WO2004008152A1

WO2004008152A1 - Method for screening ebna-1 inhibitors

Info

Publication number: WO2004008152A1
Application number: PCT/JP2003/008972
Authority: WO
Inventors: Kazuo Yanagi; Sayuri Itou
Original assignee: Fujisawa Pharmaceutical Co Ltd
Current assignee: Fujisawa Pharmaceutical Co Ltd
Priority date: 2002-07-15
Filing date: 2003-07-15
Publication date: 2004-01-22
Anticipated expiration: 2005-01-15
Also published as: AU2003281109A1

Abstract

The present application provides a method for screening for inhibitors that block the interaction between EBNA-1 and host cellular replication machinery. In the present invention, the inhibitor is selected based its ability to inhibit the localization of EBNA-1 in relation to chromatin in the cells. The candidates selected by the method are useful for therapeutic agent for treating a latent infection of Epstein-Barr Virus (EBV).

Description

DESCRIPTION

METHOD FOR SCREENING EBNA-1 INHIBITORS

Technical Field

The present invention relates to a method for selecting an agent which inhibits the binding between Epstein-Barr virus (EBV) nuclear protein 1

(EBNA-l) and chromatin; a therapeutic agent for latent infection of EBV using the selected agent; and a therapeutic agent for a disease caused by the latent infection of EBV.

Background Art

A virus belonging to the family Herpesviridae latently infects a host after primary infection and coexists without attacking the host. The depression of host immunity results in re-activation of the virus, which, for example, causes a disease, such as shingles, or recurrence of genital herpes. Such similar harboring nature is also known in the family Papillomaviridae, in addition to in the family Herpesviridae. Known therapeutic agents for treating these viruses include, for example, acyclovir and ganciclovir. These agents inhibit viral replication by inhibiting viral nucleic acid synthesis. As readily predicted based on the mechanism of action, available agents such as acyclovir which is representative of antiviral agents, have no effect on latently infected viruses. Thus, these therapeutic agents can only suppress viral propagation but cannot eliminate latent viruses.

Summary of the Invention

In order to eliminate a latent virus, it is important to establish an inhibitor of the virus replication that is believed to expand synchronously with host cell proliferation under latent state or an agent that blocks the establishment of viral latency. A complete cure for such virus infections can be achieved by suppressing the recurrence by using such an agent in combination with conventional anti-viral agents, such as inhibitors of viral DNA synthesis. With respect to a latently infected, virus in an episomal state of herpes virus, a viral protein, for example, a protein that translocates to the nucleus in the host cell, has been reported to participate in the binding between the episome and host chromatin. The protein is assumed to play an important role in episome maintenance in latent infection. The present inventors found that a singly expressed viral nuclear-localization protein was present in the newly replicated DNA region immediately after DNA replication of host chromatin. The inventors also revealed that this protein colocalized well with several host cellular proteins constituting the DNA replication machinery . The present inventors noted that these new findings correlate to the function of a previously reported viral protein that translocates to the nucleus. Then, the inventors predicted that latent virus genome could be treated with inhibiting the targeting of proteins to the newly replicated DNA region of the host chromatin. Specifically, the inventors conceived that a latent infection- blocking agent could be selected through the screening for an agent directly or indirectly inhibiting the co-localization and interaction. Accordingly, a complete cure for viral infection can be achieved by using an agent selected as described above.

The present inventors continued to study the mechanism for the localization of viral nuclear-targeted protein to chromatin in latent state. The inventors used, as a viral model, Epstein-Barr virus (EBV) belonging to the family Herpesviridae. As a result, the inventors demonstrated that EBNA-1 participated in the binding of the episome of EBV to host chromatin. Furthermore, the inventors confirmed that EBNA-1 was closely involved in the DNA replication of host cell, and revealed that EBNA-1 functionally participated and played an essential role in the replication of latent viral DNA. The functions of EBNA-1, which were identified by the present inventors, were based on an unidentified mechanism.

EBNA-1 , nuclear localization signal-containing protein coded in EBV genome, plays an essential role in the maintenance of an EBV plasmid in cells. Among EBNAs, only EBNA-1 has a sequence-specific DNA-binding activity, and has been believed to participate in the replication and maintenance of the EBV plasmid through the binding to a specific sequence located at the EBV replication origin, OriP.

EBV is a virus known to be involved in lymphoma (Burkitt's lymphoma, etc.), Hodgkin's disease, infectious mononucleosis, nasopharyngeal cancer and other diseases. EBNA-1 (GenBank Accession No.:V01555) is a transcription factor and is essential for the replication and maintenance of viral genome DNA in the nucleus of infected cell. EBNA-1 is expressed in every cell type latently infected with EBV and in every EBVpositive tumor cell type. There are some reports describing analyses to identify the domains required for the binding of EBNA-1 to host chromatin (Vincent Marechal et al, J. Viol, 73:4385-4392 (1999), Siu Chun Hung et al., PNAS, 98:1865-1870 (2001)). However, there is no report on the relationship between EBNA-1 and chromatin replication in host cells. It is important to reveal the relationship between EBNA-1 and chromatin replication in order to identify the functions of EBNA-1.

Recently, EBNA-1 was reported to bind to cellar chromatin on the chromosome in M phase. This report merely states that EBNA-1 was detected in a chromatin fraction prepared biochemically. Because there is no established experimental method for isolating intact cellar chromatin, this report only suggests the possibility of binding between EBNA-1 and chromatin.

The present inventors newly demonstrated the presence of co- localization of EBNA-1 with newly replicated DNA region of cellar chromatin. According to the finding obtained by the present inventors, EBNA-1 not only binds the viral plasmid to the cellar chromosome but also contributes to the maintenance of the viral genome. In particular, EBNA-1 was estimated to function as a factor enabling a single-round of viral genome replication per cell cycle.

In other words, EBNA-1 was newly suggested to have the functions described below, in addition to the previously identified function of structurally linking the viral genome to cellar chromosome in the mitotic phase (M phase):

- interacting with the newly replicated DNA region in infected cells;

- maintaining latent infection; and - inducing tumorigenic cell transformation, which produces EBV-positive tumor cells, and maintaining the expression of the phenotypes.

Thus, an agent that inhibits the localization of EBNA-1 to the newly replicated DNA region can ehminate the latent infection. Such an agent can be used as an effective pharmaceutical agent to treat the intractable diseases caused by EBV. Brief Description of the Drawings

Figure 1. Construction and expression of GFP-EBNAl. (A) The construct of GFP-fusion of EBNA-1 lacking the Gly Ala copolymer. The regions of EBNA-1 that are essential for its binding to cellular metaphase chromosomes (Marechal et al., 1999) are indicated between the maps of EBNA-1 and GFP-EBNAl. (B) Immunoblots of EBNA-1 from whole cell extracts of CHO-K1 cells expressing full-length B95-8 EBNA-1 (lane l), and GFP-EBNAl (lane 2) using the anti-EBNA-1 mouse monoclonal antibody OTlx and alkaline phosphatase- conjugated rabbit anti-mouse IgG antibody. The GFP-EBNAl of 84 kDa and B95-8 EBNA-1 protein of 78 kDa is produced in CHO-K1 cells. (C) Laser scanning microscopy images of GFP-EBNAl- expressing -CHO-K1 cells in mitotic phase and interphase. Shown are profiles of GFP-EBNAl (al), and DNA (a2; stained with Hoechst dye 33258), and their merged image (a3), and Nomarski image (a4), in a hving round GFP-EBNAl-expressing CHO-K1 cell in mitotic phase. Also shown are profiles of the GFP-EBNAl protein in hving GFP- EBNAl-expressing CHO-K1 cells in interphase (bl) and its Nomarski image (b2), and profiles of non-fused full-length EBNA-1 in CEW21N4 cells that were immunostained using the anti-EBNAl rat monoclonal antibody 2B4-1 and FITC- conjugated secondary antibody: immunostaining (cl) and Nomarski image (c2). Figure 2. Localization of GFP-EBNAl in relation to chromatin being condensed in interphase GFP BNAl-expressing CHO-K1 cells by the

'premature chromosome condensation' (PCC) reagent calyculin A. (A) Photomicrographs of a living cell taken immediately before the addition of calyculin A show GFP-EBNAl (al), and chromosomal DNAs (a2; stained using Hoechst dye but shown in pseudo-color red); the merged image is shown in (a3). Also shown are GFP-EBNAl and chromatin undergoing PCC, and their merged images taken, respectively, 20 min (bl-b3) and 60 min (cl^_c3) after the addition of calyculin A. Cells shown in the photograph sets (a), (b), and (c) are different ones in the same culture dish. Photomicrographs of control GFP in CHO-K1 cells taken 0 and 40-60 min after the addition of calyculin A are shown in (d) and (e, f), respectively. (B) Localization of GFP-EBNAl in relation to me thanol- acetic acid-fixed chromatin prematurely condensed by Calyculin A. Calyculin A-induced prematurely condensed chromosomes (PCCs) were fixed with Carnoy's fixative and spread on glass slides. GFP fluorescence was restored by renaturation with borate buffer (pH 8.5) and PBS. Merged images of GFP-EBNAl and DNA of PCCs (stained using Hoechst dye but shown in pseudo-color red) are shown in (a-f); The observed profiles are typical of Gl/S-phase PCCs (a), S-phase PCCs (b, c), and G2-phase PCCs (d, e, f). A Nomarski image of calyculin A-induced PCCs that were Giemsa- stained is shown in (g). This Nomarski image taken from the same glass slide that was examined by confocal microscopy in (a-f) shows various forms of PCCs indicating interphase stages of the cell cycle; solid arrowhead indicates PCCs typical of early S, arrow indicates PCCs typical of middle S, open arrowhead indicates PCCs typical of G2 (g). Similarly merged laser scanning photomicrographs of CHO-Kl cells expressing GFP as a negative control are shown in (h), indicating no association of GFP with PCCs. Figure 3. Localization of GFP-EBNAl in relation to pulse-labeled or replicated regions of chromatin that was prematurely condensed. (A)

Bromodeoxyuridine pulse-chase-labeled and prematurely condensed chromatin in GFP-EBNAl-expressing CHO-Kl cells. BrdU pulse- chase labeled PCCs were prepared as described in Materials and Methods. Images of GFP- EBNAl and of BrdTJ-labeled chromatin are shown in (al) and (a2), respectively! the merged image is shown in

(a4). The image in (a2) is merged with an image stained for DNA in (a3). Arrow indicates PCCs typical of S phase. Images of GFP- EBNAl and BrdTJ-labeled chromatin of another set of PCCs typical of G2-phase are shown in (bl) and (b2), respectively, and their merged image is shown in (b3). (B) Localization of GFP-EBNAl in hving cells in relation to Cy3-dUTP pulse-labeled chromatin and PCC. Cy3-dUTP-labeling of chromatin and PCC were performed as described in the text, (a) Images of GFP- EBNAl, Cy3-labeled chromatin in a GFP-EBNAl-expressing CHO-Kl cell, the merged image, and a Nomarski photograph of the same cell are shown. Sequential photographs of PCCs in the same living cell that were taken at the indicated time after the addition of calyculin A for PCC are shown below, (b) From left to right are shown images of GFP- EBNA1, Cy3-labeled chromatin in another GFP-EBNAl-expressing CHO-Kl cell, the merged image, the merged image overlayed on the

DNA stained with Hoechst dye 33258, and the co-localization image that shows the highly co-localized spots in white: the white spots selected by the LSM510 Co-localization option indicate the ones whose intensities of GFP-EBNAl (green) and Cy3"dUTP (red) are both high. An enlarged image of a portion indicated by a bar in the merged image is shown in (bl). Also shown is a figure drawn by the LSM510 Profile option; an arrow in magenta in the same merged image indicates the line and direction of the Profile option (b2). Photographs of PCCs in the same living cell that were taken at 10 and 25 min after the start of PCC are shown below.

Detailed Description of the Invention

The present invention provides a method selecting an agent that inhibits the binding between host chromatin and EBNA-1. The present invention also provides a method selecting an agent that inhibits the localization of EBNA-1 to the newly replicated DNA region. Furthermore, the present invention relates to therapeutic agents for treating the latent infection of EBV, which comprises an agent selected by the selection method, and therapeutic agents for lymphoma, Hodgkin's disease and nasopharyngeal carcinoma caused by EBV infection. Specifically, the present invention relates to a method for selecting an agent that inhibits the binding of EBNA-1 of EBV to the chromatin of interphase cells in vitro, which comprises the steps of (l) to (3) described below:

(1) contacting a test substance with a host animal cell into which EBNA-1, or a peptide containing the functionally active portion thereof, has been introduced;

(2) determining the localization of EBNA-1 or the peptide containing the functionally active portion thereof in the interphase cell of the host animal; and (3) determining the frequency of localization of EBNA-1 or the peptide containing the functionally active portion thereof on the chromatin. There is no limitation on the type of animal cell containing EBNA-1 introduced, which is to be used in the present invention, so long as the cell expresses EBNA-1. For example, such cells include an EBVinfected cell and an animal cell which has been transformed with an expression vector for EBNA-1. The term "expression vector for EBNA-1" refers to an animal cell containing and capable of expressing a gene encoding EBNA-1 or a peptide containing the functionally active portion thereof. Such expression vectors for EBNA-1 include inducible expression vectors, as the matter of course. As used herein, the phrase "a peptide containing the functionally active portion of EBNA-1" refers to an EBNA-1 -derived peptide that has activity of binding to the host animal cell chromatin. For example, a peptide lacking the glycine-alanine co-polymer portion of EBNA-1 is an example of a peptide containing the functionally active portion of EBNA-1. The localization of EBNA-1 in animal cells can readily be observed, for example, by using a gene encoding a fusion protein consisting of EBNA-1 and a reporter protein. The proteins listed below can be used as the reporter proteins. It is preferable to use a fluorescent protein among others, because of the convenience of detection. - luciferase derived from fire fly;

- fluorescent proteins, such as GFP and EGFP;

- alkaline phosphatase;

- horse radish peroxidase!

- enzyme proteins, such as beta-galactosidase; - various antigen proteins and peptides, such as FLAG tag, which can be detected based on the antigen-antibody reaction.

An expression plasmid containing such a gene as an insert can be prepared by conventional methods. An expression vector for EBNA-1, which has been prepared as described above, can be introduced into an appropriate animal cell, for example, by using a transformation technique, such as electroporation or lipofectamine method. Such animal cells include, for example, the cell lines listed below^:

- animal cells: CHO cell, COS7 cell, Vero cell, MDCK cell, etc.; and

- human cell lines: HeLa cell, HEK293 cell, Raji cell, etc. Various cells derived from human tissues, such as primary cells, can be used in addition to the cell lines. It is also possible to use cells isolated from blood, such as lymphocytes, mononuclear cells and macrophages.

A test substance is contacted with a host animal cell obtained by introducing EBNA-1 into it by the procedure as described above. There is no limitation on the type of test substance to be used, so long as the substance is a compound whose activity can be assessed by the assay system of the method according to the present invention. Specifically, any compounds, for example, lowmolecular-weight organic compounds, lo -molecular- weight inorganic compounds, macromolecules including proteins and nucleic acids, and saccharides, can be used as the test substances. Furthermore, mixtures of the compounds described above, natural products, synthetic products, extracts of animals, plants, fungi, algae, microorganisms can be used as the test substances.

There is no limitation on the method for contacting an animal cell with a test substance. Typically, the two can be contacted by incubating the cell culture medium in the presence of the test substance for several minutes to several hours, before or after transformation. Alternatively, when the test substance is a protein, the protein can be contacted with the animal cell by co- transfecting an expression vector containing the gene encoding the protein into the above-mentioned animal cell.

Methods for observing the intracellular localization of EBNA-1 include a method of detecting EBNA-1 by itself using an anti-EBNA-1 antibody and a method of detecting a reporter protein. When a fluorescent protein, such as GFP, is used as the reporter protein, living cells or cells fixed by a conventional method can be observed, for example, using a fluorescent microscope, such as a confocal fluorescent laser microscope. When an enzyme protein, such as alkaline phosphatase, is used as the reporter protein, or alternatively when the antigen-antibody reaction is utilized, typically the localization can be observed using a light microscope, fluorescent microscope or such, after staining by a conventional method.

The interphase chromatin is not condensed, and thus is difficult to observe. Hence, an efficient method used to observe interphase cells preferably includes the step of pre-condensing the chromatin of animal cells containing EBNA-1 introduced before observing the cells. There are many methods for condensing chromatin, including, for example, a method using a phosphatase inhibitor, such as calyculin, fostriecin and okadaic acid. Such treatment results in premature chromosome condensation in the cell nucleus, and thus facilitates the detection. In the absence of the test substance, molecules of EBNA-1 are found as spots on chromatin, when the intracellular localization of EBNA-1 is studied using such a method. Changes resulting from the presence of the test substance are assessed using micrographs obtained through microscopic observation or results of densitometric assay based on the micrographs. Specifically, when the localization of EBNA-1 onto chromatin is observed to be inhibited, the test substance can be concluded to inhibit the localization of EBNA-1 onto chromatin.

The term "interphase" normally refers to a period between a first M phase and the next M phase. The interphase is subdivided to GI phase, S phase and G2 phase. The interphase according to the present invention includes all the phases described above. Furthermore, the above-mentioned selection method of the present invention, preferably, further comprises the steps of (4) to (5) described below:

(4) determining the intracellular localization of newly replicated DNA region on the chromatin of interphase cells of the host animal; and

(5) assessing the degree of identitybetween the two types of localization described above.

The phrase "newly replicated DNA region" in the step described above refers to a region where DNA is newly synthesized to replicate the host chromosome. The "newly replicated DNA region" can also be defined as a region where labels are detected immediately after pulse-labeling. The DNA pulse-labeling can be carried out using, as substrates for DNA synthesis, nucleic acids labeled with a fluorescent dye or radioisotope. The DNA replication is believed to be achieved by the chromosome -replication machinery (replisome) which comprises the DNA polymerase complex and others derived from the host cell. The interphase chromatin is not condensed, and therefore, like the binding of EBNA-1 to the chromatin, the newly replicated DNA region is difficult to observe during interphase. Thus, in the present invention, it is preferable to observe the region after the chromatin is condensed. There are many methods for condensing chromatin, which include, for example, a method using a phosphatase inhibitor, such as calyculin, fostriecin and okadaic acid. Such treatment results in premature chromosome condensation in the cell nucleus, and thus facilitates the detection.

Specific examples of the labeled nucleic acid to be used to label the newly replicated DNA region in animal cells by the procedure described above include BrdU, Cy3-dUDP and Cy5-dUDP. Radioisotopes can be used as labels, but fluorescently labeled nucleic acids are preferred for their convenience of detection and sensitivity. When BrdU or the like is used, the detection can be achieved by using an anti-BrdU antibody after cell fixation. The sensitivity can be improved by using a secondary antibody.

Such animal cells containing newly replicated DNA region labeled, which are obtained by the procedure described above, can be observed by any suitable method known in the art. For example, fluorescently labeled cells can be observed, as living cells or after fixation, for the localization of newly replicated DNA region, by using a fluorescent microscope, such as a confocal fluorescent laser microscope. The intracellular localization of EBNA-1 and the localization of newly replicated DNA region in the cells can be compared, for example, by assessing the degree of agreement of localization on the photo-images obtained through observing the cells under a microscope. In addition, the degree of agreement can be quantified by densitometry or such. According to such assessment procedure, the test substance is concluded to inhibit the direct or indirect binding of EBNA-1 to the newly replicated DNA region when the degree of agreement is low. Such a test substance can be used as an agent for inhibiting the latent infection of EBV or as a therapeutic agent for lymphoma or such caused by the latent infection of EBV. The present invention revealed that a protein of a herpesvirus is localized in the newly replicated region of host chromatin. A protein, encoded by genome of a herpesvirus which translocates into the cellular nucleus and which localizes in the newly replicated region of host chromatin, when introduced and expressed in the cell, can be used as a target molecule of a therapeutic agent for the latent virus.

Such a protein can be identified through studying the localization of viral proteins to the newly replicated DNA region. Specifically, the present invention provides a screening method for a protein localized in newly replicated region, which is encoded by a gene of a virus belonging to the family of herpesvirus, and which translocates into the cell nucleus and is localized in - li ¬

the newly replicated region of host chromatin, when introduced and expressed in the cell, wherein the method comprises steps of (l) to (4) described below:

(1) separately introducing each of genes encoding proteins having the nuclear localization signal, which are genes of viruses belong to the family Herpesviridae, into a host animal cell;

(2) determining the localization of the expressed protein in the interphase cell;

(3) determining the localization of the newly replicated DNA region of chromatin DNA in the interphase cell; and (4) assessing the degree of colocarization of the above3) and 4)-.

In the present invention, there is no limitation on the gene encoding a protein having the nuclear localization signal. The nuclear localization signal comprises a specific amino acid sequence. Thus, it can be assessed through amino acid sequence analysis whether a protein has the nuclear localization signal. Like EBNA-1 described above, such a protein having the nuclear localization signal can be expressed in any animal cell, when an expression vector containing the gene encoding the protein is introduced into the cell. As is the case with EBNA-1 described above, various reporter genes can be used to observe the localization of the protein. The localization of a newly replicated DNA region can also be determined by the same procedure as described above. As described above, condensing chromatin is advantageous to observe the localization.

A protein suitable for the screening method of the present invention can be used in a method for selecting an agent which inhibits the binding between a target protein derived from a virus belonging to the family Herpesviridae and the chromatin of interphase cell of the host animal. The screening method of the present invention can be practiced using the same principles as the above-described screening method for an agent, which inhibits the binding between EBNA-1 and chromatin. More specifically, the screening method of the present invention can be practiced by using, instead of EBNA-1, a protein derived from a virus in family Herpesviridae, which is obtained by the screening method of the present invention. Specifically, the present invention provides a method for selecting an agent which inhibits the binding between the chromatin of the host animal interphase cell and a target protein derived from a virus in the family Hrepesviridae. The protein selected by the screening method described above, can be substitutive instead of EBNA-1.

This method comprises the steps of (l) to (3) described below. In each of the steps described below, the term "target protein" refers to a protein selected by the method of the present invention, which is encoded by a gene of a virus belonging to the family of herpesvirus and localized in newly replicated DNA region and which translocates into the cell nucleus and localizes in the newly replicated DNA region of host chromatin, when introduced and expressed in the cell: (l) contacting a test substance with a host animal cell into which the target protein has been introduced;

(2) determining the localization of the target protein in the interphase cell of the host animal; and

(3) determining the frequency of localization of the target protein on the chromatin.

The present invention also provides a method for selecting an agent which inhibits the binding between the chromatin of the host animal interphase cell and a target protein derived from a virus in the family of Herpesviridae, which is selected by the screening method described above, which additionally comprises the following steps of (4) and (5):

(4) determining the intracellular localization of the newly replicated DNA region in chromatin of the host animal interphase cell; and

(5) assessing the degree of agreement between the localization of the target protein and the newly replicated DNA region of chromatin. When the localization of the target protein is observed to be inhibited in comparison with that occurring in the absence of the test substance, the test substance is deemed to have the activity of inhibiting the target protein. Like EBNA-1 of EBV, a protein selected by the method of the present invention, which is encoded by a gene of a virus in the family Herpesviridae, and which translocates into the cell nucleus and is localized in the newly replicated DNA region of host chromatin, when introduced and expressed in the cell, plays an important role in the maintenance of latent infection. Thus, a therapeutic effect on the infection of virus in the family Herpesviridae can be provided by inhibiting the localization. The present invention also provides an inhibitor of the latent infection of EBV or a therapeutic agent for treating a disease caused by the latent infection of EBV, which comprises as an active ingredient a compound obtained by the selection method described above. Furthermore, the present invention relates to an inhibitor of the latent infection of EBV or a therapeutic method for treating a disease caused by the latent infection of EBV, which comprises the step of administering a compound obtained by the selection method described above. In addition, the present invention relates to the use of a compound obtained by the selection method described above in producing an inhibitor of the latent infection of EBV or a therapeutic agent for treating a disease caused by the latent infection of EBV.

Furthermore, the present invention provides an inhibitor of the latent infection of a virus in the family Herpesviridae or a therapeutic agent for treating a disease caused by the latent infection of herpesvirus, which comprises as an active ingredient or a compound obtained by the selection method described above. The present invention also relates to an inhibitor of the latent infection of a virus belonging to the family of Herpesviridae or a therapeutic method for treating a disease caused by the latent infection of a virus belonging to the family Herpesviridae, which comprises the step of administering a compound obtained by the selection method described above. In addition, the present invention relates to the use of a compound obtained by the selection method described above in producing an inhibitor of the latent infection of a virus belonging to the family of herpesvirus or a therapeutic agent for treating a disease caused by the latent infection of a virus belonging to the family Herpesviridae. When a compound obtained by the selection method of the present invention described above is used as a pharmaceutical product, the compound may be used by itself as a pharmaceutical product or can be pharmaceutically formulated. The compound can be used, for example, in appropriate combination with a pharmaceutically acceptable carrier or medium, specifically, known material, such as sterilized water, physiological saline, vegetable oil, emulsifier and suspension, in a format of pharmaceutical composition, for example, solid, semi-solid or liquid (for example, tablet, pill, troche, capsule, suppository, cream, ointment, aerosol, powder, solution, emulsion, suspension, etc.). Such a preparation can be given to a patient, suitably by the nasal route or ophthalmic route of administration, externally (locally), by the rectal route or pulmonary route of administration (nasal or oral injection), orally or by a parenteral route of administration (including subcutaneous, intravenous and intramuscular injections) or by inhalation. An injection can be given, for example, by a known method, such as intraarterial injection, intravenous injection and subcutaneous injection. The dose depends on patient's weight, age and condition, the type of administration method, etc.; one skilled in the art can readily determine what constitutes an adequate dose.

Example

The present invention is illustrated in more detail below with reference to Examples, but should not be construed as being limited thereto.

Cells and construction ofEBNA -1 -GFP fusion protein. The CHO-Kl cell line was obtained via the Japanese Cancer Research Resources Bank (Tokyo), originally from ATCC (U.S.A.). A truncated EBNA-1 lacking the Gly- Ala copolymer was cleaved from plasmid p205 (Yates et al., 1985) with BamΗI and Hώdlll, and inserted into the pEGFP"C3 vector (CLONTECH, U.S.A.) at its Bgl L and Hind III sites (Fig.lA).

Premature chromosome condensation. For laser scanning microscopy (LSM) of PCC in living cells, cells were grown in a glass-bottomed dish (Iwaki, Japan), calyculin A (Wako, Japan) was added to 50 nM after incubation in the presence of 20 μM Hoechst dye 33258, and the cells were kept under microscopy to observe PCC continually. For spreading the individual prematurely condensed chromosomes (abbreviated as PCCs), calyculin A- treated cells were swollen in 0.075 M KC1, fixed using Carnoy's fixative or me thanol- acetic acid (3 to l), and then dropped on to glass slides. The GFP fluorescence that was lost through using the acidic Carnoy's fixative was restored by incubating PCCs in 0.1 M borate buffer (pH 8.5) at room temperature for 30 min, followed by incubation in PBS at 4°C overnight according to Ward and Bokman (Ward & Bokman, 1982). Fixed PCCs were stained with 1 μg /ml Hoechst dye 33258 in PBS. Giemsa staining has been previously described (Gotoh, 1995).

Laser scanning and confocal microscopy. Digital images of the fluorescent profiles were acquired on an Axiovert 100M laser scanning microscope equipped with argon and helium-neon laser light sources using a software of the LSM510 system including the palette option (Zeiss, Germany). The peak UV excitation wavelengths were 351 and 364 nm. An objective lens of 63 x /w corr. was used.

BrdU labeling and indirect imm unoΔuorescence antibody analyses. Cells were incubated in medium containing 10 μM BrdU (Sigma) for 30 min, and then in BrdU-free medium for 3 h, at 37°C . These BrdU pulse-chase labeled cells were treated with calyculin A, swollen under hypotonic conditions, fixed with methanol-acetic acid (3:i), and spread on glass slides. Their fluorescence of GFP was recovered as described above. The glass slides were treated with 100 units/ml DNase I (Takara, Japan) in 100 mM sodium acetate (pH 5.3), 5 mM magnesium sulfate, at 37°C for 60 min according to Carayon and Bord (Carayon & Bord, 1992). The PCCs were then incubated with an anti-BrdU mouse monoclonal antibody (MBL, Japan) and then with Texas red- conjugated donkey anti-mouse IgG antibodies (Jackson Laboratories, U.S.A.). Cy3-dUTP labeling. Cy3-dUTP (Pharmacia, Sweden) was incorporated into monolayer cells according to the methods of McNeil and Warder (McNeil & Warder, 1987) and Manders et al. (Manders et al., 1999). Briefly, Cy3"dUTP was added at 10 μM to cells, and 425-600 μm diameter glass beads (SIGMA chemicals, U.S.A.) were immediately sprinkled onto the cells. The cell culture covered by beads was tapped, the beads were washed out immediately, and the cells were incubated in fresh medium.

GFP -EBNA-1 fusion protein co-localizes with cellular chromatin that is prematurely' condensed during interphase. The GFPΕBNA-1 fusion protein lacking the Gly-Ala copolymer (designated GFP-EBNAl), which is not required for replication/maintenance of EBV DNA (Yates et al., 1985) or for metaphase chromosome binding (Marechal et al., 1999) was expressed in CHO-Kl cells; chromosomes of CHO-Kl cells are more clearly visible owing to their small number (20 per cell) and correspondingly large size. The GFP-EBNAl protein of 84 kDa was detected in extracts from GFP-EBNAl-expressing CHO-Kl cells by western blotting (Fig. IB). GFP-EBNAl was localized in intranuclear granules/spots, which are similar to those of full-length B95-8 EBNA-1 that was expressed in CHO-Kl cells (Fig. 1C). Laser scanning microscopy (LSM) confocal images of GFP-EBNAl and DNAs showed that GFP-EBNA-1 co- localized with mitotic chromosomes in living cells, in agreement with the results of Marechal et al. (Marechal et al., 1999) (Fig. 1C, a). To visualize chromatin in interphase cells, we took advantage of the method of PCC. PCC was discovered following the cell fusion of interphase cells with mitotic ones (Johnson & Rio, 1970, Rio, 1977), and recently chemical procedures have been developed to induce PCC using the phosphatase inhibitors calyculin A, fostriecin, and okadaic acid (Coco-Martin & Beg, 1997, Gotoh, 1995, GAO et al., 1995). These reagents induce PCC at a given stage of the cell division, bringing about various forms of PCCs that are characteristic of each phase or stage of cell cycle (Albeit & Rapports, 1999, Coco-Martin & Beg, 1997, Gotoh, 1995). We analyzed the distribution pattern of EBNA-1 throughout the process of calyculin A-induced PCC in living GFP-EBNAl-expressing CHO-Kl cells by maintaining a dish of cell culture under the microscope. GFP-EBNAl protein was concentrated together with chromosomal DNAs onto PCCs throughout the PCC process, as shown in the early (Fig. 2A, b) and the final stages (Fig. 2A, c) of PCC (note that cells shown in (a), (b), and (c) are different cells from the same dish). In contrast, control GFP remained diffuse after treatment with calyculin A (Fig. 2 A, e, f).

To analyze more precisely the association of GFP-EBNAl with the PCCs, we spread the PCCs onto glass slides to observe the individual PCCs; spread PCCs from GI, S, and G2 phases are clearly distinguishable from each other. The fluorescence profiles of GFP-EBNAl associated with the spread PCCs showed that GFP BNA-1 was present in fibers and strands (Fig. 2B, a- f). We processed the same glass-slides for Giemsa staining, and re-examined their Nomarski images to obtain more distinct profiles (Fig. 2B, g). These images exhibited fibrous PCCs typical of early S phase (Fig. 3B, g, solid arrowheads), partly thin and partly thick PCCs typical of middle S phase (Fig. 2B, g, arrow) and bivalent thick PCCs typical of G2 phase (Fig. 2B, g, open arrowhead), as described previously (Gotoh, 1995, Johnson & Rio, 1970, Rio, 1977). Together, these photomicrographs show that GFP-EBNAl co-localized with PCCs that were most probably formed from Gl/S phase chromatin (Fig. 2B, a), S phase chromatin (Fig. 2B, b and c), and G2 phase chromatin (Fig. 2B, d-f); in contrast, control GFP was not detected on the spread PCCs (Fig. 2B, h). The intensity of GFPΕBNAl relative to that of the Hoechst dye varied greatly across the spread PCCs (Fig. 2B, d-f), suggesting that EBNA-1 is not associated uniformly with interphase chromatin. In addition, there was no difference in the profiles of Giemsa stained and spread PCCs between control CHO-Kl and GFP-EBNAl-expressing CHO-Kl cells was observed, suggesting that GFP-EBNAl does not affect the chromosome condensation process. These results show that GFP-EBNAl co-localized not only with cellular metaphase chromosomes but also with prematurely condensing chromatin and or chromosomes in these calyculin A treated-interphase cells.

GFP-EBNA-1 co-localizes with BrdU-pulse-chase-labeled regions and with Cy3-dUTP pulse -labeled regions of cellular chromatin. To analyze further the co-localization of EBNA-1 with chromatin in S-phase, we pulse- chase-labeled GFP-EBNAl-expressing CHO-Kl cells with BrdU, treated them with calyculin A, and fixed and spread the PCCs on glass slides. GFP-EBNAl was highly co-localized with the fibrous BrdU-labeled and immunostained PCCs that were typical of S phase (Fig. 3A, a, arrow) and typical of G2 phase (Fig. 3A, b), suggesting that EBNA-1 is associated with replicated regions of chromatin.

Next, we pulse-labeled cellular DNAs with Cy3-dUTP to examine the localization of GFP-EBNAl on PCCs in living cells. The Cy3"dUTP-labeled patterns were similar to those reported by Sedona et al. (Sedona et al., 1999). Confocal LSM of Cy3-dUTP-pulse-labeled GFP-EBNAl-expressing CHO-Kl cells, which were maintained in culture medium throughout the microscopic observations, demonstrated that EBNA-1 co-localized well with the Cy3-dUTP- labeled regions of cellular chromatin in living cells (Fig. 3B, a, b, the top rows); the same cells were treated with calyculin A, continually observed under microscope, and sequential photographs of PCCs of the same cells were taken. The photographs taken at the indicated time after the addition of calyculin A indicated that GFP-EBNAl remained associated with the Cy3-dUTP-labeled recently replicated re ions of chromatin throughout the PCC process (Fig. 3B). The binding of EBNA-1 to metaphase chromosomes is considered to be involved in the segregation of a low copy number of EBV plasmids into dividing cells (Hung et al., 2001, Marechal et al., 1999). EBNA-1 of herpesvirus papio and LANA of Kaposi's sarcoma- associated herpesvirus, which are necessary for the persistence of these viral episomes also bind to mitotic chromosomes (Ballestas et al., 1999, Piolot et al., 2001).

In the studies presented here, we have shown that EBNA-1 co- localizes with cellular chromatin that is condensed during interphase in the absence of αri DNA, by using a combined approach of confocal microscopy of a GFP-EBNA-1 fusion protein, PCC, and pulse labeling of cellular replicating DNAs. The results of our studies in living cells expand previous biochemical data showing that EBNA-1 is found in chromatin fractions from both EBV latently infected cells (Petti et al., 1990) and cells containing no αri -plasmids (Kanda et al., 2001), because isolation or purification of intact chromatin has difficulties (reviewed in (Cook, 2001)). Moreover, we have shown that GFP- EBNAl is highly co-localized with recently replicated regions of cellular chromatin in S phase in particular. EBNA-1 might bind to a cellular chromatin chromosome protein during normal condensation in the mitotic prophase, but it seems less likely because our observations in this study showed that GFP-EBNAl highly co-localized with not only the PCCs that were formed during G2 phase but also those formed during GI- and S phases. Another possibility that GFP-EBNAl bound to PCCs only after the induction of premature condensation is also less likely, because GFP-EBNAl was as highly co-localized with Cy3-dUTP-labeled regions of cellular DNAs even before the induction of PCC as during and after the PCC process (Fig. 3B). Thus, we conclude that EBNA-1 is associated with interphase chromatin in living cells, and in particular with its newly replicated regions. Further studies on the interaction of EBNA-1 with chromatin/chromosomes and their components during the course of the cell division cycle should provide better understanding of the molecular mechanism of the multiple functions of this key protein in EBV latency.

Industrial Applicability

An agent which inhibits the binding of EpsteήrBarr virus (EBV) nuclear protein 1 (EBNA -l) to chromatin can be identified using the screening method of the present invention. An agent so identified finds utility in the treatment of the latent infection of EBV. It cannot be anticipated that an inhibitor of DNA synthesis has a therapeutic effect on a virus during the latent period. The agent provided by the present invention can be used to treat infections of a virus, such as EBV, which are difficult to treat only with a DNA synthesis inhibitor.

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Claims

1. A method for selecting an agent which inhibits the binding of EBNA-1 of EBV to the chromatin of the host animal interphase cell, which comprises the following steps of (l) to (3):

(2) determining the localization of EBNA-1 or the peptide containing the functionally active portion thereof in the interphase cell of the host animal; and

(3) determining the frequency of localization of EBNA-1 or the peptide containing the functionally active portion thereof on the chromatin.

2. The method according to claim 1 for selecting an agent which inhibits the binding of EBNA- 1 of EBV to the chromatin of the host animal interphase cell, which comprises the following steps of (l) to (5): (l) contacting a test substance with a host animal cell into which EBNA-1, or a peptide containing the functionaUy active portion thereof, has been introduced; (2) determining the localization of EBNA-1 or the peptide containing the functionally active portion thereof in the interphase cell of the host animal; and (3) determining the frequency of localization of EBNA-1 or the peptide containing the functionally active portion thereof on the chromatin. (4) determining the intracellular localization of the newly replicated DNA region in chromatin of the host animal interphase cell; and (5) assessing the degree of identtiy between the localization of the target protein and the newly replicated DNA region of chromatin.

3. The method according to claim 1 or 2, wherein the peptide containing the functionally active portion of EBNA-1 is a peptide lacking the glycine- alanine co-polymer of EBNA-1.

4. The method according to claim 1 or 2, which comprises the step of condensing chromatin before determining the localization of EBNA-1.

5. An inhibitor of the latent infection of EBV or a therapeutic agent for a disease caused by the latent infection of EBV, which comprises as an active ingredient the agent selected by the method according to claim 1 or 2.

6. A screening method for a protein that is encoded by a gene of a virus belonging to the family of herpesvirus, and which translocates into the cell nucleus and is localized in the newly replicated region of host chromatin when introduced and expressed in the cell, which comprises the following steps of (l) to (4):

(1) separately introducing each of genes encoding proteins having the nuclear localization signal, which are genes of viruses belong to the family Herpesviridae, into a host animal cell,;

(2) determining the localization of the expressed protein in the interphase cell of the host animal,;

(3) determining the localization of the newly replicated DNA region of chromatin in the interphase cell of the host animal,; and

(4) assessing the degree of colocalization of the above 3) and 4).

7. A method for selecting an agent which inhibits the binding between the chromatin of the host animal interphase cell and a target protein derived from a virus belonging to the family of herpesvirus selected by the screening method according to claim 6, wherein a target protein derived from a virus belonging to the family of herpesvirus selected by the method according to claim 4 is used instead of EBNA-1 in the screening method according to claim 1 or 2.