CZ255397A3 - Mutagenic proteins, process of their preparation, pharmaceutical preparations containing thereof and their use - Google Patents

Abstract

eIF-5A mutant proteins which are normally devoid of substantial eIF-5A activity and which inhibit Rev function and thus possess an inhibitory phenotype with respect to Rev function, and genes or DNA or cDNA sequences coding therefor.

Description

Mutant proteins, processes for their preparation, pharmaceutical compositions containing them and their use

Technical field

The invention relates to mutant proteins, processes for their preparation, pharmaceutical compositions containing them and their use.

BACKGROUND OF THE INVENTION

Replication of the human immunodeficiency virus (HIV) depends on the function of the viral transactivating protein Rev. The Rev protein accumulates in the nuclear space of the host cell and binds directly and specifically to a highly structured RNA sequence, called the Rev Response Element (RRE), which occurs on every incomplete viral mRNA. Upon binding, Rev protein molecules conglomerate to RRE and associate with a host cofactor required for Rev transactivation, called eukaryotic initiation factor 5A (eIF-5A; older name: eIF-4D). This linkage results in nuclear cytoplasmic translocation of incomplete spliced viral mRNA, subsequently allowing expression of viral structural proteins (eg, Gag, Pol, Env) and further viral replication.

Mutant Rev proteins with transdominant (dominant-negative) phenotypes have been described (see, eg, WO 90/144227). These mutants will also be capable of binding to RRE RNA, but are unable to functionally associate with their cellular cofactor due to a mutation in the activation domain of Rev.

eIF-5A is an endogenous protein required for normal cell function. Several of its different variants have been prepared (eg J. Biol. Chem. 264 (1989) 18527 - 18530; Mol. Cell. Biol. 11 (1991) 3105 - 3114) and all of these variants exhibit an adverse effect on normal cellular functions, e.g., cell growth.

SUMMARY OF THE INVENTION

It has now been found that mutant forms of eIF-5A can be prepared that do not block the endogenous function of native eIF-5A and normally do not exhibit eIF-5A activity but inhibit Rev function; therefore, they have a phenotype inhibiting function of Rev protein.

This finding is very surprising since it was not assumed that a factor that is inactive in the cellular environment natural for its unmutated form could exhibit an inhibitory effect against metabolic processes initiated by an external microorganism such as a virus. It was also not believed that such mutant forms of the eIF-5A protein, including its genes, could exist at all, since the eIF-5A protein is essential for cellular regulation (Biofactors 4 (1993) 95-104; TIBS 18 (1993) 475-479) . It was believed that such proteins, including their genes, would be toxic to the cell, while the genes and proteins of the present invention (especially M13, M14, and Μ13Δ) are inactive, non-toxic and inhibit Rev protein, such as its function in HIV and thus HIV replication.

The present invention provides mutant forms of the protein, -e-IF-5A, which lacks its "natural" cellular activity! eIF-5A (referred to herein as - endogenous function of eIF-5A) while inhibiting Rev protein function and thus have an inhibitory phenotype with respect to Rev function; furthermore, the subject genes and the corresponding DNA and cDNA sequences encoding them are provided.

Mutants that are substantially devoid of endogenous eIF-5A function are preferred. The eIF-5A referred to herein is preferably

human eIF-5A and its mutants are preferably based on human native form of eIF-5A. The HIV virus disclosed herein is preferably an HIV-1 form thereof.

Mutant proteins according to the invention are understood to mean polypeptides including their modified forms, such as allelic modifications or fragments or derivatives thereof, capable of inhibiting Rev.a protein function, which preferably have almost no endogenous function of eIF-5A. For pharmaceutical purposes, they are preferably used in highly pure form, i.e. substantially free of substances from the intracellular environment.

The mutations preferably comprise a stretch or multiple stretches on the polypeptide chain, beginning approximately at the level corresponding to amino acid # 130 in the native eIF-5A genome (FIG. 3, SEQ ID NO: 17), particularly in the stretch between about amino acid # 135 and end molecules, especially between No. 135 and No. 139.

The corresponding genes or DNA or cDNA sequences are shown, for example, in Table 2, in particular for the mutants elF5AM13, eIF-5AM14 and eIF-5AM13A; furthermore, they may be those genes or sequences which hybridize under stringent conditions to the sequences coding for said proteins or genes or sequences which do not hybridize thereto solely as a result of the degeneracy of the genetic code, and preferably do not hybridize at the same time! with genes and sequences corresponding to the native forms.

The invention further provides prokaryotic and eukaryotic host cells into which they are transformed or _ 1 _ X _ y _ t _1 1 1 TV »-f TI ^_1 UC ± iiuvdi E · · = execute invention further provides a biologically functional plasmid or viral DNA vectors comprising a gene or DNA or cDNA sequence as defined above. Also contemplated by the invention are prokaryotic or eukaryotic host cells which are stably above such that the host cell is capable of expressing these genes or DNA sequences or cDNAs transformed or transfected with the above-mentioned DNA vectors.

The present invention further provides a method for preparing the genes or DNA or cDNA sequences defined above, including isolating the respective native gene from the corresponding expression system, leaning it into the appropriate system, performing the desired genetic mutation, and isolating the resulting mutant from clones with the desired mutation.

The invention further encompasses a method of producing the above defined proteins, which comprises culturing prokaryotic or eukaryotic host cells in the presence of suitable nutrients and under appropriate culture conditions, transformed or transfected with the genes or DNA or cDNA sequences defined above, in such a way as and optionally isolating the resulting polypeptide expression product.

The invention also encompasses pharmaceutical compositions comprising the above defined proteins, together with at least one pharmaceutically acceptable carrier, excipient or diluent only. The carrier may also be cells isolated from the patient's body and treated in vitro prior to administration. The invention further encompasses the use of the above defined proteins or genes or DNA or cDNA sequences encoding these proteins in the treatment of diseases caused by retroviruses in which the function of their Rev protein depends on the function of eIF-5A such as HIV, HTLV-I and HTLV-II. It is also an object of the present invention to provide them from C x-C x = C x C-X x-P x O-P x i-x. The treatment of diseases caused by retroviruses in which the function of their Rev protein depends on the function of eIF-5A, such as HIV, HTLV-I and HTLV-II.

The methods and methods necessary for carrying out the present invention are known in the art. Although their preparation is not specifically mentioned herein, the compounds, reagents, vectors, cell lines and the like that are required for implementation

of the present invention are generally known and commercially available or can be obtained by conventional methods from known and commercially available sources or similar sources can be prepared by conventional methods from other generally known and commercially available sources.

DETAILED DESCRIPTION OF THE INVENTION

a) Preparation of eIF-5A mutants

The human cDNA encoding eIF-5A (J. Cell Biol. 123/6, 1309 1320 / (1993)) was inserted as a HindIII fragment into a single HindIII restriction site of the M13mp18 vector (Pharmacia, Sweden). The corresponding single-stranded DNA was used for in vitro oligonucleotide-directed mutagenesis mutagenesis, version 2 (Amersham, UK). The sequences of all mutants were determined by DNA sequencing using Sequenase 2.0 from United States Biochemicals. Furthermore, all mutated genes encoding eIF-5A (see Table 1) were expressed in COS cells, followed by immunoprecipitation analysis specific for eIF-5A to confirm the presence of the desired expression product.

·· ···· · · ee β o 999

Table 1

Mutants of eIF-5A

Mutant Position (s)

Binding mutation Rev complementation (ak) in vitro in yeast

peF-5AM1 4,5,6 DLD— »EDL nz. Yes peF-5AM2 9.10 TG— »DL nz. Yes peF-5AM3 15,16,17 SAT—> ADL nz. Yes peF-5AM4 22,23,24 CSA—> GDL - No peF-5AM5 43.44.45 MST—> IDL - No peF-5AM6 46.47.48 SKT—> LDL - No peF-5AM7 64.65 FT—> DL - No peF-5AM8 69, 70 YE—> DL - No peF-5AM9 75.76 ST—> DL ' - No peF-5AM10 98,99,100 YLS—> LDL - No peF-5AM11 104,105,106 DSG— »QDL nz. Yes peF-5AM12 126,127 KY— »DL nz. Yes peF-5AM13 135.136 IT—> DL + No peF-5AM14 138.139 LS—> DL + No peF-5AM15 141,142,143 MTE IDL nz. Yes peF-5AM16 149,150 IK—> DL - No · = · - - = · - =: ··· . .. =, ===, .. peF-5AM13A * 135 DLCCLP-STOP + No

* peIF-5A13A is a 140 amino acid (if) protein after deletion of the C-terminal of peIF-5A. Its sequence is: native from amino acid # 1 to # 134 and amino acids: DLCCLP. All other mutants are due to amino acid substitutions.

esceββ + denotes native activity, - denotes no activity, nz. indicates not determined.

The nucleotide sequences of the specific oligonucleotides used to prepare the 17 mutants are shown in Table 2 (SEQ ID NOs 1 to 16). The last mutant, Μ13Δ, was prepared using the same oligonucleotide as M13; however, an error in the mutagenesis procedure caused the formation of the mutant Μ13Δ.

It will be appreciated that the nucleotide sequences shown are merely representative examples of the 17 mutants prepared, and that identical mutants can be prepared from a variety of other sequences, taking into account, for example, deviations from the standard genetic code.

• eeee β ··

Table 2

Oligonucleotides used to prepare eIF-5A mutants

Ml: 5 ' - GGC AGA TGA AGA TCT CTT CGA GAC AGG - 3 ' M2: 5 ' - CTT GGA CTT CGA AGA TCT AGA TGC AGG - 3 ' M3: 5 - GCA GGG GCC GCA GAT CTC TTC CCA ATG C - 3 ' M4: 5 ' - CCC AAT GCA GGG AGA TCT ATT ACG TAA G - 3 ' M5: 5 ' - CGT CGA GAT AGA TCT TTC GAA GAC TGG - 3 ' M6: 5 ' - CGA GAT GTC TRAY TTT AGA TCT TGG CAA GCA CG - 3 ' M7: 5 ' - GGT ATT GAC ATA GAT CTT GGG AAG AAA TAT G - 3 ' M8: 5 ' - GGG AAG AAA GAT CTA GAT ATC TGC CCG - 3 ' M9: 5 ' - GAT ATC TGC CCA GAT CTT CAT AAT ATG G -3 ' M10: 5'- GGC ATC CAG GAT GGG TTA GAT CTA CTG CTC CAG AGC G -2 i ' Mil: 5 ' - CTG CTC CAG GAA GAT CTG GAG GTA CGA G -3 ' M12: 5 ' - GAG ATT GAG CAA GAT CTC GAC TGT GGA G -3 ' M13: 5 ' - GAA GAG ATC CTA GAT CTG GTG CTG TCT GCC - 3 M14: 5 ' - CCT GAT CAC GGT AGA TCT TGC CAT GAC AGA GG - 3 ' M15: 5 ' - GCT GTC TGC CAT AGA TCT GGA GGC AGC TG - 3 ' Ml 6: 5 ' - GCA GCT GTT GCA GAT CTG GCC ATG GC - - 3 '

b) Testing of cellular activity of eIF-5A mutants of eIF-5A in yeast Saccharomyces cerevisiae

The sequences encoding eIF-5A were inserted into the BamHI and XbaI restriction sites of the yeast shuttle vector pRSGAL using polymerase chain reaction (PCR) technology. For testing the complementation capacity of various eIF-5A mutants in a yeast strain of Saccharomyces cerevisiae not containing eIF-5A, plasmid technology published in Mol. Gene. Genet. 244, 646-652 (1994).

·· ····

The results are summarized in Table 1; several eIF-5A mutants, particularly from peIF-5AM4 to peIF-5AM10, and further peIF-5AM13, peIF-5AM16 and peIF-5AM13A have been identified as non-functional mutants of the eIF-5A protein.

c) In vitro binding of non-functional mutated eIF-5A proteins to HIV transactivating protein using a gel mobility change assay

The genes of native and all non-functional forms of eIF-5A were inserted between the BamHI and EcoRI restriction sites of the prokaryotic expression vector pGEX-3X (Pharmacia) using a polymerase chain reaction, and expressed as fusion proteins containing glutathione-S-transferase (GST) ) in Escherichia coli, followed by purification. Various GST-eIF-5A fusion proteins were further used in gel mobility assays with RRE RNA labeled with ³² P isotope and recombinant Rev protein (Nature 342, 816-819 (1989)) according to the procedure described in Biochemistry 32, 10497 -10505 (1993).

The effect of the native GST-elF5A protein on the RRE: Rev. As shown in Figure IA, the addition of GST-eIF-5A protein to the Rev-containing RRE RNA reaction mixture allowed the detection of a complex of higher molecular weight and clearly reduced mobility (Fig. IA, lanes 5 and 4).

This was not detectable by incubating the RRE RNA together with GST or with GST-elF-5A alone (Fig. 1A, lanes ⁵ 2 and 3). '- - · ™

Furthermore, it was tested whether the eIF-5A specific response depends on the presence of the activation domain in the transactivating protein Rev. The previously described mutant proteins Rev9A14 and Rev11A14 (Biochemistry 32, 8945-8954 (1993)) were used for this finding. Both of these proteins contain an internal deletion near the C-terminal and exhibit specific binding affinity and specificity for RRE RNA that is comparable to the native form of Rev. Importantly, the activation domain was completely removed from the Rev9A14 protein, but was retained in the Rev11A14 protein. The lower molecular weight of the two proteins caused a shift of the RRE: protein bands, which was difficult to determine in the gel mobility test. However, the results clearly show that the mutant with the deleted Rev9A14 activation domain is not able to interact with GST-eIF-5A (Fig. 1A, lanes 6 and 7). In contrast, the addition of GST-eIF-5A to the RRE: Rev11A14 reaction mixture produced a new result (Fig. 1A, lanes 8 and 9; slower moving point in lane 9), indicating the interaction of eIF-5A with the activation domain of Rev.

Finally, the binding capabilities of various non-functional mutant eIF-5A proteins were tested according to the same procedure.

As shown in Figure 1B, most of the GST-E1F5A mutant proteins lost the ability to discriminate the RRE: Rev complex (lanes 4-10 and lane 13). However, the three non-functional mutant proteins: GST-1F5AM13, GST-eIF-5AM14 and GST-eIF-5AM13A, show binding that is comparable to that of native eIF-5A (Fig. 1B, lanes 3 and 11, 12 and 14).

Thus, the results show that the eIF-5AM13, eIF-5AM14 and eIF-5AM13A mutants meet the requirements for inhibitors of HIV-1 Rev function due to:

(1) loss of endogenous function of eIF-5A (see.

... yeast complementation, Table I), a. . ..

(2) for HIV-1 Rev protein binding activity (see Rev in vitro binding; Table I and Figure 1B).

d) Constitutive inhibition of HIV-1 replication in human T cells by retroviral-mediated gene transfer of eIF-5A transdominant mutants.

For testing the inhibitory phenotypes of eIF-5AM13, eIF-5AM14 and eIF-5AM13A, the respective eIF-5A coding sequences were inserted into the Xho I restriction site of the pBC 140 retroviral vector, packaged in vitro using the Am 12 cell line, and transferred to human T - CEM cells for constitutive expression as described in Proc. Nati. Acad. Sci. 89, 9870-9874 (1992). Expression of the native eIF-5A gene, the gene encoding eIF-5AM9, and the retroviral vector itself was used for reference comparison in these experiments.

HIV-1 experiments were also performed using a 2000 / ml TCID of HIV-1 SF2 strain. On the 10th day after SF2 infection, samples were taken from the culture medium for the HIV-1 replication assay by determining the p24 Gag protein using an ELISA assay. As shown in Figure 2, HIV-1 replication was substantially reduced in each of the three independently cloned CEM cell lines (designated A, B, C) producing eIF-5AM13, eIF-5AM14 or elF55ΜΜΔ (Figures 2C, 2D and 2E; left), compared to CEM cells producing the mutant eIF-5AM9 protein (CEM-eIF-5AM9; Fig. 2B). Maximum viral replication was found in CEM cells producing the parent vector pBC140 (CEM vector; Figure 2A) or native eIF-5A protein (CEM-eIF-5Awt; Figure 2A). The observed inhibitory properties were not due to a general inhibition of cell proliferation, as evidenced by comparable cell numbers in the experiments (Fig. 2; right).

In conclusion, these experiments demonstrate that (1) the constitutive expression of eIF-5AM13, eIF-5AM14 or eIF-5AM13A does not have toxic effects in human C-cell TEM cells, and (2) that eIF-5AM13, eIF-5AM14 and eIF-5AM13A are HIV-1 replication inhibitors.

·· ···

HIV is the predominant etiological agent of AIDS (acquired immunodeficiency syndrome); HTLV-I causes inter alia ATL (adult T cell leukemia); HTLV-II is etiologically related to some cases of variant hair T cell leukemia. HIV Rev and HTLV-I Rex transactivators have been found to be important for viral replication in media. Furthermore, it has been shown that HTLV-I Rex is able to functionally replace the Rev protein in HIV. In addition, Rev and Rex are therefore potential targets for therapeutic intervention in affected patients. The 6IF-5A mutant forms of the invention act as effective competitive inhibitors of the native form of Rev and / or the function of native Rex due to the ability of Rex to functionally replace Rev in HIV. Accordingly, these mutant forms of eIF-5A may also be effective inhibitors of HIV and / or HTLV-I and / or HTLV-II replication. Thus, they can be used to protect the lymphoid and / or myeloid cells of patients exposed to infection and therefore can be used to treat diseases caused by HTLV-I and HTLV-II or HIV, including AIDS and ARS or ARC (AIDS-related syndrome or complex) . They can also be used to treat diseases caused by AIDS because they are able to inhibit the activity of HTLV-I Rex and HIV Rev. This property may be particularly beneficial in patients infected with more than one viral pathogen, and in those who are not found to know which of these two agents are infected.

Thus, the present invention is directed to the prophylaxis and therapy of viral and in particular retroviral diseases such as ATL, AIDS, ARS (or ARC), infections caused by viruses such as SIV (monkey immunodeficiency virus) such as SIV _mac , FIV (feline immune deficiency virus). ), EIAV (equine infectious anemia virus), visna virus, bovine immune deficiency virus, and in particular human retroviral diseases, particularly human retroviral diseases caused by pathogens regulated by rex or rev genes or equivalents thereof, such as ATL, AIDS and ARS (or ARC). The multivalent use of the repressor effect deserves special attention as it is useful in the treatment of multiple, particularly dual viral infections, as can often be observed, for example, in persons using intravenous drugs and co-infected with HIV and HTLV-I or the risk of another infection, such as HIV infection or in the case of prophylaxis in situations where protection against infection caused by a spectrum of different viral strains is needed.

The therapeutic possibilities of the present invention are obvious, since suppression of, for example, Rex function in HTLV-I and Rev function in HIV blocks viral replication, thus reducing viral load by preventing the formation of infectious viral particles and is therefore believed to prolong the latent period of infection. Cells of persons infected with virus. HIV, having an integrated gene encoding the mutant eIF-5A repressor in its genome, will remain functional and will be indefinitely protected from the manifestation of disease symptoms without the need for further long-term therapy.

In this regard, the genes of the invention per se are medicaments intended for single or multi-phase dosing either directly in vivo or indirectly in vitro, preferably as part of a vector, for example retroviral or plasmid, in a form suitable for achieving transfer in functional form to target mammalian species. cells. For example, insertion of genes encoding these viral replication inhibitors may be performed in vitro into patient cells by direct implantation into the genome of lymphoid and / or myeloid cells taken from these patients and these cells may be delivered to the patient after insertion. Since HTLV-I and HTLV-II as well as HIV replicate in different types of T cells, the diseases they cause will be treated appropriately. T cells are therefore the desired target cells for genetic modification using the genes of the invention. Furthermore, monocyte and macrophage cells, which are target cells for HIV infection, are also suitable target cells for this modification. Preferably, hematopoietic stem cells are modified to provide long-term protection of the cell progeny of a multiple hematopoietic genus.

One use of this is related to the concept of gene therapy published by T. Friedman in Science, 244, (1989),

1275 or P.M. Lehne in Bone Marrow Transplant 1 (1987) 243. Hematopoietic stem cells are isolated from, for example, AIDS / ATL affected patients, cultured in vitro, and the mutant genes of the invention encoding an inhibitor of the function to be suppressed, namely Rev / Rex function, are implanted. into these cells using retroviral vectors. These cells, now resistant to viruses, are returned to the original patient's immune system where they are expected to proliferate due to their acquired selective advantage over other stem cells.

At the required time, the hematopoietic cell population will consist exclusively of cells producing the repressive factor and will be resistant to viruses.

Methods to achieve this are already known in the art, see, eg, USP 4'868'116. Vectors such as retroviral or plasmid vectors for transferring the mutated genes of the invention to target mammalian cells, such as bone marrow cells, are disclosed or referenced, for example, in Science 244 (1989) 1275. For example, various transdominant genes encoding Rev and / or and Rex have already been inserted into retroviral vector systems. Upon transfer of the gene by retroviruses to human cell lines infected with, e.g., HIV, the inhibitory effect of the mutants is evident on the inhibition of virus production. Said therapeutic concept is an example of intracellular immunization explained in D. Baltimore in Nature, 335, (1988), 3'95. In summary, this concept involves inserting a gene that encodes a repressor of some vital function of a selected virus into the respective target cells that the virus infects (eg, certain T cells and monocyte and macrophage cells in the case of AIDS-inducing virus).

The use of this concept in repressive therapy of any virus is subject to the condition that the vectors used for the genes encoding these mutant proteins and the methods used to insert these vectors into appropriate cells, identified as models, constitute an efficient and safe intracellular transfer system for human and human use. of animals. Thus, the experimental verification of this concept is confronted only by ethical barriers that currently prevent gene therapy. However, the first such experiments in humans have already been initiated, with a focus on safety and self-therapy (see G.T. Nabel, Human Gene Therapy, 5, (1994), 79-82).

It is expected that in a very short time after verifying the harmlessness of this procedure, these pioneering experiments will be followed by similar experiments with therapeutic targets, namely. especially in life-threatening conditions such as AIDS and that the present invention will prove very suitable for use during these experiments (see, e.g., T. Friedman, Science 244 (1989), 1279, second column: "Infectious Disease").

. ________ ". Another one. the method of utilizing the invention is to insert not the gene but directly the repressive protein of the invention into the target cells. Oral or parenteral administration is accomplished by conventional means allowing intracellular penetration, e.g., through liposomes. In such cases, the precise dose will of course depend on the compound employed, the mode of administration and the treatment desired; the determination of the most appropriate dosage in a given situation is within the skill of the skilled artisan.

• ·· · · · · · · ·

It is to be understood that the foregoing invention may be subject to various combinations or changes in its form or individual details without departing from the scope of the invention. It is also to be understood that other mutants outside the scope of the present invention, including mutants belonging to the mutants already constructed and mentioned herein, but which have not yet been characterized but may be found to be inhibitory and / or in more detail, are within the scope of this invention. and multivalent, and further mutants that meet the above principles but are not specifically disclosed herein.

In vitro gel shift RNA experiment.

FP = free probe; eIF-5A (-) = no eIF-5A.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·. · I · · · t. · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Description of the picture:

Figure 1

Figure 2; Experiments with HIV-1. P24Gag values are shown on the left. Cell numbers for the respective experiments are shown on the right:

A: CEM-vector / eIF-5A wt C: CEM-eIF-5AM9 C: CEM-eIF-5AM13 D: CEM-eIF-5AM14

E: CEM-eIF-5AM13A

Figure 3: (SEQ ID NO: 17): cDNA and amino acid sequences of native human eIF-5A (J. Bioi. Chem. 264 (1989) 1581) (SEQ ID NO: 17); the amino acid lysine shown at position 50 is post-translationally modified to hypusin. Native eIF-5A amino acids that have been mutated as outlined in Table 1 are shown in italics. The specific oligonucleotides used (Table 2, SEQ ID NO: 1 to 16) correspond to the following nucleotide positions in Figure 3 (SEQ ID NO: 17):

Ml: 3-29 (27 nucleotides) M2: 12-38 (27 nucleotides) M3: 34-61 (28 nucleotides) M4: 54-81 (28 nucleotides) M5: 120-146 (27 nucleotides) M6: 123-154 (32 nucleotides)

M7: 178-208 (31 M8: 196-222 (27 M9: 211-238 (28 M10: 277-316 (40 Mil: 301-328 (28 M12: 364-291 (28 M13: 391-420 (30 M14: 399-430 (32 M15: 411-439 (29 M16: 433-458 (26 Μ13Δ : 391-421 (30 is without translation, STOP in positions 4

(31 nucleotides) (27 nucleotides) (28 nucleotides) (40 nucleotides) (28 nucleotides) (28 nucleotides) (30 nucleotides) (32 nucleotides) (29 nucleotides) (26 nucleotides) (30 nucleotides, position # 409 is followed by codon -424), resulting in amino acid changes shown in Table 1 in the native protein sequence.

·· ····

Sequence list

Sequence # 1 (Ml) information:

(i) Sequence characteristics:

(A) Length: 27 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: Sequence # 1:

GGC TGA AGA TCT CTT CGA GAC AGG

Sequence # 2 (M2) information:

(i) Sequence characteristics:

(A) Length: 27 base pairs (B) Type:

_. . (C) String: double_th_, ______ ₅ (D) Topology: linear (ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic · «. ···· ······· · (x) Information ο Disclosure:

(Η) Document Number: (I) Filing Date: (J) Publication Date: (xi) Sequence Description: Sequence # 2:

CTT AGGA CTT AGGA TCT AGA TGC AGG

Sequence # 3 (M3) information:

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 3:

GCA GGG GCC GCA GAT GTC CTC TTC CCA ATG C

Sequence # 4 (M4):

(i) Sequence characteristics:

(A) Length: 28 (B) Type:

(C) String: double (D) Topology: linear

(ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: Sequence No. 4:

CCC AAT GCA GGG

Sequence # 5 (M5):

(i) Sequence characteristics:

(A) Length: 27 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: ₌ , (.I-.). _{i =} Da-tum =: administration _! : ^ (J) Date of publication: (xi) Sequence description: Sequence No. 5:

CGT CGA GAT TTC TTC GAA GAC TGG

9 · ····

Information ο Sequence No. 6 (M6):

(i) Sequence characteristics:

(AND) Length: 32 base pairs '(B) Type: (C) String: double (D) Topology: linear (ii) Type of molecule : cDNA (iii) Hypothetical: No (iii) Antisense: No.

(vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document Number: (I) Filing Date: (J) Publication Date: (xi) Sequence Description: SEQ ID NO: 6:

CGA GAT GTC TAC TTT

Sequence # 7 (M7):

(i) Sequence characteristics:

(A) Length: 31 base pairs (B) Type:

(C) String: double (D) Topology:. linear (ii). Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: 23

(I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 7:

GGT ATT GAC ATA GAT

Sequence # 8 (M8):

(i) Sequence characteristics:

(AND) Length: 27 base pairs (B) Type: (C) String: double (D) Topology : linear

(ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x)

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: Sequence No. 8:

GGG AAG AAA GAT CTA GAT ATC TGC CCG

Sequence # 9 (M9):

(i) Sequence characteristics:. .

(AND) Length: 28 base pairs (B) Type: (C) String: double (D) Topology: linear

(ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 9:

GAT ATC TGC CCA

Sequence # 10 (M10):

(i) Sequence characteristics:

(A) Length: 40 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date:. (. Xi.) __ Pppijs _w , sequence l 'SsJsvendé ď. _m , 1Q:.

GGC ATC CAG GAT GGG

Sequence # 11 (Mil):

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 11:

CTG CTC CAG GAA GAT

Sequence # 12 (M12):

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type: '' (C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) · Organism: Synthetic = ((x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: Sequence No. 12:

·· ····

GAG ATT GAG CAA

Sequence # 13 (M13):

(i) Sequence characteristics:

(AND) Length: 30 base pairs (B) Type: (C) String: double (D) Topology : linear

(ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 13:

GAA GAG ATC CAT GAT CTG GTG CTG TCT GCC

Sequence No. 14 (M14):

(i) Sequence characteristics:

(A) Length: 32 base pairs (B) Type:

(C) String: double. .

(D) Topology: linear (ii) Type of molecule: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic

?

(x) Publication Information:

(H) Document number: (I) Filing date: (J) Date published (xi) Sequence description: Sequence No. 14:

CCT GAT CAC GGT AGA

Sequence # 15 (M15):

(i) Sequence characteristics:

(A) Length: 29 base pairs (B) Type:

(C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x)

(H) Document number: (I) Filing date: (J) Publication date: (xi) Sequence description: SEQ ID NO: 15:

GCT GTC TGC CAT AGA

Sequence No. 16 (M16):

(i) Sequence characteristics:

(A) Length: 26 base pairs (B) Type:

(C) String: double (D) Topology: linear ·· ···· (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Synthetic (x) Publication Information:

(H) Document number: (I) Date of completion: (J) Date of publication: (xi) Sequence description: Sequence No. 16:

GCA GCT GTC ATG GC

Sequence information No. 17 (wt):

(i) Sequence characteristics:

(A) Length: 465 base pairs (and 154 amino acid residues) (B) Type: cDNA (C) Chain: double (D) Topology: linear (ii) Molecule type: cDNA (iii) Hypothetical: No (iii) Antisense: No (vi) Original source:

(A) Organism: Homo sapiens (x) Publication Information:

(H) Document number: J. Biol. Chem. 264 (1989) 1578 (I) Submission Date: (J) Date Published: 1989 (xi) Description of Sequence: SEQ ID NO: 17:

·· · «·· • ··· ··· e eee

ATG GCA 10 TTG GAC TTC GAG ACA 30 GGA GAT GCA GGG GCC TCA GAT GAC Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser 1 10 50 70 90 GCC ACC TTC CCA ATG CAG TGC TCA GCA TTA CGT AAG AAT GGC TTT Ala Thr Phe For Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe 20 May 110 130 GTG GTG CTC AAA GGC CGG CCA TGT AAG ATC GTC GAG ATG TCT ACT Wall Wall Leu Lys Gly Arg For Cys Lys Ile Wall Glu Met Ser Thr 40 150 170 TCG AAG ACT GGC AAG CAC GGC CAC GCC AAG GTC CAT CTG GTT GGT Ser Lys Thr Gly Lys His Gly His Ala Lys Wall His Leu Wall Gly 60 190 210 ATT GAC ATC TTT ACT GGG AAG AAA MELT GAA GAT ATC TGC CCG TCA Ile Asp Ile Phe Thr Gly Lys Lys Tyr Glu Asp He Cys For Ser

230 250 270

ACT CAT AAT 'ATG GAT GTG CCC AAC ATC AAA

Thr His Asn Met Asp Asn Ile Lys Arg Asn Asp Phe Gin

90

290

CTG ATT GGC ATC CAG GAT

Leu Gle Ile Gin Asp Gly Tyr Leu G Leu Le Gin Asp Ser

100 ·· ····

330

GGG GAG GTA CGA GAG GAC CTT CGT Gly Glu Wall Arg Glu Asp Leu Arg 110 370 AAG GAG ATT GAG AAG TRAY GAC TGT Lys Glu Ile Glu Lys Tyr Asp Cys 10 430 GTG CTG TCT GCC ATG ACA GAG GAG Wall Leu Ser Ala Met Thr Glu Glu

140

350

CTC CCT GAG GGA GAC CTT GGC Leu For Glu Gly Asp Leu Gly 390 ⁼ 4 GGA GAA GAG ATC CTG ATC ACG Gly Glu Glu Ile Leu Ile Thr

130

450 GCA GCT GTT GCA ATC AAG GCC Ala Ala Wall Ala Ile Lys Ala

150

Claims (13)

PATENT CLAIMS A mutant eIF-5A protein that inhibits Rev function and thereby exhibits an Rev phenotype with respect to Rev function or a gene or DNA sequence or cDNA sequence encoding that protein. The protein of claim 1, which does not possess a substantial endogenous function of eIF-5A or a gene or DNA sequence or cDNA sequence encoding the protein. The protein of claim 1 or 2 which is the protein elF5AM13, eIF-5AM14 or eIF-5AM13A, or a gene or DNA sequence or cDNA sequence encoding the protein. A prokaryotic or eukaryotic host cell, characterized in that it is transformed or transfected with a DNA or cDNA gene or sequence according to claim 1 or 2, in a manner that allows the host cell to express the gene or DNA or cDNA sequence. A biologically functional plasmid or viral DNA vector comprising a gene or DNA sequence or a cDNA sequence according to claim 1 or 2. Sixth Pr of r k t yo, or EU karyotic hostite1s ka ^"and" Bu-ňkav ⁼ ^ = - - ~ · · characterized in that it is stably transformed or transfected with a biologically functional plasmid or viral vector containing the gene or DNA sequence The DNA or cDNA sequence of claim 1 or 2. 7. A method for producing a gene or DNA sequence or cDNA sequence according to claim 1 or 2, comprising isolating the corresponding native gene from the appropriate expression system, inserting the gene into the appropriate cloning system, performing the desired genetic mutation, and obtaining the resulting mutant from the clones containing the desired mutation. A method of producing a protein according to claim 1 or 2, comprising culturing prokaryotic or eukaryotic cells transformed or transfected with the gene or DNA sequence or cDNA sequence according to claim 1 or 2 under appropriate nutrient conditions, such that the host cell is allowing the protein to be expressed and optionally also isolating the desired polypeptide expression product. A pharmaceutical composition comprising a protein according to claim 1 or 2 together with at least one pharmaceutically acceptable carrier, excipient or diluent or in the form of cells taken from the patient's body and processed in vitro prior to their return to the body. Use of a protein according to claim 1 or 2 or a gene or DNA sequence or cDNA sequence encoding the protein in the treatment of diseases caused by retroviruses whose function of Rev depends on eIF-5A. Use of a protein according to claim 1 or 2 or of the - or gene. . . a DNA sequence or cDNA sequence encoding this protein in the treatment of diseases caused by HIV, HTLV-I and / or HTLV-II. The protein of claim 1 or 2 or the gene or DNA sequence or cDNA sequence encoding the protein for use as a medicament. 99,9999 • · · 9,999 9 9 «99 9 13. for Roar Use of a protein according to claim 1 or 2 for the preparation of a medicament for the treatment of diseases caused by retroviruses whose function