JP2009506025A - Antiviral agent and viral replication inhibitor - Google Patents

Abstract

  The present invention relates to an antiviral agent comprising a zinc finger protein and / or a nucleic acid encoding the zinc finger protein for inhibiting the binding of an animal DNA virus-derived replication protein to the viral replication origin.

Description

  The present invention relates to an antiviral agent and a viral replication inhibitor against animal DNA viruses.

  One of the conventional methods for inhibiting the binding of the virus replication protein to the replication origin is, for example, a method using a “dominant negative mutant” of the protein, which is a mutant having only the DNA binding site of the virus replication protein. (Orozco et al. (2000) J. Biol. Chem., 275: 6114-6122). Such dominant negative mutants inhibit protein binding to the origin of replication which, when bound to the viral origin of replication, induces viral replication initiation.

  Since the binding capacity of the dominant negative mutant is almost the same as that of the target virus replication protein, a large amount of dominant negative mutant is required to effectively inhibit binding to the replication origin by competition between the mutant and the wild type replication protein. Mutants must be expressed. However, there is a problem that it is extremely difficult to express a sufficient amount of the mutant protein in vivo to inhibit the binding to the replication origin. Also, even if a dominant negative mutant can be expressed at a high level, such a high level causes cytotoxicity due to non-specific binding of the mutant to a sequence other than the desired target sequence. It may be. There is a need for DNA binding proteins with high affinity because they can be used at lower expression levels.

  The present inventor has established a design and production method of an artificial DNA binding protein (zinc finger protein: ZFP) that recognizes a specific genomic sequence {Special Table 2004-519211 Publication; Sera, US Patent Application Publication 2003 / 0082561A1 Sera et al. (2002) Biochemistry, 41: 7074-7081}. These reported ZFPs seek to provide a means to control viral infections in plants using reasonably designed ZFPs. For example, prevention of plant DNA virus infection by artificial ZFP has been reported by Sera (2005) J. Virology, 79: 2614-2619.

  The Sera patent application discloses that ZFP functions as an antiviral agent and a viral replication inhibitor against DNA and RNA viruses. Information on the designed ZFP and its use is also given in WO 03/62455. However, none of the data in these applications demonstrates or challenges the design of ZFPs with sufficient specificity to inhibit DNA virus replication in the presence of wild-type (or natural) viral replication proteins. Absent.

  An object of the present invention is to provide an antiviral agent and a viral replication inhibitor against viruses harmful to animals including humans. In order to achieve the above object, the present invention provides that the virus replication can be effectively suppressed by inhibiting the binding of the replication protein of animal virus to the replication origin of the virus. Based on such binding inhibition, an excellent antiviral agent can be obtained.

  According to the present invention, the following antiviral agent and viral replication inhibitor are provided, which contain, as an active ingredient, a substance that inhibits the binding of a replication protein derived from an animal DNA virus to the replication origin of the virus. It is a protein. In particular, according to one aspect of the present invention, there is provided an antiviral agent comprising an artificial zinc finger protein (ZFP) and / or a nucleic acid encoding the ZFP, wherein the ZFP is an origin of replication of the virus of a replication protein derived from an animal DNA virus. Can be inhibited. In some embodiments, the replication protein can have more than one binding site at the origin of replication of the DNA virus. In such cases, the ZFP can bind to one or more of the binding sites and inhibit binding of the replication protein at the origin of replication.

In some embodiments, the antiviral agent is for human papillomavirus, preferably HPV18, and may be for other high risk HPVs such as HPV16, 31, 35, 39, 45, 51, 52, 58, or 59. Good. A preferred target replication protein for HPV is E2 protein, and the artificial ZFP binds at, near or overlaps with the position of E2 binding site 3 (E2BS3) and / or E2 binding site 4 (E2BS4). In a preferred embodiment, the antiviral agent against HPV targets DNA represented by GAAAACGGTCGGGACCGAA or GGTCGGGACCGAAAACGGT.
In some embodiments, the antiviral agent ZFP comprises six zinc finger domains, wherein the domains are covalently linked by 0 to 10 amino acid residues. For these ZFPs, the amino acids at positions 1, 2, 3, and 6 of the α-helix of the zinc finger domain (which may be referred to as recognition amino acids) are in this order Gln, Thr for domain 1, respectively. , His and Glu;
Glu, Ser, Asn and Arg for domain 2;
Arg, Asp, His and Glu for domain 3;
Thr, Ser, His and Arg for domain 4;
Glu, Asp, Asn and Gln for domain 5; and Gln, Thr, Asn and Arg for domain 6
It is. Or the amino acids at positions 1, 2, 3, and 6 of the domain are Arg, Asn, His and Glu for domain 1 in this order;
Gln, Ser, Asn and Gln for domain 2;
Gln, Thr, His and Glu for domain 3;
Glu, Ser, Asn and Arg for domain 4;
Arg, Asp, His and Glu for domain 5; and Thr, Ser, His and Arg for domain 6
It is. For example, these recognized amino acids have the following formula:
_{-X 3 -Cys-X 2-4 -Cys-} X 5 -Z -1 -XZ 2 -Z 3 -X 2 -Z 6 -His-X 3-5 -His-X 4 -
(Wherein Z ^-1 , Z ² , Z ³ and Z ⁶ represent amino acids at positions ¹ , ² , ³ and ⁶ of the α-helix of the zinc finger domain). .
In a most preferred embodiment, the ZFP comprises the amino acid sequence of SEQ ID NO: 2 or 3.

In addition, the antiviral agent of the present invention contains ZFP, which ZFP contains at least three zinc finger domains covalently bound to each other by 0 to 10 amino acid residues, and -1,2, The amino acids at positions 3 and 6 are selected as follows:
The amino acid at position -1 is arginine, glutamine, threonine, methionine, or glutamic acid;
The amino acid at position 2 is serine, asparagine, threonine, or aspartic acid;
The amino acid at position 3 is histidine, asparagine, serine, or aspartic acid; and
The amino acid at position 6 is arginine, glutamine, threonine, tyrosine, leucine, or glutamic acid.

Similarly, the antiviral agent of the present invention comprises ZFP, which ZFP comprises at least three zinc finger domains, each zinc finger domain independently having the formula:
_{-X 3 -Cys-X 2-4 -Cys-} X 5 -Z -1 -XZ 2 -Z 3 -X 2 -Z 6 -His-X 3-5 -His-X 4 -
(X independently represents any amino acid, _Xn represents the number of occurrences of X in the polypeptide chain;
Z ^-1 is arginine, glutamine, threonine, methionine, or glutamic acid;
Z ² is serine, asparagine, threonine, or aspartic acid;
Z ³ is histidine, asparagine, serine, or aspartic acid; and
Z ⁶ is arginine, glutamine, threonine, tyrosine, leucine, or glutamic acid. The domains are independently covalently linked to each other at 0 to 10 amino acid residues.
In the above ZFP, the ZFP may further comprise a nuclear localization signal (NLS) and / or a cell penetrating peptide (CPP), which are located at the N- or C-terminus of the ZFP open reading frame (ORF) be able to.

In another aspect of the present invention, any of the above ZFPs or nucleic acids encoding these ZFPs is an antiviral agent, and the antiviral agent is a ZFP or nucleic acid according to any one of claims 1 to 17.
Yet another aspect of the invention relates to the use of any ZFP of the invention and / or a nucleic acid encoding the ZFP for the manufacture of an antiviral agent.
According to another aspect of the present invention, the prevention and / or prevention of animal viral infections wherein the viral infection is treated, reduced or ameliorated by administering to the animal a prophylactic and / or therapeutically effective amount of the antiviral agent of the present invention A method of treatment is provided.

  Furthermore, another aspect of the present invention is a method for inhibiting replication of an animal virus in the body of an animal, comprising administering an effective amount of the ZFP of the present invention and / or a nucleic acid encoding the ZFP of the present invention. It relates to a method comprising the step of inhibiting replication. Preferably the method is used to inhibit human DNA viruses in humans, which have high cancer such as HPV, preferably HPV 16, 18, 31, 35, 39, 45, 51, 52, 58, or 59. HPV types with risk are included.

The terminology and the design of ZFP used in the present invention are described below.
In the present invention, animal viruses include various animal DNA viruses that can infect humans, mammals including domestic animals, birds including chickens, fish and shellfish to be cultured, and the like. Examples thereof include human viruses such as papilloma virus, herpes virus, EB virus, rotavirus, HBV, and other hepatitis viruses, adenovirus, pox virus, and particularly human papilloma virus (HPV). Among HPVs, especially HPV-16, HPV-18, etc. are said to cause cervical cancer, and it is expected that the prevention or treatment effect of cervical cancer can be obtained by suppressing the replication of papillomavirus. Accordingly, human papillomavirus is a preferred target for the antiviral agents of the present invention.

Several zinc finger proteins having antiviral activity or viral replication inhibitory activity are disclosed in the Sera application and the corresponding non-US applications, the 2002 Sera literature, and the like. These ZFPs are generally for inhibiting plant viruses.
The ZFP that can be used as an active ingredient of the antiviral agent of the present invention preferably recognizes and specifically binds to specific four bases of double-stranded DNA per finger, for example, at the origin of replication of an animal DNA virus. . The zinc finger protein may bind to the binding site of one replication protein, but may have two or more units capable of binding to the binding sites of two or more different replication proteins. In order to produce the antiviral agent of the present invention, any number of binding sites at the origin of replication may be targeted for ZFP binding.

  Animal DNA viruses have one or more unique replication proteins that can bind to the origin of replication and initiate viral replication. For example, human papillomavirus has replication proteins E1 and E2, and inhibition of binding of one or both of these replication proteins inhibits replication of human papillomavirus. When ZFP is used as the antiviral agent of the present invention, an E2 binding site is more preferable as its target. The E2 binding site contains four binding sites (E2BS-1, E2BS-2, E2BS-3, and E2BS-4) at the origin of replication. Of these, E2BS-3 and E2BS-4 shown in FIG. 4 are more preferred as ZFP targets. E2BS-3 and E2BS-4 correspond to positions 42-53 and 58-69 on the entire 7857 base pairs of the HPV-18 genome, respectively, and those skilled in the art can compare the appropriate DNA sequences to those other than HPV-18. The corresponding position in the papillomavirus can be easily identified.

For antiviral agents targeting HPV, it is preferable to design the ZFP sequence so that it can bind to both E2BS-3 and E2BS-4. For example, if ZFP has 6 domains (each domain recognizes 4 bases) and 19 consecutive base sequences, 42-60, 43-61 on the HPV-18 genome , 44-62, 45-63, 46-64, 47-65, 48-66, 49-67, 50-68, and 51-69 to recognize 10 different base sequences It is preferred to design zinc finger proteins that target E2 replication proteins. In this regard, ZFP _HPV- 1 and ZFP _HPV- 2, which are described in detail in the examples, were designed for 44-62 and 51-69 target sequences, respectively.
If the replication protein has two or more binding sites at the origin of replication, the ZFP can be designed to bind across these two or more binding sites to make binding of the replication protein to the replication origin more specific. Can inhibit.

  The zinc finger protein that can be suitably used as the antiviral agent of the present invention has, for example, at least one zinc finger domain using a 4-base pair target sequence. Multifinger (multidomain) ZFPs designed for longer target sequences are useful as antiviral agents of the present invention as described below.

“Zinc finger protein” (ZFP) means a polypeptide having a DNA binding domain stabilized by zinc. Each DNA binding domain is typically referred to as a “finger” or “zinc finger domain”, and the ZFP is at least one finger, more typically at least two fingers, more preferably at least three fingers, more preferably It has at least 4 or 5 fingers, or at least 6 or more fingers. In general, ZFPs specifically bind to a nucleic acid sequence called a target nucleic acid sequence, with each finger binding to 3 or 4 DNA base pairs. Each finger usually contains a zinc-chelating DNA binding subdomain of about 30 amino acids. The typical motif in one group, Cys ₂ -His ₂ group, is -Cys- (X) _2-4 -Cys- (X) ₁₂ -His- (X) _3-5 -His (where X is ZFP containing this motif consists of an α helix containing two invariant histidine residues and two cysteine residues in one β turn (Berg et al., Science 271: 1081). -1085 (1996)) binds to the zinc cation.

According to a preferred embodiment, the zinc finger protein has the formula:
_{-X 3 -Cys-X 2-4 -Cys-} X 5 -Z -1 -XZ 2 -Z 3 -X 2 -Z 6 -His-X 3-5 -His-X 4 -
(Where X is any amino acid, X _n indicates the number of occurrences of X in the polypeptide chain, and thus X indicates the framework of the Cys ₂ -His ₂ zinc finger domain) 1 or more, preferably 3 to 40, more preferably 3 to 15.

Preferred embodiments of the invention for a four base target sequence are as follows:
(i) when the first base is G, Z ⁶ is arginine;
When the first base is A, Z ⁶ is glutamine,
When the first base is T, Z ⁶ is threonine, tyrosine, or leucine;
When the first base is C, Z ⁶ is glutamic acid,
(ii) when the second base is G, Z ³ is histidine;
When the second base is A, Z ³ is asparagine,
When the second base is T, Z ³ is serine,
When the second base is C, Z ³ is aspartic acid,

(iii) when the third base is G, Z ^-1 is arginine;
When the third base is A, Z ^-1 is glutamine,
When the third base is T, Z ^-1 is threonine or methionine;
When the third base is C, Z ^-1 is glutamic acid,
(iv) when the complement of the fourth base is G, Z ² is serine;
When the fourth base complement is A, Z ² is asparagine;
When the fourth base complement is T, Z ² is threonine;
When the fourth base complement is C, Z ² is aspartic acid.
In a more preferred embodiment, when the first base is T, Z ⁶ is threonine, and when the third base is T, Z ^-1 is threonine (Table 1).

As another preferred embodiment, the following embodiment can be exemplified.
(i) when the first base is G, Z ⁶ is arginine or lysine;
When the first base is A, Z ⁶ is glutamine or asparagine;
When the first base is T, Z ⁶ is threonine, tyrosine, leucine, isoleucine, or methionine;
When the first base is C, Z ⁶ is glutamic acid or aspartic acid,
(ii) when the second base is G, Z ³ is histidine or lysine;
When the second base is A, Z ³ is asparagine or glutamine;
When the second base is T, Z ³ is serine, alanine, or valine;
When the second base is C, Z ³ is aspartic acid or glutamic acid,

(iii) when the third base is G, Z ^-1 is arginine or lysine;
When the third base is A, Z ^-1 is glutamine or asparagine,
When the third base is T, Z ^-1 is threonine, methionine, leucine, or isoleucine;
When the third base is C, Z ^-1 is glutamic acid or aspartic acid,
(iv) when the complement of the fourth base is G, Z ² is serine or arginine;
When the fourth base complement is A, Z ² is asparagine or glutamine;
When the fourth base complement is T, Z ² is threonine, valine, or alanine;
When the fourth base complement is C, Z ² is aspartic acid or glutamic acid.

  The zinc finger protein used as an active ingredient of the antiviral agent of the present invention may be a multi-domain ZFP in which each zinc finger domain is independently represented by the above formula. In this case, the target nucleic acid sequence has a length of 3N + 1 base pairs (where N is the number of overlapping 4 base pair segments in the target and is obtained by dividing the target nucleic acid sequence into overlapping 4 base pair segments. Yes, the fourth base of each segment up to the N-1 segment is the first base in the immediately following segment).

Another preferred embodiment of the zinc finger protein used as an active ingredient of the antiviral agent of the present invention comprises at least three zinc finger domains covalently bonded to each other at 0 to 10 amino acid residues, and the zinc finger α -The amino acids at positions -1, 2, 3, and 6 of the helix can be selected from the preferred embodiments described above. ZFPs of preferred embodiments are each independently represented by the following formula:
_{-X 3 -Cys-X 2-4 -Cys-} X 5 -Z -1 -XZ 2 -Z 3 -X 2 -Z 6 -His-X 3-5 -His-X 4 -
Wherein the domains are covalently linked to each other with 0 to 10 amino acid residues (wherein X is any amino acid and X _n is X in the polypeptide chain). When this polypeptide is not known in the art, Z ⁻¹ , Z ² , Z ³ , and Z ⁶ are determined by the recognition codes in Table 1 (for a list of known ZFPs) See Sera application)).

As indicated above, X represents an amino acid in the framework of the Cys ₂ -His ₂ zinc finger domain and is obtained by changing the sequence of a known zinc finger framework, consensus framework, or any of these frameworks It may be a framework or any artificial framework that maintains the overall structure of the zinc finger domain. Preferably, the identity of each X can be determined using a known framework. Preferred frameworks are those derived from Sp1c and Zig268. A more preferred framework is domain 2 derived from Sp1C.

  ZFP suitably used for the antiviral agent of the present invention has 3 to 40 zinc finger domains, preferably 3 to 15 domains, 3 to 12 domains, 3 to 9 domains, Or a ZFP comprising 3 to 6 domains or having 3, 4, 5, 6, 7, 8, or 9 domains.

Further preferred ZFPs, independently or in any combination, in at least one zinc finger domain, Z- ¹ is methionine; in at least one zinc finger domain, Z- ¹ is glutamic acid; at least one zinc finger In the domain Z ² is threonine; in at least one zinc finger domain Z ² is serine; in at least one zinc finger domain Z ² is asparagine; in at least one zinc finger domain Z ⁶ is glutamic acid ; Z ⁶ in at least one zinc finger domain located at threonine; Z ⁶ in at least one zinc finger domain has tyrosine; Z ⁶ in at least one zinc finger domain is a leucine, and / or Without even in one zinc finger domains Z ² is aspartic acid, and a ZFP Z ^-1 is not arginine in the same domain.

According to yet another aspect of the present invention, the ZFP used as the antiviral agent of the present invention may contain one or more nuclear localization signals (NLS) and / or one or more cell membrane permeation peptides (CPP). . It is particularly useful to add these CPPs and NLS when the antiviral agent is a protein. The active ingredient of the antiviral agent of the present invention preferably contains both a nuclear localization signal and a cell penetrating peptide, and is located at either the N-terminus or C-terminus of the ZFP ORF. Specific examples thereof include the following configurations.
CPP-NLS-ZFP;
NLS-CPP-ZFP;
CPP-NLS-CPP-ZFP;
CPP-ZFP-NLS;
ZFP-CPP-NLS;
ZFP-NLS-CPP etc.

The ZFP and the nucleic acid encoding the ZFP preferably contain both CPP and NLS.
NLS is an amino acid sequence necessary for localizing a protein in the nucleus of a cell through the nuclear membrane structure of a eukaryotic cell. The type of NLS is not particularly important as long as NLS has a function of localizing proteins in the nucleus. For example, SV40 large-T antigen-derived NLS (polynucleotide encoding the region of amino acids 126 to 132 of large-T antigen, Proc. Natl. Acad. Sci. (1989) 86: 9327-9331), HIF- 1α NLS and the like are preferred.

The CPP can be any sequence that promotes intracellular penetration of ZFP. For example, peptides containing many basic amino acids such as Tat peptide and polylysine are preferable.
Specific examples of the membrane-permeable peptide are listed below, but are not limited thereto.
Natural protein derived:
HIV TAT (amino acids 47-57)
--- TyrGlyArgLysLysArgArgGlnArgArgArg
Antp homeodomain (amino acids 43-58)
--- ArgGlnIsoLysIsoTrpPheGlnAsnArgArgMetLysTrpLysLys
VP22 (amino acids 267 to 300)
--- AspAlaAlaThrAlaThrArgGlyArgSerAlaAlaSerArgProThr
GluArgProArgAlaProAlaArgSerAlaSerArgProArgArgProValGlu;
And artifacts:
PTD4 --- TyrAlaArgAlaAlaAlaArgGlnAlaArgAla;
R9 --- ArgArgArgArgArgArgArgArgArg.

  The method for linking CPP and / or NLS to ZFP is not particularly limited. For example, in E. coli, insect cells, animal cells, or any cell used as an expression system, these domains can be used with or without a cleavable peptide linker (which may have a protease cleavage site). The encoding DNA can be directly bound to the DNA encoding ZFP. Alternatively, CPP and / or NLS domains can be chemically linked to ZFP via a bifunctional or polyfunctional linker containing succinimide and / or maleimide. Such proteins can also be prepared as non-covalent associations utilizing protein interactions such as cJun-cFos. For example, a conjugate in which CPP is bound to cJun and a conjugate in which cFos is bound to ZFP can be used to adjust the association.

  In addition to the zinc finger protein described above, the active ingredient of the antiviral agent and viral replication inhibitor of the present invention is a polynucleotide capable of expressing a zinc finger protein such as an expression vector containing a nucleic acid encoding the zinc finger protein. May be. An expression vector can be constructed by a known method using a DNA or RNA virus vector or plasmid vector capable of expressing a protein in an animal cell such as a mammalian cell [Molecular Cloning, Cold Spring Harbor Laboratory, See the Laboratory Manual (1989) or other literature on recombinant DNA]. Expression vectors include plasmids derived from E. coli such as pCR4, pCR2.1, pBR322, pBR325, pUC12, and pUC13, plasmids derived from yeast (pSH19, pSH15, etc.), bacteriophages such as λ phage, retroviruses, vaccinia viruses, adenoviruses, etc. Animal viruses and the like can be used. These expression vectors can be introduced into animal (particularly human) cells using liposomes (particularly cationic liposomes) or other techniques known to those skilled in the art, if necessary. When an antiviral agent containing zinc finger protein as an active ingredient is administered to an animal, it is preferably administered as a fusion protein with at least CPP.

ZFP Terminology and Design Overview The four residues that nucleic acid contacts in the zinc finger domain are primarily responsible for determining specificity and affinity, and for the first consensus histidine and the second consensus cysteine. In the same position. The first residue is 7 residues on the N-terminal side of the first consensus histidine and 6 residues on the C-terminal side of the second consensus cysteine. Hereinafter, this residue is referred to as the “-1 position” residue. The other three residues are residues 2, 3, and 6 away from the C-terminus of the residue at position -1, hereinafter referred to as `` position 2 '', `` position 3 '', respectively. And called the “position 6” residue. Alternatively these positions Z ^-1 position, Z ^2-position, Z ^3-position, and also referred to as a Z ^6-position. These amino acid residues are called “base contact amino acids” (sometimes called recognition amino acids). Position -1 is the starting point of the α helix in the zinc finger domain. The positions of the amino acids at positions -1, 2, 3, and 6 in the zinc finger domain and the bases they contact in a 4 base pair DNA target sequence are schematically shown in FIG.

  The zinc finger nucleic acid recognition codes are shown in Table 1 and this combination is based on known and possible base-amino acid interactions (Figure 2). Some of the interactions listed in Figure 2 have also been identified in different proteins such as H-T-H protein, cro, and lambda inhibitors. Amino acids containing longer side chains can be selected for recognition of the first and third DNA bases in the 4 base pair region. In addition, amino acids containing shorter side chains can be selected for recognition of the second and fourth bases. For example, in the case of guanine base recognition, arginine can be selected as the amino acid at the -1 position and the 6 position, histidine can be selected as the amino acid at the 3 position, and serine can be selected as the amino acid at the 2 position. All amino acids shown in Table 1 stably interact with specific DNA bases of the replication origin targeted by hydrogen bonding (for example, the E2 protein binding site of papillomavirus in the examples). For thymidine base recognition, an amino acid having a hydrophobic side chain is also selected (ie, leucine for the first thymidine base and methionine for the third thymidine base). Other DNA base-amino acid interactions are possible, and amino acids with higher affinity can be selected. For example, lysine binds to guanine, but arginine can be preferably selected for further hydrogen bond formation.

  The recognition of four bases in a four base pair DNA sequence (first base near the 3 ′ triplet DNA) by the amino acid at position 2 is shown in FIG. Asp, Thr, Asn, and Ser at position 2 of the zinc finger domain bind preferentially to C, T, A, and G, respectively. The fourth base is present in the antisense nucleic acid strand.

  In Table 1 (and for each 4 base pair portion of the target sequence) the bases are always shown in the 5 ′ to 3 ′ direction. The fourth base in the table is always the complement of the fourth base in the target sequence. For example, when the target sequence is described in ATCC, it means the sense strand target sequence of 5′-ATCC-3 ′ and the antisense strand of 3′-TAGG-5 ′. Therefore, when translating the sense strand sequence ATCC from the above table into amino acids, the first base A means that there is glutamine at position 6, and the second base T means that there is serine at position 3. , The third base C means that there is glutamine at position -1, but the fourth base, which is described as C, is the complement of C (ie the identification of the amino acid at position 2 found in the table) G) used to mean. In this case, the amino acid at position 2 is serine.

It is also preferred that Z ⁶ is threonine when the first base is T and Z ^-1 is threonine when the third base is T. Table 2 below shows the recognition codes expanded to generally indicate more conservative amino acids for those present in the recognition codes in Table 1. In Table 2, the order of the amino acids listed in each column indicates from left to right the least preferred amino acid from the most preferred amino acid at that position.

  The framework determined by the identity of X can be any known zinc finger framework, consensus framework, or any of these frameworks as long as the framework changes maintain the overall structure of the zinc finger domain. One variant may be used. Preferred frameworks are those derived from Sp1C and Zif268. A more preferred framework is domain 2 derived from Sp1C.

  Any of a variety of techniques known in the art can be used to prepare a protein comprising a zinc finger domain designed either synthetically or recombinantly, preferably recombinantly. If the protein is prepared recombinantly (eg, via DNA encoding ZFP), codon usage can be optimized for high expression in the organism in which the ZFP is to be expressed. Such organisms include, but are not limited to bacteria, fungi, yeast, animals, or insects. For example, when the antiviral agent should be a ZFP protein, the protein can be expressed in a large amount in the protein expression of any organism, and the protein can be produced and used for administration to animals. .

  Multidomain (ie, multi-finger) ZFP design may follow the single domain design method described above (eg, using a preferred framework such as Sp2 derived domain 2), especially when the domains are not contiguous. . For ZFPs that have multiple consecutive domains (or domains separated by the linkers described herein) for target sequences greater than 4 base pairs, particularly when there are more than 3 zinc finger domains in the ZFP Designed by splitting the target sequence into overlapping 4 base pair segments gives a content-independent zinc finger recognition code, which results in ZFPs, typically ZFPs with high binding affinity It was found.

In this method, the target sequence has a length of 3N + 1 base pairs (N is the number of overlapping 4 base pair segments in the target, determined by dividing the target sequence into overlapping 4 base pair segments, N-1 The fourth base in each segment up to the segment is the first base in the immediately following segment). The rest of the design method for each 4 base pair segment follows the single domain design method for determining the identity of each X, Z ⁻¹ , Z ² , Z ³ , and Z ⁶ . This method involves 3 to 15 domains (N is any number from 3 to 15), more preferably 3 to 12 domains, 3 to 9 domains, or 3 to 6 domains. It is useful for the design of ZFPs with ZFPs with more than 40 domains are known in the art and N may range up to at least 40 if desired.

The designed zinc finger domains can be covalently linked directly to each other or separated by a linker region of 1 to 10 amino acids or peptides. The amino acids of the linker can provide flexibility or some structural rigidity. Although not necessary, the choice of linker can be determined by the desired affinity of the ZFP for the homologous target sequence. One skilled in the art can test and optimize various linker sequences to improve the binding affinity of ZFPs for homologous target sequences. Methods for measuring the binding affinity between ZFP and its target are well known. Typically, a gel shift assay can be used, but for example, a transient expression assay can also be used as shown in the examples herein. In one embodiment, it is preferred that the amino acid linker be flexible so that each of the three finger domains binds independently to their target sequence and avoids steric hindrance of binding to each other.
The ZFP that is the antiviral agent of the present invention includes any ZFP having one or more combinations of amino acids at positions 1, 2, 3, and 6 shown by the recognition codes in Table 1.

Pharmaceutical formulation
An antiviral agent or viral replication inhibitor containing the ZFP of the present invention with or without CPP and NLS domains or a nucleic acid encoding these ZFPs as an active ingredient is a physiologically acceptable carrier, excipient, or stable. It can be prepared, for example, in the form of a lyophilized preparation or an aqueous solution, using additives for preparation such as an agent. Pharmaceutical additives include, for example, buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, chloride) Benzalkonium, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (approximately 10 residues) Less than) polypeptide; protein (serum albumin, gelatin, immunoglobulin, etc.); hydrophilic polymer (polyvinylpyrrolidone); amino acid (glycine, glutamine, asparagine, histidine, arginine, lysine, etc.); monosaccharide, disaccharide, And other carbohydrates (including glucose, mannose, or dextrin); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (eg, , Zinc-protein complexes); and / or nonionic surfactants (TWEEN®, PLURONICS®, or polyethylene glycol (PEG)), etc., but are not limited to these Absent. Sterilization of the preparation can be performed, for example, by filtration using a sterile filtration membrane.

  The antiviral agent or viral replication inhibitor of the present invention may be blended with other pharmaceutical ingredients as long as the active ingredients provided by the present invention are not adversely affected. Such pharmaceutical ingredients can be combined in an amount effective for the intended prophylactic and / or therapeutic purpose. For example, you may combine with a known antiviral agent etc.

  The active ingredients of the antiviral agent provided by the present invention include, for example, microcapsules prepared by coacervation technology or interfacial polymerization (eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively), Colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions can be carried. Such techniques are disclosed in "Remington's Pharmaceutical Science", 16th edition, edited by A. Osole, (1980) or a new edition.

  The antiviral agent or viral replication inhibitor of the present invention may be prepared as a sustained release preparation. Examples of suitable sustained release substrates for preparing sustained release formulations include, for example, semi-permeable substrates of solid hydrophobic polymers containing polypeptide variants, which are molded products (eg, films Or microcapsules). Examples of sustained release substrates include polyester hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactide (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, Non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPRONDEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D- ( -)-3-Hydroxybutyric acid, etc. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for 100 days, but some hydrogels can release proteins in a short time. For stabilization, for example, the encapsulated antibody stays in the body for a long time. In some cases, antibodies may denature or aggregate as a result of contact with moisture at 37 ° C., thereby losing biological activity and altering immunogenicity. For example, if the aggregation mechanism is found to be intercellular SS bond formation by thio-disulfide interchange, modification of sulfhydryl residues, from acidic solution Can be stabilized by means such as lyophilization, adjustment of moisture content using appropriate additives, and development of specific polymer matrix compositions.

The dosage of the antiviral agent or viral replication inhibitor of the present invention is not particularly limited, but those skilled in the art will know the type of active ingredient, the type and weight of the animal to be administered, the type of animal virus, the type and symptoms of viral infection The dose can be selected according to the above.
It will be understood by those skilled in the art that various omissions, additions, and modifications may be made to the invention described herein without departing from the scope thereof, and any such modifications or changes are within the scope of the appended claims. It is intended to be included within the scope of the invention as defined. All references, patents, patent applications, and other publications are hereby incorporated by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to these examples.
In the examples below, inhibition of viral genome replication was evaluated using a transient expression vector incorporating an origin of replication. This type of test system has already been established in the study of HPV DNA replication, and it can be used to demonstrate the ability to inhibit DNA replication in fully HPV that infects humans.

Example 1
(1) Plasmid preparation In order to prevent E2 binding to the replication origin of human papillomavirus, two types of ZFPs (ZFP _HPV- 1 and ZFP _HPV- 2) were designed against the DNA sequence shown in FIG. 2002) 41: 7074-7081). FIG. 5 shows the amino acid sequences of the zinc finger domains of ZFP _HPV- 1 and ZFP _HPV- 2 each having 6 mains. These proteins were cloned into pET-21a (Novagen) for expression in E. coli. A control ZFP (referred to as ZFP _Ala ) was constructed in a similar system, except that all recognized amino acids were replaced with alanine (ie, positions 1, 2, 3, and 6 in each zinc finger domain are alanine).

The Escherichia coli expression plasmid encoding HPV-18 E2 is an E2 open reading frame (ORF) derived from pHPV-18 (American Type Culture Collection) (nucleotide (nt) number 2817-3914 of HPV-18) of pET-21a. Prepared by cloning into EcoRI / HindIII sites.
ZFPs with CPP and NLS domains for ZFP _HPV- 2 were prepared as follows. The first ZFP called the PTD4-ZFP _HPV -2, which PTD4 sequence (TyrAlaArgAlaAlaAlaArgGlnAla ArgAla), 3 pieces of glycine linker, NLS sequence (ProLysLysLysArgLysVal), additional linkers (GlyGlyGlyGlySer), ZFP _HPV -2 ORF in pET-21a It has a T7 tag (MetAlaSerMetThrGlyGlyGlnGlnMetGly) in front of the N-terminus. This T7 tag is used to detect proteins by Western blotting and to protect PTD4 from degradation by exoproteases.

The second ZFP is called ZFP _HPV- 2-R9, which is a ZFP _HPV- 2 ORF linked at the C-terminus with a linker (GlyGlyGlyGlySer), the same NLS sequence, three glycine linkers, an R9 sequence (ArgArg ArgArgArgArgArgArgArg), And the V5-tag ((GlyLysProIleProAsnPro LeuLeuGlyLeuAspSerThr) in pET21a. It has the same T7 tag in front of the N-terminus of ZFP _HPV- 2 ORF. The presence of V5 protects R9 from degradation by intracellular exoproteases. .

The structures of PTD4-NLS and NLS-R9 are shown below.

  pRL-E1, pRL-E2 and pUC-Ori177 (for transient viral DNA replication experiments) were constructed as follows. The pRL-E1 and pRL-E2 vectors represent the E1 gene (nucleotides 904 to 2887 derived from HPV-18 gene) and the E2 gene (nucleotides 2817 to 3914 derived from HPV-18 gene), respectively, from the NheI / of pRL-SV40 vector (Promega). It was constructed by cloning into the XbaI site. PUC-Ori177, a vector with the HPV-18 origin of replication, is called Ori177 and contains a 177 base pair AluI / BamHI fragment containing the HPV-18 origin of replication (nucleotides 7800-7857 and 1-119 of the HPV-18 gene). Was constructed by cloning into the HincII / BamHI site of pUC19.

Mammalian expression vectors pCMV-ZFP _HPV- 1, pCMV-ZFP _HPV- 2 and pCMV-ZFP _Ala for expressing ZFP _HPV- 1, ZFP _HPV- 2 and ZFP _Ala in animal cells, respectively, modify their respective ZFP ORFs pcDNA3 It was constructed by cloning into .1 (Invitrogen). This modified plasmid contains an N-terminal T7 tag, a nuclear localization signal from SV40 large T antigen, and a multiple cloning site for ZFP insertion.

(2) Protein purification
Proteins used for DNA binding experiments (ZFP _HPV- 1, ZFP _HPV- 2 and E2) and PTD4-ZFP _HPV- 2 and ZFP _HPV- 2-R9 used for replication inhibition experiments are E. coli strains BL21 (DE3) or BL21. (DE3) pLysS (Novagen) was used for expression. When the OD ₆₀₀ value reached 0.6 after 37 ° C. culture, 1 mM IPTG was added to induce protein expression. After culturing for 3 hours, Escherichia coli was sonicated and the protein of interest was purified using a Bi-Rex 70 cation exchange resin column (Bio-Rad) as generally described in Sera (2002). The purity of the obtained protein was 95% or more on SDS-PAGE, and the protein concentration was determined by Protein Assay ESL (Roche Molecular Biochemicals).

(3) DNA binding
(a) Kd estimation
DNA binding assays two E2 binding sites duplex of 56 base pairs having the (E2 are underlined in the binding site) DNA probes _{5 '- (TA) 4 GGAGTA} ACCGAAAACGGT CGGG ACCGAAAACGGT G TATAT (TA) 4 - For 3 '(SEQ ID NO: 4) or 40 base pair double-stranded DNA probe having one E2 binding site 5'-(TA) ₄ GGAGTA ACCGAAAACGGT GTATAT (TA) ₄ -3 '(SEQ ID NO: 5) It was. Each probe was labeled with [α- ³² P] dATP or [α- ³² P] dTTP and Klenow fragment.

For the binding reaction with ZFP, 0.03 fmol of labeled 56 base pairs and the indicated concentration of purified ZFP were added on ice to 10 μl of binding reaction buffer (10 mM Tris-HCl (pH 7.5) / 100 mM NaCl / 5 mM MgCl ₂ / Incubated with 1 μg of poly (dA-T) _{2 in} 0.1 mM ZnCl ₂ /0.05% BSA / 10% glycerin) for 1 hour. For the binding reaction with E2, 0.03 fmol of labeled 40 base pair DNA probe and the indicated concentration of purified E2 were incubated under similar conditions.
After completion of the reaction, the reaction mixture was separated and analyzed by gel shift assay by non-denaturing polyacrylamide gel electrophoresis (6% gel, 45 mM Tris-borate buffer, 140 V, 2 hours at 4 ° C.).

Figure 6 shows the results of ZFP _HPV -1 and ZFP _HPV -2 binding to DNA (left two figures), indicating that these ZFPs bind to the HPV E2 binding site at a concentration of approximately 10 pM. . Under the same conditions (ie, in the presence of an excess of unlabeled poly (dA-dT) ₂ ), the apparent dissociation constant (K _d ) of the E2 protein at which half the maximum binding is observed was about 40 nM. It was. Thus, both ZFP _HPV- 1 and ZFP _HPV- 2 have about 4000 times higher affinity for DNA than E2.
Another DNA binding experiment was performed and using the 9 mutant probes shown in FIG. 10a, ZFP _HPV- 1 (10, 100, and 1000 pM concentrations) and ZFP _HPV- 2 (10, 100, and 1000) The DNA binding specificity (pM concentration) was examined. These mutants are for a 56 base pair DNA probe. The results are shown in FIG.

Finally, the control protein ZFP _Ala was unable to shift the target DNA even at 1 μM in the presence of an excess of competing DNA, unlabeled (dA-dT) 2, but this was 19 base pairs. It shows that specific recognition amino acids in both ZFP _HPV- 1 and ZFP _HPV- 2 are required to recognize target DNA.
The results of the above experiment proved that ZFP _HPV- 1 and ZFP _HPV- 2 specifically bind to the HPV origin of replication (target DNA sequence).

(b) Competitive binding In competitive binding experiments, labeled 56 base pair DNA probes were labeled with E2 (final concentration 100 nM) and ZFP _HPV -1 or ZFP _HPV -2 (final concentrations 0.1, 1, 10) under the same conditions as above. Or 100 nM) at the same time. 5 lanes 8 in FIG. 8 shows the results of the ZFP _HPV -1, 9 lanes 12 shows the results of a ZFP _HPV -2, ZFP _HPV -1 and ZFP _HPV -2 both inhibit DNA replication of HPV This indicates that ZFP _HPV- 2 has a strong replication inhibitory action. Lanes 1 to 4 show the band positions of free DNA, E2 binding DNA, ZFP _HPV- 1 binding DNA, and ZFP _HPV- 2 binding DNA, respectively.

Mutation by leaving only the binding site of the unmodified E2 replication protein and mutating only the CGGT (MT2), CTGT (MT10), ATAT (MT11), or ATATATAT (MT12) from the binding site CGGG (WT) in the linker site of ZFP A transient viral replication system (described below) with an origin of replication was prepared. These mutant probes are also shown in the upper part of FIG. The result of the gel shift assay for the DNA probe co-transfected with pCMV-ZFP _HPV- 2 is shown in FIG. 11b. The result of the gel shift assay for the mutant ori plasmid co-transfected with pCMV-ZFP _HPV- 2 is shown in FIG. 11c. No inhibition of replication of the transient mutant virus origin was observed (FIG. 11c), suggesting that ZFP _HPV- 2 does not bind to this mutant replication origin.

Therefore, it was confirmed that the binding selectivity of ZFP _HPV- 2 in vitro is also exhibited in cells. This result suggests that the above-described zinc finger protein replication inhibitory action observed in a transient virus replication system containing a natural origin of replication is caused by the specific binding of ZFP to the origin of restoration.

(4) Transient viral DNA replication
(a) Inhibition of replication by ZFP gene A total of 8 × 10 ⁵ human cells 293H (Invitrogen) per well were plated on a biocoated poly-D-lysine 12-well plate (Becton Dickinson), and 0.1 mM non-essential amino acids and 10 Maintained in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with% fetal bovine serum (Invitrogen). Three vectors required for transient replication of cells after seeding: 1.5 μp pRL-E1, 0.17 μg pRL-E2, and 0.17 μg pUC-Ori177, and 0.17 for expression in animal cells μg of the appropriate ZFP vector: pCMV-ZFP _HPV- 1, pCMV-ZFP _HPV- 2, pCMV-ZFP _Ala , or pcDNA3.1 (ZFP expression control; Invitrogen) using Lipofectamine 2000 (Invitrogen) Co-transfected according to After culturing at 37 ° C under 5% CO ₂ for 3 days, the plasmid was isolated from the cells by the Hirt extraction method (Hirt (1967) J. Mol. Biol. 26: 365-369) and linearized by digestion with HindIII did.

  To distinguish between replicated and non-replicated DNA, half of each sample was treated with excess DpnI to remove non-replicated methylated insert DNA PUC-Ori177. One percent of the remaining half of each linearized sample was used to confirm that the same amount of plasmid used in each transient replication assay was introduced into 293 cells. The DNA samples were separated by electrophoresis (in 0.8% agarose using 0.5 × Tris-borate-EDTA buffer) and subjected to Southern blot hybridization.

For this analysis, a 200 base pair DNA probe was used to detect the ampicillin resistance gene in pUC-Ori177 labeled with digoxigenin (DIG), and the signal was using anti-DIG-AP and CDP-Star (Roche Molecular Biology). Detected. The DNA band intensity on the X-ray film was digitized and quantified with UN-SCAN-IT (Silk Science, Inc.). The average intensity of the replicated pUC-Ori derivative DNA band was calculated from four independent experiments and the input pUC-Ori derivative DNA band intensity was normalized. ZFP _ALA is a negative control.

The results shown in FIG. 7 indicate that ZFP _HPV- 1 and ZFP _HPV- 2 inhibited DNA replication. In particular, ZFP _HPV- 2 reduced the replication level to about 12% of the control pcDNA3.1. Under the same conditions, the control protein ZFP _ALA did not inhibit DNA replication (FIG. 7, lane 4) and specific recognition amino acids in ZFP _HPV- 1 and ZFP _HPV- 2 (ie -1, 2, 3 and 6) were shown to be necessary for inhibition.

(b) Inhibition of replication by ZFP For these experiments, PTD4-ZFP _HPV- 2 or ZFP _HPV- 2-R9 was added to 8 × 10 ⁵ (per well of 12-well plate) 293H cells at the final concentration indicated. It was. Cells were cultured for 1 hour and transfected with pRL-E1 (1.5 μg), pRL-E2 (0.17 μg) and pUC-Ori177 (0.17 μg). The replication of pUC-Ori177 was analyzed in the same manner as (a) above.

The results shown in FIG. 9 indicate that when ZFP _HPV- 2-R9 is added to the transient virus replication system, it can enter the cell and migrate into the nucleus to inhibit virus replication. Therefore, this protein can act effectively as an antiviral agent or a viral replication inhibitor.
Prepare the E2C-R9 protein by recombinantly fusing the R9 domain (giving cell permeability) to the DNA binding domain located at the C-terminus (amino acids 283 to 365) of the E2 protein (ie dominant-negative mutant) did. When inhibition of transient virus replication by E2C-R9 was examined, it was shown that ZFP _HPV- 2-R9 has a stronger replication inhibitory ability than dominant-negative mutant E2C-R9. It was also suggested that ZFP having high DNA binding ability can exert high effectiveness as an antiviral agent (FIG. 12).

(c) Verification of ZFP uptake 4 × 10 ⁵ 293H animal cells per well were seeded in a 24-well plate, and ZFP _HPV- 2-R9 or PTD4-ZFP _HPV- 2 was added to the cell culture medium at a final concentration of 250 nM. . Cells were cultured at 37 ° C. under 5% CO ₂ concentration for the specified time. At each time, the cells were washed twice with 1 ml of 1 × PBS and lysed by adding 100 μl of 1 × Passive Lysis Buffer (Promega). Furthermore, the wells were washed with 100 μl of 1 × Passive Lysis Buffer, and the buffer was added to the cell lysate. Cell debris was removed by centrifugation, and the amount of ZFP in the supernatant was Western blotted with ZFP _HPV- 2-R9 and PTD4-ZFP _HPV- 2 as primary antibodies using anti-V5 tag antibody and anti-T7 tag antibody, respectively. Measured by the method. The secondary antibody detected the signal using ECL Plus (Amersham) as anti-mouse Ig-HRP. The results are shown in FIG. ZFP _HPV- 2-R9 and PTD4-ZFP _HPV- 2 were added to the cell culture medium, indicating that large amounts of the protein could be detected in the cells within 10 minutes. Thus, ZFP _HPV- 2-R9 and PTD4-ZFP _HPV- 2 permeated the cell membrane and were thus taken up by the cells. ZFP _HPV- 2-R9 was taken up more efficiently, consistent with the higher replication inhibitory ability exhibited by this protein.

  The antiviral agent and viral replication inhibitor of the present invention are useful for the prevention and / or treatment of animal viral infections. In particular, an antiviral agent or viral replication inhibitor containing a peptide having cell membrane permeability and a zinc finger protein linked to a nuclear localization signal as an active ingredient has a significantly higher effect than conventional antiviral agents. It is extremely useful as a medicine.

FIG. 1 is a schematic diagram showing the binding of one unit of zinc finger domain to a 4-base pair DNA target site. Residues at positions -1, 2, 3, and 6 are each independently in contact with one base. Position -1 is the starting point of the α-helix in the zinc finger domain. FIG. 2 shows the interaction of known and possible bases with amino acids. An interaction similar to that between guanine (G) and histidine (His) is also formed by other amino acids (such as serine and lysine) that serve as hydrogen bond donors. An interaction similar to that between thymidine and threonine is also formed by other hydrophobic amino acids. An interaction similar to that formed between thymidine and threonine / serine is also formed by other amino acids that serve as hydrogen bond donors. FIG. 3 is a diagram showing recognition of the fourth base in a 4-base pair DNA target sequence by the amino acid at position 2 of the zinc finger domain. FIG. 4 shows a ZFP (zinc finger domain) target sequence, in particular, the constitution of the HPV-18 replication origin and the amino acid sequence for ZFP to inhibit DNA replication. Squares indicate E2-binding sites (E2BS) E2BS-3 and E2BS-4, respectively, and indicate the 12 base pair DNA sequence recognized by the E2 protein. The number below the square indicates the position (nucleotide number) of the sequence in the HPV-18 genome. A 19 base pair target DNA selected for ZFP _HPV- 1 and ZFP _HPV- 2 was also shown. FIG. 5 shows the amino acid sequences of the six zinc finger domains of each ZFP _HPV- 1 and ZFP _HPV- 2. The underlined amino acid in each finger domain indicates the recognized amino acid at positions -1, 2, 3, and 6 in the α-helix of the finger domain. FIG. 6 shows autoradiographs of DNA binding assays of ZFP _HPV- 1 and ZFP _HPV- 2 (left two figures) and E2 (right figure). For ZFP _HPV- 1 and ZFP _HPV- 2, probes of 56 base pairs labeled with ³² P containing E2BS-3 and E2BS-4 were used. For the E2 protein, a 40 P probe labeled with ³² P containing a single E2BS was used. The positions of DNA-ZFP complex and free DNA are shown. FIG. 7 shows inhibition of HPV-18 DNA replication in a transient replication assay using the ZFP gene. Transient replication assays were performed using pRL-E1, pRL-E2, and pUC-Ori177 with the ZFP expression plasmid shown below each lane. Hirt-extracted samples were digested with DpnI prior to Southern blot hybridization using a probe specific for the ampicillin resistance gene pUC-Ori177. FIG. 8 shows inhibition of E2 binding by ZFP. Competitive binding experiments between ZFP _HPV- 1 or ZFP _HPV- 2 and E2 use a constant concentration of E2 protein (100 nM) together with a 56 P probe labeled with ³² P containing E2BS-3 and E2BS-4. Lanes 8 to 5 for _HPV- 1 and lanes 12 to 9 for ZFP _HPV- 2 were performed in the presence of increasing concentrations (nM) of ZFP. Lanes 1 to 4 show the band positions of free DNA, E2 binding DNA, ZFP _HPV- 1 binding DNA, and ZFP _HPV- 2 binding DNA used as markers in lanes 5 to 12, respectively. The concentrations of ZFP and E2 protein used are indicated below each lane. FIG. 9 shows inhibition of HPV-18 DNA replication using a transient replication assay with ZFP having a cell penetration domain and a nuclear localization signal. FIG. 10 shows in vitro specific binding of ZFP _HPV- 1 or ZFP _HPV- 2 DNA bound to the replication origin using a mutant DNA probe. The top figure (a) shows the DNA sequence of the mutation probe. Mutations are underlined and lane numbers correspond to those shown in Figures a to g. Figures b, d and f show autoradiographs of ZFP _HPV- 1 at 10, 100 and 1000 pM. Figures c, e, and g show autoradiographs of ZFP _HPV- 2 at 10, 100, and 1000 pM. The positions of DNA-ZFP complex and free DNA are shown. FIG. 11 shows the specific binding of the artificial DNA binding protein to the origin of replication in the cells as measured in a transient replication assay. The mutant probe and mutant ori plasmid are shown in FIG. In order to minimize the negative impact of mutations on HPV-18 DNA replication, mutations were introduced into a 4 base pair DNA spacer located between E2BS-3 and E2BS-4 (ie 5_-CGGG-3_). Figure b shows the gel shift assay of the mutant probe and Figure c shows the transient replication of the HPV-18 mutant ori plasmid. In transient replication assays, pRL-E1, pRL-E2, and pUC-Ori177 derivatives were used with pCMV-ZFP _HPV- 2 or pcDNA3.1 as shown below the lane. FIG. 12 shows a comparison of inhibition of virus replication by E2C-R9 and ZFP _HPV- 2-R9 in a transient replication assay. FIG. 13 is a view showing Western blot analysis of the amount of the protein taken up by cells after adding ZFPHPV-2-CPP (cell membrane permeation peptide) fusion protein to the cell culture medium.

Claims (25)

An antiviral agent comprising an artificial zinc finger protein (ZFP) and / or a nucleic acid encoding the ZFP, wherein the ZFP can inhibit binding of an animal DNA virus-derived replication protein to the viral replication origin Viral agent. The antiviral agent according to claim 1, wherein the replication protein has two or more binding sites at the origin of replication of the DNA virus, and the ZFP binds to the two or more binding sites. An antiviral agent that inhibits the binding of the replication protein to the origin. The antiviral agent according to claim 1 or 2, wherein the animal DNA virus is a human papilloma virus. The antiviral agent according to claim 3, wherein the human papillomavirus is human papillomavirus 18 (HPV18). The replication protein is an E2 protein of human papillomavirus, and the ZFP binds to the position of E2 binding site 3 (E2BS3) and E2 binding site 4 (E2BS4) in the vicinity or in an overlapping manner. The antiviral agent according to any one of the above. The antiviral agent according to claim 5, wherein the ZFP targets a DNA represented by GAAAACGGTCGGGACCGAA or GGTCGGGACCGAAAACGGT. The ZFP contains 6 zinc finger domains, the domain is covalently linked to 0 to 10 amino acid residues, and the amino acids at positions -1, 2, 3, and 6 of the domain are in this order. Gln, Thr, His and Glu for domain 1, respectively;
Glu, Ser, Asn and Arg for domain 2;
Arg, Asp, His and Glu for domain 3;
Thr, Ser, His and Arg for domain 4;
Glu, Asp, Asn and Gln for domain 5; and Gln, Thr, Asn and Arg for domain 6
The antiviral agent according to claim 6. The antiviral agent according to claim 7, wherein the ZFP comprises the amino acid sequence of SEQ ID NO: 2. The ZFP contains 6 zinc finger domains, the domain is covalently linked to 0 to 10 amino acid residues, and the amino acids at positions -1, 2, 3, and 6 of the domain are in this order. Arg, Asn, His and Glu for domain 1 respectively
Gln, Ser, Asn and Gln for domain 2;
Gln, Thr, His and Glu for domain 3;
Glu, Ser, Asn and Arg for domain 4;
Arg, Asp, His and Glu for domain 5; and Thr, Ser, His and Arg for domain 6
The antiviral agent according to claim 6. The antiviral agent according to claim 9, wherein the ZFP comprises the amino acid sequence of SEQ ID NO: 3. The ZFP comprises at least three zinc finger domains covalently bonded to each other at 0 to 10 amino acid residues, and the amino acids at positions 1, 2, 3, and 6 of the α-helix of the zinc finger domain are as follows: Selected:
The amino acid at position -1 is arginine, glutamine, threonine, methionine, or glutamic acid;
The amino acid at position 2 is serine, asparagine, threonine, or aspartic acid;
The amino acid at position 3 is histidine, asparagine, serine, or aspartic acid; and
The antiviral agent according to any one of claims 1 to 5, wherein the amino acid at position 6 is arginine, glutamine, threonine, tyrosine, leucine, or glutamic acid. The ZFP includes at least three zinc finger domains, each zinc finger domain independently having the formula:
_{-X 3 -Cys-X 2-4 -Cys-} X 5 -Z -1 -XZ 2 -Z 3 -X 2 -Z 6 -His-X 3-5 -His-X 4 -
(Wherein X independently represents any amino acid, and X _n represents the number of occurrences of X in the polypeptide chain;
Z ^-1 is arginine, glutamine, threonine, methionine, or glutamic acid;
Z ² is serine, asparagine, threonine, or aspartic acid;
Z ³ is histidine, asparagine, serine, or aspartic acid; and
Z ⁶ is arginine, glutamine, threonine, tyrosine, leucine, or glutamic acid. The antiviral agent according to claim 11, wherein the domains are independently covalently bonded to each other at 0 to 10 amino acid residues. The antiviral agent according to any one of claims 1 to 12, wherein the ZFP further comprises a nuclear localization signal (NLS) and / or a cell penetrating peptide (CPP). The antiviral agent according to claim 13, wherein the NLS and the CPP are located at the N-terminus of the ZFP. The antiviral agent according to claim 13, wherein the NLS and the CPP are located at the C-terminus of the ZFP. The antiviral agent according to any one of claims 1 to 15, which is a ZFP protein. The antiviral agent according to any one of claims 1 to 15, which is a nucleic acid encoding the ZFP protein. A virus replication inhibitor which is the ZFP or nucleic acid according to any one of claims 1 to 17. Use of an artificial zinc finger protein (ZFP) and / or a nucleic acid encoding the ZFP for the production of an antiviral agent, wherein the ZFP inhibits the binding of an animal DNA virus-derived replication protein to the viral replication origin The above use is something that can be done. A method for preventing and / or treating animal viral infection, wherein the viral infection is treated by administering to the animal a prophylactic and / or therapeutically effective amount of the antiviral agent according to any one of claims 1 to 17. , Reduce or improve. A method for inhibiting replication of an animal virus in an animal body, wherein the artificial zinc finger protein (ZFP) (however, the ZFP can inhibit the binding of an animal DNA virus-derived replication protein to the virus replication origin) And / or administering an effective amount of a nucleic acid encoding the ZFP, thereby inhibiting replication of the virus. The method according to claim 20 or 21, wherein the animal is a human. The method according to claim 22, wherein the animal virus is human papillomavirus (HPV). 24. The method of claim 23, wherein the HPV is HPV type 16, 18, 31, 35, 39, 45, 51, 52, 58, or 59. 24. The method of claim 23, wherein the HPV is HPV18.

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