IE913993A1 - Antiviral activity of the rep gene encoded by adeno-¹associated virus type 2 - Google Patents

Antiviral activity of the rep gene encoded by adeno-¹associated virus type 2

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Publication number
IE913993A1
IE913993A1 IE399391A IE399391A IE913993A1 IE 913993 A1 IE913993 A1 IE 913993A1 IE 399391 A IE399391 A IE 399391A IE 399391 A IE399391 A IE 399391A IE 913993 A1 IE913993 A1 IE 913993A1
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Ireland
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hiv
rep
aav
virus
gene
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IE399391A
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Behringwerke Ag
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Publication of IE913993A1 publication Critical patent/IE913993A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
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  • Biophysics (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the use of a rep-gene which originates from the adeno-associated virus type 2 and has an antiviral activity towards the HIV-1 virus.

Description

This invention relates to the use of the rep gene encoded by adeno-associated virus type 2 (AAV-2) for the preparation of a specific antiviral agent.
The invention furthermore relates to plasmids with antiviral activity and to a diagnostic kit.
Human immunodeficiency syndrome (AIDS) has in recent years become a serious problem for human society. Although there are some applications which deal with the control of the human immunodeficiency virus (HIV) which is (causally) associated with the AIDS disease, control of this virus has to date had only limited success. Some agents are able to reduce spread of the virus in vivo and thus increase the life expectancy of the affected patients. However, no medicament or treatment which completely destroys the virus and thus restores the health of an affected person has yet been found. All the pharmaceuticals used to date against HIV have a serious disadvantage: they have very severe side effects.
The inventors were aware of these great disadvantages in the treatment of HIV and have therefore looked for alternative antiviral factors which ought not to display the serious side effects of known medicaments.
Inhibition of the replication and of the potency for transformation of adenovirus (5) and of herpes simplex virus (6) by the adeno-associated virus (AAV) as well as the inhibition of cellular transformation which is assisted by bovine papillomavirus (Virology, 172, pp. 253-261 (1989)) and the inhibition of the gene amplification induced by HSV (Journal of Virology, 64. - 2 pp. 3012-3018 (1990)) have likewise been observed.
AAV is a parvovirus which is dependent on helper viruses and has a single-stranded DNA genome with an approximate length up to 5 kb (Advances in Virus Research, 32. pp. 243-307 (1987)).
The virus has a wide host range and depends on helper functions of other viruses. However, this virus does not appear to depend on tissue factors or species-specific cellular factors. The genome contains two non-overlapping open reading frames, that on the 3' side encoding three viral coat proteins, and the second, which is called rep, encoding for four known rep proteins with molecular weights of 78 kd, 68 kd, 52 kd and 40 kd (Journal of Virology 60, pp. 823-832 (1986)). The 68 kd rep protein has been shown to have DNA-binding capacity and an ATP-dependent endonuclease activity and DNA helicase activity (Cell 61, pp. 447-457 (1990)). It is known that the products of the open reading frame (rep) regulate the autologous promoters p5 and pl9 in trans (J.
Virol. 63., pp. 4450-4454 (1989); Mol. Cel. Biol. 6, pp. 2884-2894 (1986)).
The inventors have investigated a possible negative interference evident on replication of HIV type 1 in the presence of intact AAV-2 DNA.
The aim of the present invention is to provide factors which have an antiviral activity. Another aim of the present invention is specifically to find a factor which has HIV-l-inhibiting activity.
The aim of the invention is met by the subject-matter of claim 1. The inventors have found that the so-called rep gene which is encoded by adeno-associated virus type 2 (AAV-2) has an antiviral activity. This means that the rep gene or a part-sequence of the gene can be used to prepare a specific antiviral factor.
In particular, it is possible to control HIV virus type 1 with the rep gene or part-sequences of this gene.
The effects of the AAV rep open reading frame, which is integrated in the complete AAV genome, on HIV-1 replication have been investigated in a transient assay system (Biochem. Biophys. Res. Comm. 169. pp. 643-651 (1990)). This system is based on comicroinjection of wild-type AAV or of a mutant DNA (J. Virol. 64. pp. 30123018 (1990)) together with an infectious HIV-1 proviral DNA (clone pNL4-3) (J. Virol. £2., pp. 284-291 (1986)) into the nuclei of human epitheloid SW480 cells. These cells permit HIV-1 replication after transfection but cannot be infected with HIV-1 virus (J. Virol. £9., pp. 284-291 (1986)). HIV-1 virus was initially prepared in comicroinjected cells and was grown by cocultivation with human T-lymphoid MT-4 cells. HIV-1 was then measured in a cell-free coculture supernatant with the aid of commercial HIV-1 antigen ELISA (Biochem. Biophys. Res. Comm. 169. pp. 643-651 (1990)). It is evident from these results, which are summarized in Fig. 1, that the strong inhibition of HIV-1 replication at a molar ratio of HIV-1 DNA to AAV DNA of 1:10 in the initially comicroinjected cells is correlated with the presence of an intact AAV rep open reading frame. The plasmids mentioned in Fig. 1 (pTAV) have been described (Heilbronn et al 1990; J.
Virology 64: 3012-3018 and AAV-2 wild-type sequence in Laughlin et al. (1983), Gene 23: 65-73). Inactivation of the rep open reading frame while, at the same time, retaining the integrity of the cap open reading frame permits completely normal HIV-1 replication. Inhibition is not observed when the rep open reading frame is inactivated. Furthermore, it is found that the rep+, rep' mutant (pTAV2-3) which ought not to be able to produce AAV virus particles - even if it is possible to assume a hypothetical helper function of the HIV-1 virus for AAV replication in SW480 cells - has the same anti-HIV-1 activity as the wild-type AAV (rep+, cap+). This finding makes it clear that the inhibition of HIV-1 replication actually takes place in the microinjected SW480 cells and that this effect is by no means caused by an artefact occurring in a late stage of coculture. The products of translation of the AAV rep open reading frame are involved in the inhibition of HIV-1 replication. This is why another preferred element of the invention is that the polypeptides which are produced after translation of the open reading frame of the AAV rep gene can be used to prepare a specific antiviral factor. These proteins can also form part of a diagnostic kit which contains at least one of the four proteins obtained after translation of the open reading frame of the AAV rep gene.
The inhibition, mediated by the rep gene, of HIV-1 replication is not the result of a possible cytotoxicity of the rep proteins and is therefore not based on the affected cells' incapacity for HIV-1 replication owing to destruction of the cells' own synthesis apparatus either. Comicroinjection of a Rous sarcoma virus LTR-driven £-galactosidase gene with rep+ (pTAV2) or rep' (pTAV2-3) DNA leads to no difference in the percentage of microinjected cells with 0-galactosidase gene expression when this expression is measured 24 h after injection. This clearly shows that SW480 cells very probably permit gene expression in the presence of the rep gene and that, consequently, the microinjected cells retain their potency for HIV-1 gene expression and for virus production.
The AAV-dependent inhibition of HIV-1 virus replication depends on an intact rep gene. However, it remains to be determined which elements of the HIV-1 virus are involved in the negative interference. To analyze whether sequences which from HIV-1 5'-LTR are involved in the repdependent inhibition of HIV-1 replication, the HIV-1 LTR (U3/R portion) driven expression of chloramphenicol acetyltransferase (CAT) was observed as an indicator gene. The CAT expression level was measured in the presence of an intact AAV rep gene using the assay described above. The results show that pTAV2-6 (rep+), but not pTAV2-3 (rep‘), inhibits CAT expression (Fig. 2a) in a dose-dependent manner (Fig. 2b). This means that the U3/R portion of HIV-1 5'-LTR is sufficient for rep5 dependent inhibition of gene expression. However, this does not exclude an influence of the rep gene also on other steps in HIV-1 replication.
The nucleotide positions for HIV-1 accord with Adachi et al. (1986) J. Virol., 59, 284-291 and for AAV-2 accord with Berns and Bohenzky (1987) Adv. Virus Res., 32, 243307 (Fig. 3).
Fig. 4 describes the position of the AHH sequence in the rep gene.
The biochemical properties and the biological role of the 15 rep proteins suggest that there is an interaction between the rep proteins and the HIV-1-specific nucleic acids. It is possible in this connection that the DNA, or possibly the RNA, both of which are contained in the U3/R sequence of HIV-1, play an important part in the mechanism of HIV-1 inhibition. Comparison of the DNA sequence between the AAV-2 DNA and the HIV-1 LTR sequence (position 1-634, J. Virol. 55., pp. 284-291 (1986)) shows a 25 bp region with 72 % sequence identity at positions 16-40 on the AAV-2 DNA and positions 483-507 on the HIV-1 DNA, which is part of the tar sequence.
Furthermore, a region with a high degree of homology between the calculated local secondary structure of the HIV-1 tar region and the AAV terminal sequence is to be found. This shows that secondary structure recognition is involved in a possible direct or indirect interaction between rep protein and HIV-1 RNA sequence.
The AAV rep gene inhibits HIV replication due to the interaction with the HIV-1 LTR sequence. As shown above, it is possible by comparing the sequences of AAV-2 and of - 6 the HIV-1 LTR region to find a sequence which is 25 bp long, this region being highly conserved. This region was called AHH, and this region also shows great homology between AAV-2 and HIV-1 at the level of the two-dimen5 sional structure. Competition experiments with HIV-1 LTR fragments show that the inhibition (in this case the repdependent inhibition of HIV-1 LTR-driven CAT expression) depends on the presence of the AHH region. It was possible to show that with a double-stranded DNA fragment which is 25 base-pairs long and has the AHH sequence there is an even more drastic rise in the rep-mediated inhibition of HIV-1 LTR-driven CAT expression. This unambiguously shows the involvement of the AHH region in the inhibitory mechanism.
This result shows in an unambiguous manner the antiviral potential of the AAV-encoded rep genes. The circumstance that the rep gene is non-toxic for human SW480 cells and that an exceptionally high inhibitory effect on HIV replication is observed in the presence of the rep gene shows in a very clear manner the outstanding properties of the subject-matter of the present invention and the favorable prerequisites which are required for a use as antiviral agent.

Claims (8)

1. Patent Claims
1. The use of a rep gene derived from adeno-associated virus type 2 (AAV-2) or of a part-sequence of this gene for the preparation of a specific antiviral agent for 5 controlling viral infections.
2. The use as claimed in claim 1, wherein the virus to be controlled is the HIV-1 virus.
3. A peptide which is a translation product of the open reading frame of the AAV rep gene. 10
4. A peptide as claimed in claim 3, which is a translation product of the insert of the plasmids pTAV2 or pTAV2-6.
5. A diagnostic kit containing at least one peptide of the peptides of claims 3 and 4.
6. The use as claimed in claim 1, substantially as hereinbefore described.
7. A peptide as claimed in claim 3, substantially as hereinbefore described.
8. A diagnostic kit as claimed in claim 5, substantially as hereinbefore described.
IE399391A 1990-11-17 1991-11-15 Antiviral activity of the rep gene encoded by adeno-¹associated virus type 2 IE913993A1 (en)

Applications Claiming Priority (1)

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DE4036784A DE4036784A1 (en) 1990-11-17 1990-11-17 ANTIVIRAL ACTIVITY OF THE ADENO-ASSOCIATED VIRUS TYPE 2 REP GENE

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IE913993A1 true IE913993A1 (en) 1992-05-20

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EP (1) EP0486917A3 (en)
KR (1) KR920009981A (en)
AU (1) AU8785491A (en)
CA (1) CA2055635A1 (en)
DE (1) DE4036784A1 (en)
IE (1) IE913993A1 (en)
PT (1) PT99520A (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0733122A4 (en) * 1993-05-26 1998-06-10 Us Health Fusion proteins containing adeno-associated virus rep protein and bacterial protein
US6146847A (en) * 1996-11-01 2000-11-14 Genespan Corporation Stabilized transient gene expression
CA2270137A1 (en) * 1996-11-01 1998-05-14 Genespan Corporation Stabilized transient gene expression

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* Cited by examiner, † Cited by third party
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EP0291893A1 (en) * 1987-05-19 1988-11-23 The Du Pont Merck Pharmaceutical Company Stable human cell lines expressing an indicator gene product under virus-specific genetic controls
EP0327960A1 (en) * 1988-02-11 1989-08-16 F. Hoffmann-La Roche Ag Secretable forms of alkaline phosphatase
AU1150792A (en) * 1990-12-06 1992-07-08 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Inhibition of human immunodeficiency virus by an adeno-associated virus gene for human cells

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KR920009981A (en) 1992-06-26
PT99520A (en) 1992-10-30
AU8785491A (en) 1993-01-28
EP0486917A3 (en) 1992-12-16
DE4036784A1 (en) 1992-05-21
EP0486917A2 (en) 1992-05-27
CA2055635A1 (en) 1992-05-18

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