CA2055635A1 - Antiviral activity of the rep gene encoded by adenoassociated virus type 2 - Google Patents
Antiviral activity of the rep gene encoded by adenoassociated virus type 2Info
- Publication number
- CA2055635A1 CA2055635A1 CA002055635A CA2055635A CA2055635A1 CA 2055635 A1 CA2055635 A1 CA 2055635A1 CA 002055635 A CA002055635 A CA 002055635A CA 2055635 A CA2055635 A CA 2055635A CA 2055635 A1 CA2055635 A1 CA 2055635A1
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- rep
- aav
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- rep gene
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Links
- 101150066583 rep gene Proteins 0.000 title claims abstract description 21
- 241000702423 Adeno-associated virus - 2 Species 0.000 title claims abstract description 7
- 230000000840 anti-viral effect Effects 0.000 title abstract description 11
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 241000700605 Viruses Species 0.000 claims description 13
- 108700026244 Open Reading Frames Proteins 0.000 claims description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 238000013519 translation Methods 0.000 claims description 5
- 238000009007 Diagnostic Kit Methods 0.000 claims description 3
- 239000003443 antiviral agent Substances 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 241000702421 Dependoparvovirus Species 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 230000014616 translation Effects 0.000 claims 2
- 208000036142 Viral infection Diseases 0.000 claims 1
- 230000009385 viral infection Effects 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 description 15
- 230000010076 replication Effects 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 10
- 241000725303 Human immunodeficiency virus Species 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 102000003844 DNA helicases Human genes 0.000 description 1
- 108090000133 DNA helicases Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Classifications
-
- 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)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
HOE 90/B 035 - Ma 869 Abstract of the disclosure Antiviral activity of the rep gene encoded by adeno-associated virus type 2 The invention relates to the use of a rep gene derived from adeno-associated virus type 2 and having antiviral activity against the HIV-1 virus.
Description
ZC~55~35 Behringwerke AG HOE 90/B ~35 - Ma 869 Dr. Bo Antiviral activity of the rep gene encoded by adeno-associated virus type 2 Thi6 invention relates to the use of the rep gene encoded by adeno-associated virus type 2 (AAV-2) for the prepar-ation 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 di~advantage: 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, 2~55f~
pp. 3012-3018 (1990)) have likewise been observed.
~AV 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, S pp. 243-307 (lg87)).
The virus ha~ a wide host range and depends on helper functions of other viruses. However, this virus does not appear to depend on tissue factor or species-specific cellular factors. The genome contain~ 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 D~A-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-1-inhibiting activity.
The aim of the invention is met by the æub~ect-matter of claLm 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-se~uence of the gene can be used to prepare a specific antiviral factor.
.
X(:'55635 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-l replication have been investigated in a transient assay system (Biochem. Biophys. Res. Comm. 169, pp. 643-651 (1990)). This system i8 based on comicroin~ection of wild-type AAV or of a mutant DNA (J. Virol. 64, pp. 3012-3018 ~1990)) together with an infectious HIV-l proviral DNA (clone pNL4-3) (J. Virol. 59, pp. 284-291 (1986)) into the nuclei of human epitheloid SW480 cells. ~hese cells permit HIV-1 replication after tran~fection but cannot be infected with HIV-1 virus (J. Virol. 59, pp.
284-291 (1986)). HIV-1 virus was initially prepared in comicroinjected cells and was grown by cocultivation with human ~-lymphoid MT-4 cells. HIV-1 was then measured in a cell-free coculture supernatant with the aid of commer-cial HIV-1 antigen ELISA (Biochem. Biophys. Res. Comm.
169, pp. S43-651 (1990)). It is evident from these results, which are summarized in Fig. 1, that the strong inhibition of HIV-l replication at a molar ratio of HIV-1 DNA to AAV DNA of 1:10 in the initially comicroin~ected cells is correlated with the presence of an intact AAV
rep open reading frame. The plasmids mentioned in Fig. 1 : 25 (pTAV) have been described (Heilbronn et al 1990; J.
Virology 64: 3012-3018 and AAV-2 wild-type seguence 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-l 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-l virus for AAV
replication in SW480 cells - has the same anti-HIV-l activity as the wild-type AAV (rep+, cap+). ~his finding makes it clear that the inhibition of HIV-l replication - 4 - ~ ~55~3S
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 o~ coculture. The product~ 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 protein~ 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-l 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 micro-in~ected cells with ~-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 microin~ected cells retain their potency for HIV-l gene expression and for virus production.
The AAV-dependent inhibition of HIV-l 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 sequen-ces which from HIV-l 5'-LTR are involved in the rep-dependent inhibition of HIV-1 replication, the HIV-1 LTR
(U3/R portion) driven expression of chloramphenicol acetyltran6ferase (CAT) was observed as an indicator gene. The C~T expression level was measured in the presence of an intact AAV rep gene using the assay Z~'~5~3S
described above. The results show that pTAV2-6 (rep~), but not pTAV2-3 (rep~), inhibit~ CAT expression (Fig. 2a) in a dose-dependent manner (Fig. 2b). This means that the ~3/R portion of HIV-l 5~-LTR iæ sufficient for rep-dependent inhibition of gene expression. However, thisdoes not exclude an influence of the rep gene al80 on other steps in HIV-l replication.
The nucleotide positions for HIV-l 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, 243-307 (Fig. 3).
Fig. 4 describes the position of the AHH sequence in the rep gene.
The biochemical properties and the biological role of the rep proteins suggest tha~ 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 po~sibly the RNA, both of which are contained in the U3/R sequence of HIV-1, play an important part in the mechanism of HIV-l inhibition. Comparison of the DNA sequence between the AAV-2 DNA and the HIV-l LTR sequence (position 1-634, J. VirolO 53, 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-l DNA, which is part of the tar sequence.
Furthermore, a region with a high degree of homology between the calculated local secondary 6tructure of the HIV-l tar region and the AAV terminal sequence is to be found. This shows that secondary structure recognition is involved ~n a possible direct or indirect interaction between rep protein and HIV-l RNA sequence.
The AAV rep gene inhibits HIV replication due to the interaction with the HIV-l LTR sequence. As shown above, it is possible by comparing the sequences of A~V-2 and of - 6 - Z~5~3~
the HIV-l 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 æhows great homology between AAV-2 and HIV-1 at the level of the two-dimen-sional structure. Competition experLments with HIV-l LTR
fragments show that the inhibition ~in this case the rep-dependent inhibition of HIV-l LTR-driven CAT expression) depends on the presence of the AHH region. It was pos-sible to show that with a double-stranded DNA fragment which is 25 base-pairs long an~ has the AHH se~uence there is an even more drastic rise in the rep-mediated inhibition of HIV-l LTR-driven CAT expression. This unambiguously shows the involvement of the AHH region in the inhibitory mechanism.
This result æhows 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 sub~ect-matter of the present invention and the favorable prerequisites which are required for a use as 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 di~advantage: 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, 2~55f~
pp. 3012-3018 (1990)) have likewise been observed.
~AV 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, S pp. 243-307 (lg87)).
The virus ha~ a wide host range and depends on helper functions of other viruses. However, this virus does not appear to depend on tissue factor or species-specific cellular factors. The genome contain~ 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 D~A-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-1-inhibiting activity.
The aim of the invention is met by the æub~ect-matter of claLm 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-se~uence of the gene can be used to prepare a specific antiviral factor.
.
X(:'55635 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-l replication have been investigated in a transient assay system (Biochem. Biophys. Res. Comm. 169, pp. 643-651 (1990)). This system i8 based on comicroin~ection of wild-type AAV or of a mutant DNA (J. Virol. 64, pp. 3012-3018 ~1990)) together with an infectious HIV-l proviral DNA (clone pNL4-3) (J. Virol. 59, pp. 284-291 (1986)) into the nuclei of human epitheloid SW480 cells. ~hese cells permit HIV-1 replication after tran~fection but cannot be infected with HIV-1 virus (J. Virol. 59, pp.
284-291 (1986)). HIV-1 virus was initially prepared in comicroinjected cells and was grown by cocultivation with human ~-lymphoid MT-4 cells. HIV-1 was then measured in a cell-free coculture supernatant with the aid of commer-cial HIV-1 antigen ELISA (Biochem. Biophys. Res. Comm.
169, pp. S43-651 (1990)). It is evident from these results, which are summarized in Fig. 1, that the strong inhibition of HIV-l replication at a molar ratio of HIV-1 DNA to AAV DNA of 1:10 in the initially comicroin~ected cells is correlated with the presence of an intact AAV
rep open reading frame. The plasmids mentioned in Fig. 1 : 25 (pTAV) have been described (Heilbronn et al 1990; J.
Virology 64: 3012-3018 and AAV-2 wild-type seguence 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-l 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-l virus for AAV
replication in SW480 cells - has the same anti-HIV-l activity as the wild-type AAV (rep+, cap+). ~his finding makes it clear that the inhibition of HIV-l replication - 4 - ~ ~55~3S
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 o~ coculture. The product~ 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 protein~ 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-l 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 micro-in~ected cells with ~-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 microin~ected cells retain their potency for HIV-l gene expression and for virus production.
The AAV-dependent inhibition of HIV-l 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 sequen-ces which from HIV-l 5'-LTR are involved in the rep-dependent inhibition of HIV-1 replication, the HIV-1 LTR
(U3/R portion) driven expression of chloramphenicol acetyltran6ferase (CAT) was observed as an indicator gene. The C~T expression level was measured in the presence of an intact AAV rep gene using the assay Z~'~5~3S
described above. The results show that pTAV2-6 (rep~), but not pTAV2-3 (rep~), inhibit~ CAT expression (Fig. 2a) in a dose-dependent manner (Fig. 2b). This means that the ~3/R portion of HIV-l 5~-LTR iæ sufficient for rep-dependent inhibition of gene expression. However, thisdoes not exclude an influence of the rep gene al80 on other steps in HIV-l replication.
The nucleotide positions for HIV-l 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, 243-307 (Fig. 3).
Fig. 4 describes the position of the AHH sequence in the rep gene.
The biochemical properties and the biological role of the rep proteins suggest tha~ 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 po~sibly the RNA, both of which are contained in the U3/R sequence of HIV-1, play an important part in the mechanism of HIV-l inhibition. Comparison of the DNA sequence between the AAV-2 DNA and the HIV-l LTR sequence (position 1-634, J. VirolO 53, 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-l DNA, which is part of the tar sequence.
Furthermore, a region with a high degree of homology between the calculated local secondary 6tructure of the HIV-l tar region and the AAV terminal sequence is to be found. This shows that secondary structure recognition is involved ~n a possible direct or indirect interaction between rep protein and HIV-l RNA sequence.
The AAV rep gene inhibits HIV replication due to the interaction with the HIV-l LTR sequence. As shown above, it is possible by comparing the sequences of A~V-2 and of - 6 - Z~5~3~
the HIV-l 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 æhows great homology between AAV-2 and HIV-1 at the level of the two-dimen-sional structure. Competition experLments with HIV-l LTR
fragments show that the inhibition ~in this case the rep-dependent inhibition of HIV-l LTR-driven CAT expression) depends on the presence of the AHH region. It was pos-sible to show that with a double-stranded DNA fragment which is 25 base-pairs long an~ has the AHH se~uence there is an even more drastic rise in the rep-mediated inhibition of HIV-l LTR-driven CAT expression. This unambiguously shows the involvement of the AHH region in the inhibitory mechanism.
This result æhows 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 sub~ect-matter of the present invention and the favorable prerequisites which are required for a use as antiviral agent.
Claims (5)
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 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.
4. A peptide as claimed in claim 3, which is a trans-lation 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4036784.3 | 1990-11-17 | ||
| DE4036784A DE4036784A1 (en) | 1990-11-17 | 1990-11-17 | ANTIVIRAL ACTIVITY OF THE ADENO-ASSOCIATED VIRUS TYPE 2 REP GENE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2055635A1 true CA2055635A1 (en) | 1992-05-18 |
Family
ID=6418520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002055635A Abandoned CA2055635A1 (en) | 1990-11-17 | 1991-11-15 | Antiviral activity of the rep gene encoded by adenoassociated virus type 2 |
Country Status (7)
| Country | Link |
|---|---|
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994028157A1 (en) * | 1993-05-26 | 1994-12-08 | The United States Government As Represented By The Secretary Of The Department Of Health And Human Services | 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 |
| JP2000509998A (en) * | 1996-11-01 | 2000-08-08 | ジーンスパン コーポレイション | Stabilized transient gene expression |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
1990
- 1990-11-17 DE DE4036784A patent/DE4036784A1/en not_active Withdrawn
-
1991
- 1991-11-11 EP EP19910119188 patent/EP0486917A3/en not_active Withdrawn
- 1991-11-15 IE IE399391A patent/IE913993A1/en not_active Application Discontinuation
- 1991-11-15 CA CA002055635A patent/CA2055635A1/en not_active Abandoned
- 1991-11-15 PT PT99520A patent/PT99520A/en not_active Application Discontinuation
- 1991-11-15 AU AU87854/91A patent/AU8785491A/en not_active Abandoned
- 1991-11-15 KR KR1019910020336A patent/KR920009981A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0486917A3 (en) | 1992-12-16 |
| EP0486917A2 (en) | 1992-05-27 |
| AU8785491A (en) | 1993-01-28 |
| KR920009981A (en) | 1992-06-26 |
| DE4036784A1 (en) | 1992-05-21 |
| IE913993A1 (en) | 1992-05-20 |
| PT99520A (en) | 1992-10-30 |
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