt cctgggtagg ggagetagte cctggaccct tggetgaggt 1407 cataggtggg gacteaaggt aeaggaceaa gactgaagtg tggctcccac aacettggae 1467 tccagctggc agctgaaatg gaaaagattg gggaagggga tttgtttgtt ctgttettgt 1527 ttttgtttat ccaaaagcaa ettcageggg taaaetgtgg atacaggtaa tccagaggca 1587 taagagteet ggtccaaagt agggaetata gctggtccag ctctccccaa aaattgggtt 1647 tttagttgca gtagctcctc agtgtttcac aatcctgcac ttcgcctcct ttttgaggaa 1707 gaataaggte aggaatggca gctgagctta etttggcttc cttataeact gtagggacaa 1767 aaggaaaaac ggggtggagc agaeaaggge tggatccaaa aagtggatgg cacctcttcc 1827 ttcctcaagg gaaaaggaag agttgtgcca gtgggtgttg tcatggccca tgtcacattg 1887 geaectgatg gcatcagctc aaatgeettt aggaggaaac ttgggtgaga gtggcccatg 1947 aagecetttt ettetttttt tggggagtcg gtcttggaga tggtatgtgg ctacctcgct 2007 gecattgaet cctaaagtga gacagatgtg ttgageaagg agtcatggaa gtcaaagtgg 2067 gttctctctc cttgtgccct ettaggaaea acccaagact ggteceaagt tggctaacag 2127 ctgeteaaga aacaaaggaa tctggggtct tetgacatge ctgttgagca gtaggagctg 2187 ttctccctcc ccaagtccta teactgtatt caggtagagg aggtagggag gggtetgtec 2247 tttggtccca aaaaacaaga teteggettg cttcct gatg gccatcccta cccaacagag 2307 tgtgggggat gggggtgggg gtgetgeaga gaaaagacta aagaagaaac etaatgtagg 2367 ttecattett aaccatattc tttgctcctc tctgctccaa taccaccacc ttgtccactc 2427 ccaaaaaagt aatcgggtgt gtgtgtgtgt gtgagagaga gtgtgtgtga gatatgaatg 2487 eatgtatttg tatgettgea ggagatggca tattattgag eeaaacectt ctctggecct 2547 cacetteagg gaggatgagt ggaaaatagc cteetettgc ceaaacatga agggtgcttg 2607 aggtctcagc aggagctgaa gaacacaatt tggggaacag aagggctttt gttttggagg 2667 gttttttttt ttaagatgaa ttcaacagcc tggtggttag gatgttteta acccaagcca 2727 gggcttgtga acatgaattt tctaaggtga aataaacctt ttetttetge taaagaaaga 2787 ateettatgg gaagttttgg ggtttggttg acccccactg taacctgacc tgacctgtgg 2847 cgggtacggg aactgtetta gtaatcatgg agactggctg aaectgagtg ccaagctgtc 2907 agtagggtct gagatsggaa actectgaaa gtatgatttc attteccaga aaatgtmaag 2967 aatggatacc ageaatgatt cctggcwgtg acctcatctg gaggggtggg gttggggagg 3027 gggttgtcca taectetgtg gtatgagtat ttcagggaaa aagaaagcag gcatggcacc 3087 cattegattt tccctgacag catetgagat ccttttgggg agaegetgag gagtgtttgc 3147 tgecatgtae tettacaget etatgetgae actcccattt gatgtggtcc agaaettaga 3207 cctcagtcct tggateagee ctgtggtccc tgeaagcagg ggeatettte tgcatgtgag 3267 ccagcc CCCT tcctgttcaa gggttctgct ggatctgggc ttttectgte cttcacttct 3327 gggatgattc accccacatc ttccagtacc ctgtaaacca ttttaaaata tttagaaaac 3387 tatcctccca aaaatgcttt tgaaaatgag agccctctgt ccctgccact tacagetagt 3447 ctctttggga taggggtgta tgtggagaga tteatgtaag teteacatga gtgacctgtg 3507 cccctatgtg taetaatgtg tgtactgggt eagaaggtge cctgggttcc cacagacctt 3567 ggtttcctgc ctgggtgggt gggagggagg gaaetteaga gagtatgtge gaacageeat 3627 ageagageca cccctgccaa ggacccagtg agggaaactc aggagcaggt ggttggttca 3687 gttcccgcgc tgatgtctgg ggcagtgcat gtgtgagaag agaggegeaa agcagtgaaa 3747 ggagtgattt ggeatecaga gggcccggga gccccacgga taccccaggg aagccactga 3807 ctcacaccgc tgeagagget cctgggccca atgcccgacc cctgctggcc agcatggggc 3867 etagcactgg aagatgeetc aggtgtgttt ggagtggggg aaaeggaagt agggeaeget 3927 ctcagagacc aggatagaga gtcagggaac ctgggaaaga gtagtgccag ttccgggtag 3987 gctgctgcac caggccctac cagctcaggc tataaacagt ctgctttgcc ceaagtettt 4047 tccgtccacc cccaacagcc ttgteateae ttagetactg atcacgccca tggettgaea 4107 ttggagggtt a cattagtgg agtccgccac agettegaae cctctcccca gatgecettt 4167 gcctctgctg tggccctggg gttttatcaa ctgtcccctt cctatcctcc tctccaccct 4227 gttttttgtt tttttttttt tttaaaataat attttgetatatgggagattatgatgggatat taccattaaa gtgcagtgta tgttccttcg tgatatattt aggatattta aataaaagga 4407 aaatggggct tttctaaaaa aaaaaaaaaa aaa 4440

<210> 3

<211> 193

<212> PRT

<213> Homo sapiens

<400> 3

Met Ala Gin Ala Ser Leu Leu Ala Cys Glu Gly Leu Ala Gly Val Ser 1 5 10 15

Leu Val Pro Thr Ala Ala Ser Lys Lys Met Met Leu Ser Gin Ile Ala 20 25 30

Ser Lys Gin Ala Glu Asn Gly Glu Arg Ala Gly Ser Pro Asp Val Leu 35 40 45

Arg Cys Ser Ser Gin Gly His Arg Lys Asp Ser Asp Lys Ser Arg Ser 50 55 60

Arg Lys Asp Asp Asp Ser Leu Ser Glu Ala Ser His Ser Lys Lys Thr 65 70 75 80

Val Lys Lys Val Val Val Val Glu Gin Asn Gly Ser Phe Gin Val Lys 85 90 95

Ile Pro Lys Asn Phe Val Cys Glu His Cys Phe Gly Ala Phe Arg Ser 100 105 110

Ser Tyr His Leu Lys Arg His Ile Leu Ile His Thr Gly Glu Lys Pro 115 120 125

Phe Glu Cys Asp Ile Cys Asp Met Arg Phe Ile Gin Lys Tyr His Leu 130 135 140

Glu Arg His Lys Arg Val His Ser Gly Glu Lys Pro Tyr Gin Cys Glu 145 150 155 160

Arg Cys His Gin Cys Phe Ser Arg Thr Asp Arg Leu Leu Arg His Lys 165 ^• 170 175

Arg Met Cys Gin Gly Cys Gin Ser Lys Thr Ser Asp Gly Gin Phe Ser 180 185 190

Leu

<210> 4

<211> 193

<212> PRT

<213> Homo sapiens

<220>

<221> PROPEP <222> (1) .. (193) <223> TB7

<220> <221> BINDING <222> (103) .. (123) <223> Zn-Finger 1

<220>

<221> BINDING <222> (131) .. (151) <223> Zn finger 2

<400> 4

Met Ala Gin Ala Ser Leu Leu Ala Cys Glu Gly Leu Ala Gly Val Ser 1 5 10 15

Leu Val Pro Thr Ala Ala Ser Lys Lys Met Met Leu Ser Gin Ile Ala 20 25 30

Ser Lys Gin Ala Glu Asn Gly Glu Arg Ala Gly Ser Pro Asp Val Leu 35 40 45

Arg Cys Ser Ser Gin Gly His Arg Lys Asp Ser Asp Lys Ser Arg Ser 50 55 60

Arg Lys Asp Asp Asp Ser Leu Ser Glu Ala Ser His Ser Lys Lys Thr 65 70 75 80

Val Lys Lys Val Val Val Val Glu Gin Asn Gly Ser Phe Gin Val Lys 85 90 95

Ile Pro Lys Asn Phe Val Cys Glu His Cys Phe Gly Ala Phe Arg Ser 100 105 110

Ser Tyr His Leu Lys Arg His Ile Leu Ile His Thr Gly Glu Lys Pro 115 120 125

Phe Glu Cys Asp Ile Cys Asp Met Arg Phe Ile Gin Lys Tyr His Leu 130 135 140

Glu Arg His Lys Arg Val His Ser Gly Glu Lys Pro Tyr Gin Cys Glu 145 150 155 160

Arg Cys His Gin Cys Phe Ser Arg Thr Asp Arg Leu Leu Arg His Lys 165 170 175

Arg Met Cys Gin Gly Cys Gin Ser Lys Thr Ser Asp Gly Gin Phe Ser 180 185 190

Leu

Claims

1. Isolated, enriched or purified nucleic acid, characterized in that it codes for a TB7 polypeptide or contains the nucleic acid coding for a TB7 polypeptide.

2. Nucleic acid according to claim 1, characterized in that the nucleic acid has a nucleotide sequence which a) codes for a polypeptide with the amino acid sequence of SEQ ID NO: 3 or 4 or a functional part thereof; b) is complementary to the nucleotide sequence of (a); c) hybridizes under highly stringent conditions with the nucleic acid of (a) and codes for a functional TB 7 polypeptide or a functional part or derivative thereof.

3. Nucleic acid according to claim 1 or 2, characterized in that it has the sequence of SEQ ID NO: 2 which corresponds to the cDNA of TB7 and it is part of the genomic DNA of SEQ ID NO: 1.

4. Nucleic acid according to one or more of the preceding claims, characterized in that it is isolated from a mammal, the mammal preferably being a human.

5. Nucleic acid according to one or more of the preceding claims, characterized in that it is present in an expression vector for expressing the TB7 polypeptide or a functional part or derivative thereof in a host cell.

6. Nucleic acid according to one or more of the preceding claims, characterized in that it has the cDNA of TB 7.

7. Nucleic acid according to one or more of the preceding claims, characterized in that one or more of the bases are modified due to the degeneration of the genetic code.

8. Nucleic acid according to one or more of the preceding claims, characterized in that it is a fragment which codes for a domain or an epitope of the TB7 polypeptide.

9. Nucleic acid probe for detecting the nucleic acid coding for a TB7 polypeptide or a functional part or derivative thereof in a sample.

10. RNA derived from a nucleic acid according to one or more of the preceding claims.

11. Isolated enriched or purified polypeptide, characterized in that it is encoded by a nucleic acid according to SEQ ID NO: 1 or SEQ ID NO: 2 or the A- amino acid sequences SEQ ID NO: 3 or 4 or a functional part or derivative thereof.

12. Polypeptide according to claim 11, characterized in that one or more of the amino acids are replaced by another amino acid, one or more amino acids are deleted and / or one or more amino acids are additionally contained, the function of TB7 as a repressor of the DNA Replication of EBV or the function of the domain or epitops is retained.

13. Polypeptide according to one or more of the preceding claims with the amino acids 103 to 151 acc. SEQ ID NO: 4.

14. Isolated, purified or enriched TB7 polypeptide, functional parts and derivatives thereof.

15. Antibody or antibody fragment with a specific binding affinity to a TB7 polypeptide or a TB7 polypeptide domain or a TB7 polypeptide epitope.

16. Antibody according to claim 15, characterized in that it is a monoclonal antibody.

17. Hybridoma, characterized in that it is an antibody with a specific binding affinity to a TB7 polypeptide or

TB7 polypeptide domain or epitope produced.

18. Recombinant cell, characterized in that it expresses a TB7 polypeptide or a functional part or derivative thereof.

19. Recombinant cell, characterized in that it contains a nucleic acid tional for a recombinant polypeptide or a func TB7 _¬ part or derivative thereof encoded.

20. Recombinant cell according to claim 18 or 19, characterized in that it is a prokaryotic cell, preferably an E. coli cell.

21. Recombinant cell according to claim 19 or 20, characterized in that it is a eukaryotic cell, preferably a B cell.

22. A method for inhibiting the replication of EBV, characterized in that a cell containing the EBV genome is brought into contact with the TB7 polypeptide or a functional part or derivative thereof.

23. The method according to claim 22, characterized in that the bringing into contact by expression of a TB7 polypeptide or functional part or derivative thereof in the infected cell.

24. Pharmaceutical composition, characterized in that it contains a TB7 polypeptide or a functional part or derivative thereof in a pharmaceutically effective amount, together with conventional carriers and auxiliaries.

25. Use of a TB7 polypeptide or a functional part or derivative thereof for inhibiting the replication of gamma herpes viruses, in particular EBV and HHV8, and beta herpes viruses, in particular cytomegalovirus.

26. Use of a TB7 polypeptide according to claim 25 for the treatment of diseases associated with gamma herpes viruses, in particular EBV and HHV8, and beta herpes viruses, in particular whose cytomegalovirus are associated.

27. Use according to claim 26 for the treatment of Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinoma.

28. A method for producing a TB7 polypeptide or a functional part or derivative thereof according to one or more of the preceding claims, characterized in that a recombinant cell which contains a nucleic acid which codes for a TB7 polypeptide or a functional part or a derivative thereof , wherein the nucleic acid coding for the TB7 polypeptide or the functional part or a derivative thereof is under the control of an inducible promoter which controls the expression of the TB7 polypeptide or the functional part or derivative, the expression of the polypeptide or its is cultivated --- functional part or derivative takes place and then the TB7 polypeptide or the functional part or the derivative of TB 7 is obtained.

29. Vector containing a nucleic acid according to one or more of the preceding claims.