WO1987007296A1 - Expression de la transcriptase inverse du virus lymphotropique des lymphocytes t humains (htlv-iii), et utilisations - Google Patents

Expression de la transcriptase inverse du virus lymphotropique des lymphocytes t humains (htlv-iii), et utilisations Download PDF

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Publication number
WO1987007296A1
WO1987007296A1 PCT/US1987/001162 US8701162W WO8707296A1 WO 1987007296 A1 WO1987007296 A1 WO 1987007296A1 US 8701162 W US8701162 W US 8701162W WO 8707296 A1 WO8707296 A1 WO 8707296A1
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plasmid
reverse transcriptase
htlv
iii
host cell
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PCT/US1987/001162
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English (en)
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Stephen P. Goff
William A. Haseltine
Naoko Tanese
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The Trustees Of Columbia University In The City Of
The Dana-Farber Cancer Institute
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Publication of WO1987007296A1 publication Critical patent/WO1987007296A1/fr

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • AIDS Acquired Immune Deficiency Syndrome
  • HTLV-III/LAV Lymphadenopathy virus
  • Efforts to arrest the spread of this virus are being made on two broad fronts: the development of antiviral vaccines which might allow immunized individuals to resist infection, and the development of antiviral drugs which would specifically retard or arrest viral replication.
  • One potentially important target of such drugs is the virion-associated enzyme reverse transcriptase (5-8).
  • RNA is copied to form a double-stranded DNA, which is integrated into host DNA to generate the provirus (for review, 1).
  • the synthesis of the proviral DNA is catalyzed by the enzyme reverse transcriptase, which may efficiently utilize either RNA or DNA templates for DNA synthesis by the elongation of a primer bearing a paired 3' hydroxyl terminus.
  • RNAse H which degrades RNA present as a duplex RNA:DNA hybrid.
  • the vira pol gene encodes many enzymatic activities which participate in various steps of the life cycle.
  • the pol gene product is initially expressed as a polyprotein Pr2009ag ⁇ pol (2,3), containing sequences encoded by the gag gene fused to sequences encoded by the pol gene; proteolytic processing is required to remove the gag sequences and to excise the mature products from the pol sequences.
  • Reverse transcriptase is widely used as a means of producing complementary DNA (cDNA) copies of messenger RNA (mRNA) molecules.
  • cDNAs may be inserted into expression vectors which are used to transform cells so that the resulting cells produce a desired polypeptide encoded by the original mRNA.
  • the HTLV-III reverse transcriptase has been purified in small quantities and some of its properties are established.
  • the enzyme has unusual template and divalent cation preferences (9-11), suggesting that specific inhibitors of the enzyme may be discovered.
  • Certain inhibitors of HTLV-III reverse transcriptase activity may be used to block the activity of this enzyme as it produced by viruses infecting human cells and tissues. Reverse transcriptase being an integral part of the viral life cycle, this will allow use of the inhibitors to treat or prevent HTLV-III related diseases. The isolation of such inhibitors would be facilitated by the availability of larger quantities of the active enzyme.
  • Reverse transcriptase produced by and isolated from, virions is commercially available. However, it is quite expensive due to the low abundance of the pol gene product in the virions.
  • the present invention uses a modified region of the HTLV-III pol gene which is inserted into a plasmid, its transcription being controlled by an inducible promoter.
  • the expression of the inserted gene fragment re suits in the production of a polypeptide with HTLV-III reverse transcriptase activity.
  • the present invention describes a method for identifying substances which inhibit HTLV-III reverse transcriptase activity.
  • the invention concerns a double-stranded DNA plasmid which, when expressed in a suitable host cell , produces a polypeptide having HTLV- III reverse transcriptase activity, the plasmid comprising in 5 ' to 3 ' order :
  • HTLV-III human T-cell lymphotropic virus
  • the plasmid of this invention may be introduced into a suitable host cell where the gene may be expressed under suitable conditions.
  • the plasmid is pHRT25 and the host cell is an Escherichia coli HB101 cell (deposited together under ATCC No. 67117.
  • Suitable inducible promoters are ones which are induced when the host cell is grown in a medium deficient in one or more amino acids.
  • One such inducible promoter is in the Trp operon of E. coli.
  • the invention also concerns a method for identifying substances which inhibits HTLV-III reverse transcriptase.
  • the method comprises isolating HTLV-III reverse transcriptase, contacting the reverse transcriptase so isolated with an RNA molecule to be reversed transcribed in the presence of a suitable amount of a mixture of deoxyribonucleotides to produce a transcription cocktail, contacting the transcription cocktail with an amount of a substance to be identified for a suitable period and detecting the amount of reverse transcriptase activity in the cocktail, a significant decrease in the amount of reverse transcriptase activity relative to the amount of activity in a cocktail not contacted with the substance indicating an inhibition of the reverse transcriptase activity.
  • the invention comprises a method for treating an HTLV-III virus-related disease.
  • the method comprises administering to the patient a suitable amount of a physiologically acceptable HTLV-III reverse transcriptase inhibiting substance for a suitable period.
  • Figure 1 Construction of active gene fusion.
  • This map shows restriction sites in the vector (pATH2, dark lines) and in the insert (pol region of HTLV-III, light lines).
  • Start of insert is at about nucleotide 1640 (Bglll site of HTLV-III) and end of insert is at about nucleotide 5368 (Sail site in HTLV-III) from HTLV-III map of Ratner, et al. (12).
  • HB101 cells carrying the indicated plasmids were used. Either 1 ul or 0.1 ul of each extract as indicated was incubated in 50 ul reaction cocktails containing 50 mM Tris-HCl pH 8.3, 30 mM DTT, 60 mM NaCl, 0.05% NP40, and 10 uM of the appropriate a- 32 P deoxyribonucleotide (1 Ci/mmole). Reactions contained either Mg++ (10mM) or Mn++ (ImM) as indicated at the left.
  • 2XSSC 0.3 M NaCl, 0.03 M Sodium Citrate
  • Samples marked V and C are assays of authentic reverse transcriptase from Moloney murine leukemia virus, and of medium from control cells.
  • Panel B HB101 (PolA+) and C2110 (PolAl-) cells carrying the indicated plasmids were used. Assays and substrates were as in Panel A.
  • a double-stranded DNA plasmid has been made which, when expressed in a suitable host cell, produces a polypeptide having HTLV-III reverse transcriptase activity.
  • the plasmid includes in 5' to 3' order: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation codon; a portion of the Human T-cell lymphotropic virus III (HTLV-III) pol gene, said portion including a DNA sequence which encodes the polypeptide having HTLV-III reverse transcriptase activity; a DNA sequence which contains a gene associated with a selectable or identifiable phenotypic trait such as drug resistance, e.g.
  • ampicillin resistance which is manifested when the vector is present in the host cell; a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell, e.g., Escherichia coli.
  • the inducible promoter of the plasmid is one which is induced when the host cell is grown upon a medium deficient in one or more amino acids.
  • the inducible promoter may be the Trp promoter Escherichia coli and the medium deficient in the amino acid tryptophan.
  • the inducible promoter is one which is induced when the host cell is subjected to increased temperature.
  • the ATG initiation codon of the plasmid may be derived from the coding sequence of the Trp E protein of Escherichia coli. e.g. a DNA sequence derived from about a 1000 nucleotide long sequence encoding a portion of the Trp E protein of Escherichia coli.
  • the origin of replication is derived from pBR322.
  • the plasmid may comprise a circular double-stranded DNA sequence such as the plasmid identified as pHRT25, having the restriction map shown in Fig. 2 and deposited in E. coli HB101 under ATCC No. 67117.
  • the portion of the HTLV-III pol gene of the plasmid may comprise the nucleotide sequence from about nucleotide 1640 to about nucleotide 5368. In one embodiment the 5' end of the central portion of the pol gene is 21 nucleotides from the start of the DNA sequence which encodes the polypeptide having reverse transcriptase activity.
  • cloning vehicles such as plasmids, e.g., pBR322
  • plasmids e.g., pBR322
  • cloning vehicles can be modified or engineered so that they contain DNA sequences, i.e., inducible promoters (Trp promoter, etc.), involved in the regulation or expression of the sequences encoding a polypeptide having reverse transcriptase activity.
  • the DNA molecules so inserted may be made by various methods ineluding enzymatic or chemical synthesis.
  • the resulting cloning vehicles are chemical entities which do not occur in nature and may only be created by the modern technology commonly described as recombinant DNA technology.
  • These cloning vehicles may be introduced into a suitable host cell, either procaryotic, e.g., bacterial (E. coli or B. subtilis. etc.) or eucaryotic, e.g., yeast, using techniques known to those skilled in the art, such as transformation, transfection and the like.
  • the one embodiment of this invention is the E. coli HB101 strain containing the plasmid pHRT25 deposited under ATCC No. 67117.
  • the cells into which the plasmid of this invention is introduced will thus contain DNA encoding a non-naturally occurring polypeptide having reverse transcriptase activity. Further, the expression of the DNA encoding the non- naturally occurring polypeptide will be under the control of the Trp promoter.
  • the resulting cells into which DNA encoding the non- naturally occurring polypeptide encoding reverse transcriptase activity and encoding the Trp promoter has been introduced may be grown under suitable conditions known to those skilled in the art so as to control and effect the expression of the genetic information encoded by the DNA and permitting the production of the polypeptide having reverse transcriptase activity and the recovery of the resulting polypeptide.
  • one embodiment of this invention concerns the polypeptide so prepared, e.g. the polypeptide having reverse transcriptase activity characterized by being encoded by the plasmid pHRT25.
  • Another embodiment of this invention is a method for identifying substances which inhibits HTLV-III reverse transcriptase.
  • the method comprises isolating HTLV-III reverse transcriptase, contacting the reverse transcriptase so isolated with an RNA molecule to be reversed transcribed in the presence of a suitable amount of a mixture of deoxyribonucleotides to produce a transcription cocktail, contacting the transcription cocktail with an amount of a substance to be identified for a suitable period and detecting the amount of reverse transcriptase activity in the cocktail, a significant decrease in the amount of reverse transcriptase activity relative to the amount of activity in a cocktail not contacted with the substance indicating an inhibition of the reverse transcriptase activity.
  • HTLV-III reverse transcriptase will be isolated from cells, i.e. bacterial or yeast cells containing plasmids comprising the HLTV-III pol gene, i.e. plasmid pHRT22 or pHRT25. HLTV-III reverse transcriptase may also be isolated from other sources, i.e., tissue culture cells infected with the HTLV-III virus. Methods used in isolating HTLV-III reverse transcriptase are known in the art and are described more fully hereinafter under Experimental Details.
  • Mixtures of deoxyribonucleotides to be used in the present invention will contain dATP, dGTP, dCTP and dTTP in relative amounts suitable to each experiment.
  • detecting the amount of reverse transcriptase activity comprises detecting by autoradiography the amount of one or mere radiolabelled deoxyribonucleotides, i.e. 32 p-ATP, incorporated by the reverse transcriptase.
  • Other methods i.e., quantitative immunoassay, may also be useful.
  • Still another embodiment of this invention is a method for treating an HTLV-III virus-related disease in a subject. The method comprises administering to the patient a suitable amount of a physiologically acceptable HTLV-III reverse transcriptase inhibiting substance for a suitable period.
  • the substance may be administered by a variety of methods, including injection in a pharmaceutically acceptable carrier, i.e. intravenously or pertioneally, topical application, oral application in a liquid or solid form or a variety of other routes well known in the art.
  • a pharmaceutically acceptable carrier i.e. intravenously or pertioneally, topical application, oral application in a liquid or solid form or a variety of other routes well known in the art.
  • Clones pHRT22 and pHRT25 have essentially similar restriction maps. Parallel constructions were performed to insert the same pol sequences into the pATH3 vector in the wrong reading frame to form the control plasmids pHRT31 and pHRT32. Bacterial cultures containing various plasmids were grown in minimal medium, starved for tryptophan, and exposed to indoleacrylic acid to derepress the trp operon (14-17). After 2h, the cells were harvested, lysates were prepared, and the crude extracts were assayed directly for reverse transcriptase activity (Fig. 3A) as described previously (16-18).
  • the extracts were tested for their ability to incorporate the appropriate radioactive triphosphate on either of two substrates: poly(rA) primed with oligo(dT), or pol(rC) primed with oligo (dG). Each substrate was tested with either of two divalent cations: Mg ++ (lOmM), or Mn ++ (ImM).
  • oligo (dT) template the plasmids were transferred into a bacterial strain defi cient in DNA Polymerase I (18). These cells do not support replication of the plasmid DNA, and transformants carry the plasmid DNA integrated into the host chromosome only in one or a few copies. Extracts were prepared and assayed as before (Fig. 3B).
  • PolyA- cells carrying the vector plasmid pATH2 showed no detectable background activity on either template, with either divalent cation. These same cells carrying the pHRT22 construct showed high levels of activity in the presence of Mg++; the activity could be detected on either poly(rC): oligo (dG) or on poly(rA): oligo (dT), but there was slightly higher activity on the poly(rC): oligo (dG) template. There was very little activity in the presence of Mn++ on either template. These properties of the activity duplicate precisely the behavior of the authentic HTLV-III reverse transcriptase enzyme as purified from virion particles (9-11).
  • Extracts of cells carrying the pHRT22 constructs showed more than a 200-fold increase in specific activity over extracts from control cells. Analysis of the time course of the reaction, using 0.1 ul of extract, showed that the reaction was linear for more than 10 minutes (Table 2). These experiments also showed large increases in the specific activity of cells carrying the pHRT22 constructs relative to controls.
  • the PolA- cells with pHRT22 yielded about 5-fold less activity that the PolA+ cells carrying the same plasmid, presumably due to the lower copy number of the plasmids in the PolA- cells.
  • the HTLV-III pal gene has been inserted into a bacterial expression plasmid and it was demonstrated that the construct induces reverse transcriptase activity.
  • the appearance of activity depends on the joining of the trpE and pol sequences in the correct reading frame, and is independent of the bacterial polA gene.
  • the resulting activity closely mimics the behavior of the authentic HTLV-III enzyme, strongly preferring Mg++ over Mn++, and differs sharply from the enzyme encoded by the murine leukemia viruses. It is expected that these constructs and more active derivatives will be useful in the genetic analysis of the functions of the HTLV-III pol gene, and in surveys for antiviral agents.
  • Extracts from the indicated cultures were prepared as before (16-17).
  • the indicated volumes of the extracts were incubated in reaction mixes (50 ul total volume) containing 50 mM Tris-HCl pH 8.3, 20 mM DTT, 60mM NaCl, 0.05% NP40, 10 mM MgCl 2 , 10 ug/ml poly (rC), 5 ug/ml oligo (dG), and 10 uM a -32 P-dGTP (1 Ci/mmole).
  • reaction mixes 50 mM Tris-HCl pH 8.3, 20 mM DTT, 60mM NaCl, 0.05% NP40, 10 mM MgCl 2 , 10 ug/ml poly (rC), 5 ug/ml oligo (dG), and 10 uM a -32 P-dGTP (1 Ci/mmole).
  • HB101 (pATH2) 4.4 mg/ml
  • HB101(pHRT22) 3.3 mg/ml
  • C2110 (pATH2) 3.9 mg/ml
  • C2110(pHRT22) 5.2 (mg/ml). Specific activities were determined as before.

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Abstract

L'invention se rapporte à un plasmide, qui, lorsqu'il est introduit dans une cellule hôte appropriée et cultivé dans des conditions appropriées effectue l'expression d'un gène sur le plasmide et la production d'un polypeptide possédant l'activité de transcriptase inverse du HTLV-III. Le plasmide est une molécule d'ADN à double brin comprenant, dans un ordre allant de la position 5' à la position 3', les éléments suivants: une séquence d'ADN comprenant un promoteur inductible; une séquence d'ADN comportant un codon initiateur d'ATG; une portion du gène pol du virus lymphotropique III des lymphocytes T humains (HTLV-III), cette portion comprenant une séquence d'ADN qui code le polypeptide possédant l'activité de transcriptase inverse du HTLV-III; une séquence d'ADN contenant un gène associé avec un trait phénotypique sélectionnable ou identifiable, se manifestant lorsque le vecteur est présent dans la cellule hôte; ainsi qu'une séquence d'ADN contenant un point d'origine de réplication provenant d'un plasmide bactérien capable de réplication autonome dans la cellule hôte. Est également décrit un procédé d'identification de substances inhibant la transcriptase inverse du HTLV-III, de même qu'un procédé de traitement d'une maladie se rapportant au virus HTLV-III.
PCT/US1987/001162 1986-05-20 1987-05-20 Expression de la transcriptase inverse du virus lymphotropique des lymphocytes t humains (htlv-iii), et utilisations WO1987007296A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0267802A2 (fr) * 1986-11-14 1988-05-18 Genetic Systems Corporation Antigène synthétique pour la détection de maladie de SIDA et maladies s'y rapportant
EP0372904A2 (fr) * 1988-12-07 1990-06-13 The Research Foundation for Microbial Diseases of Osaka University Protéase, transcriptase inverse et endonucléase des rétrovirus et procédé de production de ces enzymes
WO1990010230A1 (fr) * 1989-02-23 1990-09-07 University Of Ottawa Polypeptide avec activite immunologique destine a servir de vaccin et/ou de reactif diagnostique
WO1991011523A1 (fr) * 1990-01-31 1991-08-08 The Upjohn Company Souche ral-4- utile pour la production de proteines et de peptides de faible poids moleculaire
EP0563323A1 (fr) * 1990-12-19 1993-10-06 Epitope, Inc. Vaccin a transcriptase inverse contre le vih
US5500355A (en) * 1988-12-07 1996-03-19 The Research Foundation For Microbial Diseases Of Osaka University Protease, reverse transcriptase and endonuclease of HIV-1 and RSV method for producing these enzymes
EP0940469A2 (fr) * 1990-04-20 1999-09-08 Cold Spring Harbor Laboratory Clonage par complémentation et procédés rélatifs à celui-ci

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US4487835A (en) * 1977-12-30 1984-12-11 A/S Alfred Benzon Plasmid having temperature dependent plasmid copy number

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Title
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Cell, Volume 40, issued January 1985, (Cambridge, Massachusetts, USA) (S. WAIN-HOBSON et al.), "Nucleotide Sequence of the Aids Virus, LAV", see pages 9-17, especially pages 9 and 13. *
Journal of Virology, Volume 59, No. 3, issued September 1986 (Washington, D.C., USA), (N. TANESE et al.), "Expression of Reverse Transcriptase Activity of Human T-Lymphotropic Virus Type III (HTLV-III/LAV) in Escherichia Coli", see pages 743-745. *
Molecular General Genetics, Volume 177, No. 3, issued February 1980, (Heidelberg, Federal Republic of Germany) (W. TACON et al.), "The Construction and Characterization of Plasmid Vectors Suitable for the Expression of A11 DNA Phases under the Control of the E. Coli Tryptophan Promoter", see pages 427-438, especially pages 427, 428, 436 and 437. *
Nature, Volume 293, issued October 1981, (London, U.K.) (R. HITZEMAN et al.), "Expression of a Human Gene for Interferon in Yeast", see pages 717-722, especially pages 717 and 721. *
Nature, Volume 313, No. 6001, issued January 1985 (London, U.K.) (L. RATNER et al.), "Complete Nucleotide Sequence of Aids Virus, HTLV-III", see pages 277-284, especially pages 277 and 281. *
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Proceedings National Academy Sciences, USA, Volume 82, issued August 1985 (Washington, D.C., USA) (N. TANESE et al.), "Expression of Enzymatically Active Reverse Transcriptase in Escherichia Coli", see pages 4944-4948. *
Proceedings National Academy Sciences, USA, Volume 82, issued October 1985 (Washington, D.C., USA) (H. MITSUYA et al.), "3-Azido-3'-deoxythymine (BW A 509U): An Antiviral Agent that Inhibits the Infectivity and Cytopathic Effect of Human T-lymphotropic Virus Type III/Lymphadenopathy-Associated Virus In Vitro", see pages 7096-7100, especially pages 7096 and 7097. *
Science, Volume 228, No. 4695, issued April 1985 (Washington, D.C., USA), (N. CHANG et al.), "Expression in Escherichia Coli of Open Reading Frame Gene Segments of HTLV-III", see pages 93-96, especially pages 93 and 94. *
Science, Volume 236, No. 4799, issued April 1987 (Washington, D.C., USA) (W. FARMERIE et al.), "Expression and Processing of the AIDS Virus Reverse Transcriptase in Escherichia Coli", see pages 305-308, see especially 305 and 306. *
The Lancet, No. 8444, issued June 1985, (London, U.K.) (E. SANDSTROM et al.), "Inhibition of Human T-Cell Lymphotropic Virus Type III In Vitro by Phosphonoformate", see pages 1480-1482. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0267802A2 (fr) * 1986-11-14 1988-05-18 Genetic Systems Corporation Antigène synthétique pour la détection de maladie de SIDA et maladies s'y rapportant
EP0267802A3 (fr) * 1986-11-14 1991-03-06 Genetic Systems Corporation Antigène synthétique pour la détection de maladie de SIDA et maladies s'y rapportant
EP0372904A2 (fr) * 1988-12-07 1990-06-13 The Research Foundation for Microbial Diseases of Osaka University Protéase, transcriptase inverse et endonucléase des rétrovirus et procédé de production de ces enzymes
EP0372904A3 (fr) * 1988-12-07 1991-09-25 The Research Foundation for Microbial Diseases of Osaka University Protéase, transcriptase inverse et endonucléase des rétrovirus et procédé de production de ces enzymes
US5500355A (en) * 1988-12-07 1996-03-19 The Research Foundation For Microbial Diseases Of Osaka University Protease, reverse transcriptase and endonuclease of HIV-1 and RSV method for producing these enzymes
WO1990010230A1 (fr) * 1989-02-23 1990-09-07 University Of Ottawa Polypeptide avec activite immunologique destine a servir de vaccin et/ou de reactif diagnostique
US5858646A (en) * 1989-02-23 1999-01-12 University Of Ottawa Modified HIV-pol polypeptide having immunological activity for use as diagnostic reagent
WO1991011523A1 (fr) * 1990-01-31 1991-08-08 The Upjohn Company Souche ral-4- utile pour la production de proteines et de peptides de faible poids moleculaire
EP0940469A2 (fr) * 1990-04-20 1999-09-08 Cold Spring Harbor Laboratory Clonage par complémentation et procédés rélatifs à celui-ci
EP0940469A3 (fr) * 1990-04-20 1999-12-22 Cold Spring Harbor Laboratory Clonage par complémentation et procédés rélatifs à celui-ci
EP0563323A1 (fr) * 1990-12-19 1993-10-06 Epitope, Inc. Vaccin a transcriptase inverse contre le vih
EP0563323A4 (en) * 1990-12-19 1996-02-28 Epitope Inc Hiv reverse transcriptase vaccine

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AU7512287A (en) 1987-12-22
JPS63503356A (ja) 1988-12-08

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