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EP0497949A1 - Systeme d'essai permettant de controler l'activite de proteinases virales - Google Patents

Systeme d'essai permettant de controler l'activite de proteinases virales

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Publication number
EP0497949A1
EP0497949A1 EP19910914980 EP91914980A EP0497949A1 EP 0497949 A1 EP0497949 A1 EP 0497949A1 EP 19910914980 EP19910914980 EP 19910914980 EP 91914980 A EP91914980 A EP 91914980A EP 0497949 A1 EP0497949 A1 EP 0497949A1
Authority
EP
European Patent Office
Prior art keywords
proteinase
hrv2
proteinases
dna molecule
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910914980
Other languages
German (de)
English (en)
Inventor
Wolfgang Sommergruber
Hans-Dieter Liebig
Timothy Skern
Marion Luderer
Dieter Blass
Ernst KÜCHLER
Heinrich Kowalski
Ljerka Frasel
Herbert Auer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19904027153 external-priority patent/DE4027153A1/de
Priority claimed from DE19914112952 external-priority patent/DE4112952A1/de
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP0497949A1 publication Critical patent/EP0497949A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22029—Picornain 2A (3.4.22.29)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to an in vivo color test system for checking the activity of viral proteases.
  • proteolytic enzymes involved in replication are highly substrate-specific and usually recognize a fission region - i.e. a structurally determined identifier - rather than a precisely defined amino acid pair, as is usually used as a recognition sequence.
  • a general overview of this topic is given by H.G. Herbal and ⁇ . Wimmer (1988, Ann. Rev. Biochem. 52, 701-751) and Kay, J. and Dünn, B.M. (1990, Biochim. Biophys. Acta 1048. 1-18).
  • the viral proteinases represent a good therapeutic target due to their substrate specificity and their catalytic mechanism (Johnston, MI et al., 1989, Trends Pharmacol. Sci. 10. 305-307).
  • Rous Sarcoma virus Leis, J. et al., 1990, ASM-News üü, 77-81
  • HIV I HIV I
  • it is possible to use computer-assisted molecular "designing" of highly specific inhibitors to perform (Meek, TD et al., 1990, Nature 343, 90-92).
  • Specific proteinase inhibitors could thus represent new antiviral substances which are directed, for example, against viruses, against which the development of a vaccine is not possible for purely technical reasons; For example, well over 115 serotypes which do not cross-react with one another are currently known in rhinoviruses, of which 90 have already been classified as defined serotypes (Cooney, MK et al., 1982, Infect. Immun. 22.642-647).
  • the substrate specificity of the viral enzymes could be analyzed in more detail by means of detailed studies such as point mutation analyzes, cleavage of peptide substrates "in vitro” and amino acid sequencing of the native cleavage products. It turned out that, as already mentioned above, it is less the gap point itself and a few positions “up” or “downstream” that play a major role in the gap point detection. However, that sequence heterogeneity of the cleavage signals or their immediate surroundings leads to a kind of "hierarchy" of the cleavage events in the polyprotein (Krausslich, HG et al., 1989, Proc. Natl. Acad. Sci. 86. 807-811; Pichuantes, S .
  • the variation of the individual cleavage regions in the polyprotein thus allows a precisely determined sequence from kinetically "favorable” to kinetically “unfavorable” cleavages, which subsequently enables differentiated proteolysis of the individual cleavage products.
  • the principle of recognizing a specific secondary structure in the region of the cleavage site is not limited to picornaviruses, but should represent a general principle of viral proteinases. These properties are, for example, in the adenovirus system (Webster, A. et al., 1989, J. Gen. Virol. 7_ ⁇ , 3225-3234; Webster, A. et al., 1989, J.
  • Rhinovirus infections are among the most common human diseases. Although the disease is usually harmless, secondary infections by other viruses or bacteria can occur due to a temporary weakening of the organism, which can then lead to serious illnesses.
  • RNA of the rhinoviruses is modified shortly after infection by cleavage of the oligopeptide VPg bound to the 5 'end and subsequently serves as mRNA for the synthesis of a polyprotein which encompasses the entire continuous reading frame of the nucleic acid sequence (Butterworth, BE, 1973, Virology 5ji / 439-453; Mc Lean, C. and Rueckert, RR, 1973, J. Virol. H, 341-344; Mc Lean, C. et al., 1976, J. Virol. £ 1 , 903-914; Agol, Vl, 1980, Prog. Med.
  • the primary cleavage separation of the capsid precursors from the growing polypeptide chain; 2.) the secondary cleavage: processing structural and non-structural precursor proteins and 3.) the maturation cleavage of the capsid.
  • the first step thus serves to cleave off the precursor of the envelope proteins and (in the case of entero and rhinoviruses) is carried out autocatalytically by proteinase 2A (“cis” activity).
  • proteinase 2A (“cis” activity).
  • sequence of proteinase 2A (hereinafter referred to as 2A) lies immediately behind the section coding for the envelope proteins. 2A is the first because of its location in the polyprotein detectable enzymatic function of the virus. The separation of the coat protein region from the section responsible for replication takes place during the translation of the polyprotein "into statu nascendi".
  • this primary cleavage at the P1-P2 region can be carried out intermolecularly, ie "in trans" by the mature proteinase 2A (Krausslich, HG and Wimmer, E., 1988, loc. Cit.).
  • the cleavage signal which is recognized by proteinase 2A, was determined on the one hand by direct amino acid sequence analysis of the N-terminus of 2A and / or the C-terminus of VP1, or on the other hand derived by comparing the primary structure on the basis of homology studies. This is a Tyr / Gly- in Polio (Pallansch, MA et al., 1984, J.
  • the course of infection in Picornaviruses depends crucially on the viral enzymes. Since these enzymes are particularly well preserved and their properties are very similar in the case of various rhinoviruses, they offer themselves as the target of a chemotherapeutic intervention, such as B. the viral enzyme 2A.
  • the chemotherapeutic approach is the inhibition of enzymatic activity by specific inhibitors. If the first proteolytic activity, 2A activity, is inhibited, any further maturation process of the viral system is prevented.
  • the 2A region of HRV2 shows pronounced homology not only with other rhinoviruses, but also with representatives of other groups of the Picornaviridae.
  • An inhibitor against HRV2 2A could therefore also be applicable to other Picornaviruses.
  • HIV I human immunodeficiency virus 1
  • deletion and point mutations in the proteinase region of this type of retroviruses the essential role of the proteinase in the maturation of this virus class could be recognized (Katoh, I. et al., 1985, Virol. 145 280-292; Kohl, NE et al., 1988, Proc. Natl. Acad. Sei. USA, 85. 4686-4690; Crowford, S. and Goff, SP, 1985, J. Virol. 53, 899-907).
  • X-ray structure analyzes and molecular biological studies have also shown that the proteinase of HIV I belongs to the Asp type, can process itself on the precursor protein (also in recombinant prokaryotic systems), can cleave "into trans” specifically peptides and as an active proteinase in one homodimeric form (Navia, MA et al., 1989, loc. cit .; Meek, TD et al., 1989, loc. cit .; Katoh, I. et al., 1985, loc. cit.).
  • inorganic and organic compounds as well as peptide derivatives and proteins are known in the picornaviral system, which have an inhibitory effect on the proteolytic processing of these viruses.
  • the effect of these substances is based on the direct interaction with the proteinases (Kettner, CA et al., 1987, US Pat. No. 4,652,552; Korant, BD et al., 1986, J. Cell. Biochem. 22., 91-95 ) and / or by the indirect way of interaction with substrates of these proteinases (Geist, FC et al., 1987, Antimicrob. Agents Chemother.
  • Picornaviral proteases expand the understanding of the structure and function of these viral enzymes and thus enable step-by-step rational "designing" - starting from modified peptide substrates - up to highly specific and non-toxic peptidomimetics.
  • the object of the present invention was to provide such test systems.
  • the bacteriophage M13mpl8 (Yanish-Perron, C. et al., 1985, Gene 22, 103-119) - or also other M13 phages from the mp series - contains in its genome the gene segment for the amino-terminal part of the ⁇ -galactosidase from E. .coli.
  • the expression of this gene fragment which is under the control of the promoter of the lac operon, leads to a polypeptide, called ⁇ -fragment, which contains the first 146 amino acids of the ⁇ -galactosidase. This polypeptide is inactive.
  • ⁇ -fragment which contains the first 146 amino acids of the ⁇ -galactosidase. This polypeptide is inactive.
  • E.coli bacterial strains that can be infected by the M13 phages, for example the E.coli JM strains, in particular E.coli JM 101 and E.coli JM 109, also contain an inactive form of the ⁇ -galactosidase called ⁇ M15.
  • the ⁇ -galactosidase lacks amino acids 11-41 here. If these E.
  • coli bacteria which form the inactive ⁇ -galactosidase, are infected with M13 phages and both the expression of the host-coded, inactive ⁇ -galactosidase and the expression of the phage-coded, inactive ⁇ -galactosidase are induced via isopropylthiogalactoside (IPTG), the two inactive forms of the ⁇ -galactosidase can complement each other.
  • IPTG isopropylthiogalactoside
  • An active ⁇ -galactosidase is formed, which can either cleave its natural substrate, lactose or allolactose, or a synthetic substrate, 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (X-Gal), whose cleavage product, an indoxyl derivative, is oxidized under the influence of 0 to the blue 5,5'-dibromo-4,4'-dichloroindigo.
  • the bacteria in which the two inactive forms of ß-galactosidase complement each other to form an active enzyme appear to be stained blue and can be easily distinguished from those bacteria in which no so-called ⁇ complementation, i.e.
  • the activities of the HRV-2A proteinase and also other proteinases preferably the 3C proteinases of the picornaviruses and the HIV proteinase, can be measured.
  • the recognition sites required for inserting these proteinases into the test system can be created, for example, by inserting the recognition sequence 5'GTCGAC3 'into the DNA section of the ⁇ -fragment of the ⁇ -galactosidase.
  • This recognition sequence is recognized by 3 different restriction endonucleases (Sall, Hindi, AccI), which cut differently. In this way, the appropriate reading frame can be selected individually for each desired proteinase.
  • Another possibility of checking the activity of the HRV2-2A in particular is to use modified vectors in connection with the polyclonal antiserum against the peptide PC20 (Sommergruber, W. et al, 1989, loc. Cit.).
  • the expression product of the pure 2A proteinase gene can be recognized by the polyclonal antiserum.
  • Examples 1-5 show that the M13 color test system can be used to distinguish between active and inactive 2A proteinase molecules. This system should also be suitable for making this distinction in a large number of cases to examine clones quickly and meaningfully.
  • the genome of the phage M13-2A (Example 5), the region which codes for the C-terminus of VP1 and for the 2A proteinase, was mutagenized in vitro. The mutagenesis was carried out with the aid of the polymerase chain reaction (PCR), since it is known that the Taq DNA polymerase makes errors relatively frequently because it has no "proofreading" activity (Leung, DW et al., 1989, Technique. A Journal of Methods in Cell and Molecular Biology. 1, 11-15).
  • PCR polymerase chain reaction
  • the PCR amplification was carried out according to standard methods (Torgersen et al., 1989, J. Gen. Virol., 70, 3111-3116).
  • the mutated DNA fragments thus obtained were transformed into E. coli. Around 1000 plaques were checked for their ability to complement ⁇ . 30 different plaques were analyzed in more detail.
  • the 2A gene of phages from the fourth light blue plaque shows only one silent mutation; since a free 2A is found in the immunoblot ( Figure 13B; H3), it is likely that a mutation in another area of the ⁇ -complementation system could be responsible for the reduced ⁇ -complementation. In fact, a point mutation was found in the Lac promoter region.
  • the analysis of 17 further phage genomes after PCR mutagenesis is described in Example 9; the associated immunoblots are shown in FIG. 19 and the mutations in the 2A genes in FIG. 20. Table 2 summarizes the results. All phage genomes derived from M13 phages from five examined colorless plaques show mutations in their VP1 / 2A regions.
  • a phage genome shows a nucleotide exchange that converts an amino acid codon into a stop codon, and two genomes have deletions in the 2A gene that lead to a loss of the reading frame for the 2A gene.
  • the M13 color test system can be used to distinguish inactive mutants from active proteinases, in this case the 2A proteinase from HRV2.
  • individual regions of the proteinase gene can be selected separately by selecting the oligonucleotides for the amplification; for example, one could only amplify the cleavage site or only the region coding for the catalytic center.
  • the system can also be used to regenerate an active form from an inactive proteinase by in vitro mutagenesis.
  • Examples 1 to 7 as well as 9 and 10 describe the M13 color test system as a method with which the so-called "ice” or intramolecular cleavage of the 2A proteinase can be studied.
  • the 2A proteinases of the polio and rhinoviruses also cleave a host protein; this reaction, which therefore takes place intermolecularly or in "trans", indirectly leads to the cleavage of the p220 protein and thereby to the inhibition of the translation of the host's own mRNAs provided with a so-called "cap” structure. Since neither the mechanism of this cleavage nor the amino acid pair that is cleaved are known, it is of interest to develop a system that can be used to study this intermolecular cleavage.
  • Such a system should also be used to test the effectiveness of inhibitors that prevent intermolecular cleavage.
  • the difference between the two cleavage processes can be seen in FIG. 15.
  • the M13 color test system was modified to allow an investigation of the intermolecular cleavage.
  • three phage constructs were created, which were named M13 / ⁇ lacZ2A, M13 / ⁇ lacZ2A ⁇ 2B and M13 / ⁇ lacZ2A ⁇ 3Gly.
  • the constructs M13 / ⁇ lacZ2A and M13 / ⁇ lacZ2A ⁇ 2B contain an active 2A proteinase while the construct M13 / ⁇ lacZ2A ⁇ 3Gly contains one contains inactive 2A proteinase.
  • the plasmid pBR2A ⁇ was constructed, which after transformation into E.
  • coli and induction of the expression block with IPTG results in a lacZ / ⁇ VPl / ⁇ 2A / 8F5 fusion protein.
  • substrate-providing plasmids are those which, after transformation into E. coli and induction of the expression block, result in a fusion protein from the so-called ⁇ -fragment and a polypeptide fraction which can be cleaved from an active proteinase in trans.
  • a plasmid is also to be used which results in a fusion protein lacZ / ⁇ VPl / ⁇ 2A, lacZ / ⁇ VPl / 2A (R134: Q) ⁇ 2B or lacZ / ⁇ VPl / ⁇ 2A ( ⁇ 3Gly).
  • the fusion proteins should also be used as such to investigate the trans activity.
  • the genomic DNA of the three above-mentioned phage constructions was transformed in the presence of IPTG and X-gal into E. coli JM101 bacteria which have the plasmid pBR / 2A ⁇ .
  • the resulting plaques are shown in FIG. 17. It can clearly be seen that those from the bacteriophages M13 / ⁇ lacZ2A and M13 / ⁇ lacZ2A ⁇ 2B plaques formed are dark blue; in contrast, the plaques formed by the bacteriophage M13 / ⁇ lacZ2A ⁇ 3Gly are colorless.
  • Figure 18 A shows an immunoblot with the 8F5 antibody.
  • a band of 26 kDa is visible in the protein extracts from bacteria that either have only the plasmid ⁇ BR2A ⁇ (lane 2) or have additionally been infected with the inactive variant M13 / ⁇ lacZ2A ⁇ 3Gly (lane 5), ie with M13 phages from colorless plaques ; in contrast, the extracts from bacteria infected with an active variant (ie with M13 phage from blue plaques) have an additional band of 20 kDa (lanes 3 and 4).
  • the 26 kDa band arises from the amino acids of the ⁇ fragment, ⁇ VP1, ⁇ 2A and the 8F5 binding site ( ⁇ VP2); the 20 kDa band arises from the 26 kDa band when an active 2A molecule processes the 2A cleavage site and thereby cleaves the ⁇ -fragment and the amino acids of ⁇ VP1.
  • the antiserum PC20 was used to check whether an active 2A molecule was found in the induced fusion proteins of blue plaques.
  • a band of 18 kDa was detected in proteins from a colorless plaque (ie of the variant M13 / ⁇ lacZ2A ⁇ 3Gly), which is a fusion protein from the remaining 8 amino acids of the ⁇ fragment, the 28 w amino acids of ⁇ VP1 and corresponds to the 142 amino acids of Figure 2A.
  • a band of 15 kDa which corresponds to a free 2A protease, was found in the induced fusion proteins of blue plaques (ie of the variant M13 / ⁇ lacZ2A). No signal was found with the construction M13 / ⁇ lacZ2A ⁇ 2B, since the antiserum only recognizes a 2A molecule that has a free C-terminus.
  • a system was developed with which the proteolytic "trans" activity of 2A proteinase from bacterial extracts can also be checked in vitro.
  • a plasmid was constructed from a rabbit ⁇ -globin sequence and an HRV2 sequence, with which any DNA fragment can be transcribed and translated in vitro.
  • HRV2 and HRV14 was expressed, which corresponds to the region ⁇ VPl / ⁇ 2A and ⁇ VP3 / VP1 / ⁇ 2A, respectively.
  • the translation product of HRV2 VP1 / ⁇ 2A could be cleaved with bacterial extracts of M13-2A or pBR2A / l and pBR2A / 2, which express a mature 2A.
  • no cleavage was found in bacterial extracts with M13-2A ⁇ 3Gly or pBR2A ⁇ that do not express a mature 2A protein.
  • This system can therefore be used to check the presence of active 2A molecules in bacterial extracts.
  • No cleavage of the HRV14 ⁇ VP3 / VP1 / ⁇ 2A region from bacterial extracts with HRV2 2A was found. It was concluded that the specificity of the
  • Color test system can surprisingly be used to intermolecular cleavage
  • the invention relates in detail to the vectors characterized in the claims, the DNA molecules, the fusion proteins, the host organisms transformed with the vectors and the use of the vectors and the fusion proteins as test systems.
  • Effective inhibitors of viral proteinase activity influence the ⁇ -complementation more or less strongly when using the in vivo color test systems according to the invention, which is shown by more or less strongly colored bacteria.
  • Figure 1 shows the construction of M13-18521R and M13-18521Q.
  • Figure 2 shows the extent of ⁇ complementation with M13-18521R and M13-18521Q.
  • FIG. 3 shows the construction of M13-I-18521R and M13-I-18521Q as well as M13-I-18521R-MluI and M13-I-18521Q-M1UI.
  • FIG. 4 shows the mutation oligonucleotides used.
  • Figure 5 shows the extent of ⁇ complementation of M13-18521Q and of the return mutants to M13-18521R.
  • the arrow with the letter Q indicates a "plaque” with M13-18521Q phage and the arrow with the letter R indicates a "plaque” with M13-18521R phage.
  • FIG. 6 shows the extent of the ⁇ complementation after introduction of the Sall cleavage at amino acid position 44/45 of the ⁇ -galactosidase in M13-I-18521R-MluI (A) and M13-I-18521Q-MluI (B) and after introduction at amino acid position 42/43 at M13-I-18521R-MluI (C) and M13-I-18521Q-MluI (D).
  • Figure 7 shows the construction of M13-I-18521R-MluI / SalI and M13-I-18521Q-MluI / SalI once for (44/45) and (42/43).
  • Figure 8 shows the construction of M13-2A and M13- ⁇ 3Gly.
  • FIG. 9 shows the immunoblot of a 15% denaturing protein gel with induced 2A proteinase from various M13 clones.
  • the 2A proteinase is detected via the polyclonal antiserum against the peptide PC20 (Sommergruber, W. et al., 1989, loc. Cit.)
  • Lane 1.4 * induced 2A proteinase from M13-2A
  • Lane 2 induced 2A proteinase from M13- ⁇ 3Gly
  • Lane 3.5 induced 2A proteinase from M13-I-18521R-Mlul / Sall
  • the numbers (unit: kilodalton) with a small arrow indicate selected molecular size markers.
  • the numbers (unit: kilodalton) with a large arrow indicate the processed, active 2A proteinase (15) and - the unprocessed, inactive 2A proteinase (24).
  • the 2A proteinase from M13-I-18521R-MluI / SalI has no free carboxy terminus and is therefore not recognized by the anti-PC20.
  • FIG. 10 schematically shows the creation of restriction enzyme recognition sites around the cleavage site of HRV2-2A and in the coding part of HRV2-2A.
  • Restriction enzymes that are not printed in bold represent the recognition sequences originally present in pEx2A; Framed in bold print indicate the newly introduced detection sequences in p ⁇ x2A / 21 and pEx2A / II.
  • AS means amino acids.
  • FIG. 11 shows the double-stranded oligonucleotide for the production of pEx2A / 21, a restriction-modified variant of pEx2A.
  • the oligonucleotide is flanked by an Acc I or a Bst ⁇ II site; the restriction sites in bold were introduced into the DNA with the aid of the modified nucleotide sequence of the mutation oligo while maintaining the original amino acid sequence.
  • FIG. 12 shows the PCR amplification oligonucleotides used (1987: 5 • GTTACCCAACTTAATCG3 'and 1988: 5'GTTACGTTGGTGTAGATG3') and their position on the genome of the phage M13-2A.
  • Figure 13 shows immunoblots of phage expression proteins from 30 selected plaques.
  • the proteins induced with IPTG were separated on 15% denaturing polyacryamide gels; the resulting blots were incubated with the antiserum against the peptide PC20 (Sommergruber, W., et al., 1989, loc. cit.).
  • part A the IPTG-induced proteins from 20 phages that showed colorless plaques were examined.
  • the closed arrow indicates the position of the band that corresponds to a fusion protein from the ⁇ fragment and an inactive 2A molecule (24 kD), the open arrow indicates the position of the mature 2A molecule (15 kD).
  • the ⁇ -fragment / inactive 2A fusion protein can be seen in 15 of the 20 proteins. No fusion protein of this size can be seen in samples W1, W5, W12, W17 and W18; these samples are deletion mutants (WI, W5, W12, W17) or mutants (W18) that have an early stop codon. In such cases, the amino acid sequence recognized by the PC20 antiserum is not translated.
  • Figure 14 shows the amino acid changes found by DNA analysis of the mutants.
  • Part A shows the amino acid exchanges that can be attributed to a single nucleotide exchange in the bacteriophage genome.
  • the exchanges were found in the genomes of bacteriophages, which were derived as follows: blue plaques, closed circles; light blue plaques, crossed circles; colorless plaques, open circles.
  • Part B shows the amino acid exchanges based on multiple nucleotide exchanges in the phage genome.
  • Mutant 1 gave blue plaques, all other phage mutants led to colorless plaques.
  • the individual mutations are: mutant 1, Met 5: Val and Ile 127: Arg; Mutant 2, Ile-4: Väl and Phe 130: Ser; Mutant 3, His 8: Tyr and Lys 109: Glu; Mutant 4, Ile 28: Thr and Cys 112: Arg; Mutant 5, Asn 65: His and His 114: Arg; Mutant 6, Ile 70: Thr and Gly 99: Ser; Mutant 7, Cys 106: Arg and Lys 109: Glu; Mutant 8, Val 7: Ala, Glu 102: Gly and Phe 130: Ser.
  • Figure 15 shows the difference between intramolecular cleavage (in “ice”) and intermolecular cleavage (in “intra-trans”).
  • the ⁇ fragment is shown as a black block and the active proteinase as an open block; an inactive proteinase is shown with a block filled with slashes. Proteinase cleavage sites are identified by a thick black line.
  • FIG. 16 shows the constructions M13 / ⁇ lacZ2A, M13 / ⁇ lacZ2A ⁇ 2B, M13 / ⁇ lacZ2A ⁇ 3Gly and pBR2A ⁇ as used for the investigation of the intermolecular cleavage.
  • the manufacture of the four constructions is described in detail in the text.
  • E. coli JM101 bacteria in which the plasmid pBR2A ⁇ had previously been introduced were transfected with the construction M13 / ⁇ lacZ2A or M13 / ⁇ lacZ2A ⁇ 2B, ⁇ -complementation could be observed. These are marked with a plus. However, no ⁇ complementation was found after transfection with the construction M13 / ⁇ lacZ2A ⁇ 3Gly, characterized by - signs.
  • FIG. 17A shows the extent of ⁇ complementation in E. coli JM101 without pBR2A ⁇ (pictures 1 and 4) and with pBR2A ⁇ (pictures 2 and 3) after transfection with:
  • FIG. 17B shows the extent of ⁇ complementation in E. coli JM101 with pBR2A ⁇ after transfection with:
  • Image 1 M13 / ⁇ lacZ2A ⁇ 2B
  • Image 2 M13 / ⁇ lacZ2A
  • Image 3 M13 / ⁇ lacZ2A ⁇ 3Gly
  • FIG. 18 shows the immunoblot of two 15% denaturing polyacryamide gels, on which the proteins induced with IPTG from JM101 cells are separated.
  • Immunoblot A was incubated with the antibody anti 8F5 (Skern et al, J. Gen. Virol., 68, 315-323, 1987), immunoblot B with the antiserum against the peptide PC20 (Sommergruber, W. et al., 1989, loc. cit).
  • Lane 1 E. coli JM101 bacteria without plasmid
  • Lane 2 E. coli JM101 bacteria with pBR2A ⁇
  • Lane 3 E. coli JM101 bacteria with pBR2A ⁇ transfected with M13 / ⁇ lacZ2A
  • Lane 4 E. coli JM101 bacteria with pBR2A ⁇ transfected with M13 / ⁇ lacZ2A ⁇ 2B
  • Lane 5 E. coli JM101 bacteria with pBR2A ⁇ transfected with M13 / ⁇ lacZ2A ⁇ 3Gly
  • the numbers on the left indicate the proteins expressed by pBR2A ⁇ : the processed ⁇ 2A / ⁇ VP2-8F5 substrate (20) and the unprocessed lacZ / ⁇ VPl / ⁇ 2A / ⁇ 2B-8F5 substrate (26 ).
  • the numbers on the right indicate the processed, active 2A proteinase (15) and the unprocessed, inactive 2A proteinase (18).
  • the 2A proteinase of the construction M13 / ⁇ lacZ2A ⁇ 2B has no free carboxy terminus and is therefore not recognized by the anti-PC20.
  • Figure 19 shows immunoblots of phage expression proteins from the 17 plaques selected in Example 9. The proteins induced with IPTG were separated on 15% denaturing polyacrylamide gels; the resulting blots were incubated with the antiserum against the peptide PC20 (W. Sommergruber, loc. cit.).
  • the closed arrow indicates the position of the band, which corresponds to a fusion protein from the ⁇ fragment and an inactive 2A molecule (24 kD).
  • the open arrow indicates the position of the mature 2A molecule (15 kD).
  • Part A shows the proteins of 5 M13 phages induced with IPTG, which cause colorless plaques.
  • the ⁇ -fragment / inactive 2A fusion protein can be seen in samples W21 and W28 (closed arrow). No fusion protein of this size can be seen in samples W24, W29 and W30; these are deletion herbants or mutants that have an early stop codon. In such cases, the amino acid sequence recognized by the PC20 antiserum is not translated.
  • Part B shows the phage proteins induced with IPTG from 7 different light blue plaques. Only the ⁇ -fragment / inactive 2A fusion protein is visible in five of the seven proteins. No fusion protein of this size can be seen in samples H15 and H16; these samples are deletion mutants or mutants that have an early stop codon. For samples H6, H7, H9, H13 and H14 only one ⁇ fragment / inactive 2A fusion protein found. In these cases it is assumed that enough ⁇ -fragment is released to be able to detect an ⁇ -complementation, but the amount of mature proteinase 2A is below the detection limit of the immunoblot.
  • Part C shows the IPTG-induced phage progeny proteins from 5 different blue plaques. A mature 2A molecule is detected in all cases.
  • Figure 20 shows the amino acid changes found by DNA analysis of the mutants.
  • Part A shows the amino acid exchanges that can be attributed to a single nucleotide exchange in the bacteriophage genome.
  • the bacteriophage genomes are symbolized as follows: closed circles, phage genomes from blue plaques; crossed circles, phage genomes of light blue plaques; open circles, white plaques.
  • Part B shows the amino acid exchanges based on multiple nucleotide exchanges in the phage genome.
  • the individual mutations are: Mutant 1, Glyl: Ala and Ile 96: Thr; Mutant 2, Gly97: Val and Ilell9: Val. Both mutants gave light blue plaques.
  • Part C shows the amino acid exchanges that can be traced back to a single nucleotide exchange in the bacterophage genome and which reactivate the mutant Phel30: Tyr. All revertants have a blue phenotype.
  • FIG. 21 shows immunoblots of expression proteins from phages from 4 of those described in Example 10 Plaques.
  • the proteins induced with IPTG were separated on 15% denaturing polyacryamide gels; the resulting blots were incubated with the antiserum against the peptide PC20 (W. Sommergruber, loc. cit.).
  • the closed arrow indicates the position of the band, which corresponds to a fusion protein of the ⁇ fragment and an inactive 2A molecule (24 kD), the open arrow indicates the position of the mature 2A molecule (15 kD).
  • Phel30 Tyr
  • a mature 2A molecule was detected in the expressed proteins of the three revertants, which cause reactivation of 2A.
  • FIG. 22 shows a schematic representation of the HRV2 genome and a summary of the rabbit ⁇ -globin 5 * UTR / HRV2 hybrids described in the text.
  • the open rhombus represents the 5 'UTR of rabbit ß-globin and the closed arrows the ATG codons at 449 and 611.
  • the black block represents the translated region of HRV2; the block filled with oblique lines represents the additional translatable section that results from the initiation of translation at ATG codon 449.
  • S shows the promoter of T7 RNA polymerase.
  • Figure 23 shows the constructions GR1, GR2, GR5 and GR6.
  • the following restriction sites are given: A, AccI; B, BamHI; H, HincII; H3, Hindlll; N, Ncol; P, PstI; RI, Eco RI; RV, EcoRV; Sa, Sall; Sm, Smal.
  • the other symbols are identical to those in FIG. 22.
  • the first and last nucleotides of the respective HRV2 cDNA insert are given.
  • Figure 24 shows the oligonucleotides used.
  • Figure 25 shows constructions pß + 611 and pß-611.
  • the nucleotide sequence of the rabbit ⁇ -globin 5 'UTR in the positive orientation (pß + 611; Fig. 25A) and in the negative orientation (pß-611; Fig. 25 B) as well as the first two codons of VP4 of HRV2 are shown.
  • the other symbols are identical to those in FIG. 22.
  • Figure 26 shows constructions p20, pl, pß + 449 and pß-449.
  • the plasmid p20 is shown in part Ai; the nucleotide sequence of the rabbit ⁇ -globin 5 'UTR (open rhombus) is in the positive orientation (the sequence is identical to that of pß + 611 in Figure 25). The first 15 codons resulting from the initiation of translation at ATG codon 449 are shown. Part Aii shows the introduction of the 1.0 kb PstI fragment of GR6 in ⁇ 20; this results in the plasmid pß + 449.
  • the plasmid pl is shown in part Bi; the nucleotide sequence of the rabbit ⁇ -globin 5 'UTR (open rhombus) is in the negative orientation (the sequence is identical to that of pß-611 in Figure 25). The first 15 codons resulting from the initiation of translation at ATG codon 449 are shown. Part Bii shows the introduction of the 1.0 kb PstI fragment from GR6 in pl; this results in the plasmid pß-449.
  • Figure 27 shows the fluorogram of a 12.5% protein gel with translation products of RNAs of the four plasmids pß + 449, pß-449, pß + 611 and pß-611.
  • Lane C is the result of a translation without the addition of RNA.
  • the numbers (unit: kilodalton) indicate selected molecular markers.
  • the open arrow indicates the 35 kD translation product from ATG codon 449 and the closed arrow indicates the 30 kD translation product from ATG codon 611.
  • 8F5 the result of an immunoprecipitation of the translation products of pß + 611 RNA with the antibody 8F5 is shown; the 39kD gang was specifically felled.
  • Figure 28 shows the fluorogram of a 12.5% protein gel with translation products of RNAs of the two plasmids pßHRV14 / sub (lane 1) and pßHRV2 / sub (lane 2).
  • Track C shows the result of a translation without the addition of RNA.
  • the numbers on the left indicate selected molecular markers.
  • the open arrow points to the 40 kD translation product of the RNA of pßHRV2 / sub and the closed arrow to the 66 kD translation product of the RNA of pßHRV14 / sub.
  • Figure 29 shows fluorograms of three 12.5% protein gels with translation products of RNAs of the two plasmids pßHRV2 / sub and pßHRV14 / sub after incubation with bacterial extracts.
  • Part A shows the fluorogram the result of the incubation of the translation product of the RNA of pßHRV2 / sub with the following bacterial extracts: lane 1, no incubation; Lane 2, M13-2A; Lane 3, M13-2A ⁇ 3Giy; Lane 4, plasmid pBR2A / l; Lane 5, plasmid PBR2A / 2; Lane 6, plasmid pBR2A ⁇ .
  • the left numbers (unit: kilodalton) show selected molecular markers.
  • the closed one Arrow indicates the 40 kD translation product from the RNA of pßHRV2 / sub; the arrow marked S indicates the 32 kD cleavage product and the arrow marked SE indicates the 30 kD cleavage product. These cleavage products can only be detected after incubation with extracts containing an active 2A proteinase.
  • Part Bi shows an immunoblot of expression proteins from extracts precipitated with ammonium sulphate in Example 11.
  • the proteins were separated on 15% denaturing polyacryamide gels; the resulting blots were incubated with the antiserum against the peptide PC20 (W. Sommergruber, loc. cit).
  • the extracts are as follows: Lane 1, M13-2A; Lane 2, pBR2A / l; Lane 3, PBR2A / 2.
  • the numbers on the left indicate selected molecular size markers.
  • the open arrow indicates the position of the mature 2A molecule (15 kD).
  • Part Bii shows the fluorogram of the products of the incubation of the translation product of the RNA of pßHRV2 / sub with the following bacterial extracts: lane 1, no incubation; Lane 2, M13-2A; Lane 3, pBR2A / l; Lane 4, pBR2A / 2.
  • the right numbers indicate selected molecular markers.
  • the closed arrow shows the 40 kD translation product of the RNA of pßHRV2 / sub; the arrow marked with S the 32 kD fission product. The 30 kD fission product is no longer visible.
  • the fluorogram of the products of the incubation of the translation product of the RNA of pßHRV14 / sub shows with the following bacterial extracts. Lane 1, M13-2A; Lane 2, pBR2A / l; Lane 3, pBR2A / 2.
  • the left numbers show selected molecular markers.
  • the closed arrow points to the 66 kD translation product from the RNA of pßHRV14 / sub. No cleavage is evident after incubation with the three extracts.
  • Example 1 Development of an in vivo color test system for checking the cis activity of 2A proteinase from HRV2
  • the circular, double-stranded (RF) form of the bacteriophage genome M13mpl8 was linearized by the restriction endonuclease Pvul and the overhanging 3 'ends were removed with S1 nuclease.
  • pExl8521 and pExl8521Q are mutant of p ⁇ xl8521, whereby the triplet for Argl34 was replaced by a triplet for gin
  • an AccI- was Fragment isolated and the AccI ends filled with DNA polymerase I (Klenow enzyme).
  • the AccI fragment from pExl8521 contains the information for the last 28 carboxy-terminal amino acids of 1D (VP1), for the entire 2A proteinase, for the first 39 amino-terminal amino acids of 2B and for 19 plasmid-encoded amino acids (FIG. 1).
  • the AccI fragment from pExl8521Q contains the same information, only two nucleotide exchanges have been carried out in the gene of 2A proteinase. Codon AGA, which codes for the amino acid arginine at position 134 in FIG. 2A, has been replaced by CAA, which codes for glutamine. This amino acid exchange inactivates 2A.
  • the respective AccI fragments were ligated with the linearized M13 DNA using standard methods and transformed into E.
  • JL, 309-321) showed that after the triplet for the 45th amino acid of ⁇ -galactosidase in the same reading frame, the codons for the 28 carboxy-terminal amino acids of VP1 follow, followed by the nucleotide sequence for 2A, for the amino terminus of 2B and for the plasmid-coded amino acids (the numbering of the amino acids of the ⁇ -galactosidase does not take into account the amino acids which are encoded by the multilinker region in the coding region of the ⁇ -galactosidase).
  • the phage progeny whose recombinant genome contains the active 2A proteinase gene from pExl8521, show a clearly visible ⁇ complementation in the presence of IPTG and X-Gal. In comparison, the ability for ⁇ complementation in phage progeny with an inactive 2A proteinase from ⁇ Exl8521Q is reduced (FIG. 2).
  • the amino acids of the ⁇ fragment should remain fused to the 28 amino acid carboxy terminus of VP1; in the case of the inactive proteinase, an additional 200 amino acids from 2A, 2B and parts of the plasmid should follow, since the inactive proteinase cannot be released via a cleavage at its amino terminus.
  • the 228 amino acid long polypeptide chain remaining on the ⁇ fragment therefore leads to a reduction in ⁇ complementation.
  • the phages that can form an active 2A proteinase are called M13-18521R, and those that form an inactive proteinase are called M13-18521Q.
  • lacZ / ⁇ VPl / 2A / ⁇ 2B lacZ / ⁇ VPl / 2A (R134: Q) / ⁇ 2B.
  • Example 2 Creation of singular interfaces in the vicinity of the 2A gene
  • FIG. 5 shows the difference in the ⁇ complementation between inactive proteinase and proteinase which is active again by back mutation.
  • Figure 2 in Example 1 shows that the ⁇ -fragment from phages M13-18521Q is still able to convert X-Gal to a certain extent even after fusion with an inactive 2A.
  • the singular nucleotide sequence 5'GTCGAC3 ' was introduced in M13-I-18521R-Mlul and M13-I-18521Q-MluI by directed mutagenesis (FIG. 4), which is from the isoschizomers Sall, AccI and HincII and Hindll is recognized. This recognition sequence was used at two different positions: 1.
  • the codon sequence ACC GAT which codes for the 44th and 45th amino acid of the ⁇ -galactosidase, was converted into ACG TCG. This made the 45th Amino acid of ß-galactosidase changed from aspartic acid to serine.
  • the base sequence AC from the next codon ACT completes the sequence 5'GTCGAC3 '.
  • Figure 6 shows that the ability to complement ⁇ is not affected by this mutation.
  • the introduction of the recognition sequence 5'GTCGAC3 ' also offers the possibility of fusing other cis-active proteinases, such as 3C proteinases from Picornaviruses or the HIV proteinase, at this point with the ⁇ -fragment of the ⁇ -galactosidase, since this recognition sequence is different from 3 different ones Restriction endonucleases are recognized that cut differently. In this way, the appropriate reading frame can be selected individually for each desired proteinase.
  • the new, recombinant phages or phage genomes are given the names M13-I-18521R-MluI / SalI and M13-I-18521Q-MluI / SalI (FIG. 7).
  • the active 2A proteinase is distinguished from the inactive form on the basis of a color test system.
  • An additional possibility of distinguishing the two forms of proteinase directly at the protein level is that in the recombinant phage genomes M13-I-18521R-MluI / SalI or M13-I-18521Q-MluI / SalI the gene sequences are deleted which correspond to the carboxy terminus of FIG. 2A follow proteinase.
  • the expression product of a pure 2A proteinase gene can be recognized by the polyclonal antiserum PC20 (Sommergruber, W. et al., 1989, loc. Cit.).
  • the BstEII / HindIII fragment from pEx2A / 21 contains the gene section of the active 2A proteinase; the BstEII / Hindlll fragment from pEx ⁇ 3Gly contains the gene sequence of an inactive 2A proteinase since the triplets for the three glycine amino acids at positions 104, 107 and 108 have been deleted.
  • ⁇ Ex ⁇ 3Gly can be prepared from the DNA of pEx2A / 21 by deleting the codons for Gly 104, 107 and 108.
  • the new recombinant phage genomes are named M13-2A (active proteinase) and M13- ⁇ 3Gly (inactive proteinase).
  • the fusion proteins that can be expressed by these constructions are called lacZ / ⁇ VPl / 2A or lacZ / ⁇ VPl / ⁇ 2A (3Gly). Both forms of proteinase can be clearly identified in Western blots be distinguished from one another (FIG. 9).
  • the gene product of M13- ⁇ 3Gly shows no ⁇ -complementation in the color test system; in the case of M13-2A an ⁇ complementation is visible.
  • AccI fragment (approx. 700 bp) was isolated under standard conditions. This fragment includes the C-terminus of VP1, the entire coding region of HRV2-2A (including the stop codons after the last amino acid for HRV2-2A), as well as parts of the vector DNA (see Fig. 10).
  • the purified AccI fragment from pEx2A was then cut with BstEII. The larger BstEII / AccI fragment (approx. 635 bp) was separated from the smaller AccI / BstEII fragment (approx.
  • PCR polymerase chain reaction
  • Amplification was carried out with the oligonucleotides 1987 and 1988; the sequences of these oligonucleotides and their orientation relative to the rhinoviral sequences can be seen in FIG.
  • the amplified DNA was cut with the restriction enzymes Mlul and Hindlll, the 450bp fragment released thereby isolated from an agarose gel, ligated with the 7.0kb Mlul / Hindlll fragment from M13-2A and transformed in the presence of IPTG and X-Gal into E.coli JM101 .
  • Around 1000 plaques were checked for their ability to complement ⁇ .
  • about 5% of the phage progeny showed no ⁇ -complementation in this case and around 1% showed a reduced ⁇ -complementation.
  • nucleotide sequences of the ⁇ VP1 / 2A genes of these 30 phages were determined by DNA sequencing and compared with the wild-type sequence.
  • Example 8 Adaptation of the M13 color test system to investigate the intermolecular cleavage of the HRV22A proteinase
  • the M13 color test system was modified in such a way that it enables an investigation of the intermolecular cleavage.
  • a 1063bp Aval / HindIII DNA fragment was isolated from the genome of the M13-2A construction (Example 5); this fragment contains the entire lacZ / 2A expression block.
  • DNA polymerase I Klenow fragment
  • the DNA fragment was cloned into the 4282b ⁇ Clal / Sall fragment of the plasmid pBR328 (Boehringer Mannheim).
  • Two plasmids, pBR2A / l and pBR2A / 2 were isolated, which encode the entire lacZ / 2A expression block in each case opposite orientation included.
  • a 444bp Rsal DNA fragment which codes for amino acids 120 to 193 of the capsid protein VP2 (1B) of HRV2 and therefore the binding site for the antibody 8F5 was ligated into the unique Apal site of the plasmid pBR2A / l, which had been blunt-ended with Sl nuclease; the resulting plasmid is referred to as pBR2A ⁇ ( Figure 16).
  • the plasmid was transformed into E.
  • This fusion protein serves as a substrate for an active 2A molecule that is encoded by an M13 bacteriophage.
  • the genome of this bacteriophage was constructed in such a way that the genomic DNA of M13-2A was cut with the restriction enzymes EcoRI, which cuts right at the beginning of the polylinker, and AccI. After a replenishment reaction with DNA polymerase I (Klenow fragment), the larger of the two restriction fragments formed was religated. This treatment eliminates the amino acids of the polylinker and amino acids 7 to 43 of the ⁇ fragment; however, the reading frame remains intact. The construction is called M13 / ⁇ lacZ2A.
  • M13 / 2A ⁇ 2B corresponds to M13-I-18521R-MluI / SalI
  • the resulting construction is called M13 / ⁇ lacZ2A ⁇ 2B.
  • M13 / ⁇ lacZ2A ⁇ 3Gly a variant with an inactive 2A proteinase (named M13 / ⁇ lacZ2A ⁇ 3Gly) was constructed; M13 / ⁇ 3Gly served as the initial construction. All three constructions are shown schematically in FIG. 16.
  • the genomic DNA of the three above-mentioned phage constructions was transformed in the presence of IPTG (0.1mM) and X-gal (0.16mM) into E.
  • FIG. 16 Figure 18 A shows an immunoblot with the 8F5 antibody.
  • the PC20 antiserum was used to check whether an active 2A molecule can be found in the induced fusion proteins of blue plaques. As can be seen in FIG.
  • the bacteria were then centrifuged off, washed with 1 ml washing solution (150 mM NaCl, 50 mM Tris, 1 mM EDTA), centrifuged again, resuspended in 200 ⁇ l washing solution and with 50 ⁇ l 5 ⁇ LSB (20% SDS, 725 mM TrisHCl , pH 6.8, 25% 2-mercaptoethanol, 50% glycerin and 1% bromophenol blue) 10 min. heated at 95 ° C.
  • the expressed proteins were then examined for immunoblots with the PC20 antiserum (Sommergruber, W. et al., 1989, loc. Cit.). The result is shown in FIG. 19.
  • nucleotide sequences of the VP1 / 2A genes of these 17 phage genomes were determined by DNA sequencing and compared with the wild-type sequence. The evaluation of the sequence comparisons of these 17 different phage genomes coincides with the results from the protein studies. Table 2 and Figure 20 summarize the results.
  • the mutagenesis of the region coding for the C-terminus of VP1 and for the 2A proteinase was mutagenized using the polymerase chain reaction.
  • the PCR amplification was carried out according to standard methods. As described in Example 7 amplified the DNAs of the following inactive 2A mutants; M13-2A (Phel30: Tyr), M13-2A (Phel30: Ser), M13-2A (Phel30: Leu), (M13-I-18521Q-MluI / SalI (42/43).
  • the amplified DNA was as in the example 7, and finally ligated to the vector, a 7.0 kb Mlul / Hindlll fragment of M13-2A, and approximately 0.4% of the phage progeny turned blue again, indicating the presence of an active 2A molecule.
  • mutant M13-2A (Phel30: Tyr)
  • sequence analysis of 2 blue progeny showed the exchange Ser27: Pro, of 4 further blue progeny the exchange Hisl35: Arg and of 4 further progeny the exchange Hisl37: Arg; another blue plaque was found to be a jerk mutant or uncut M13 * 2A vector DNA.
  • An examination of the proteins induced with IPTG with the antiserum PC20 (as described in Example 7) showed that a mature 2A molecule was again present in these cases.
  • the protein studies are shown in FIG. 21 and the location of the mutations which lead to the reactivation of the inactive 2A mutant M13-2A (Phel30: Tyr) in FIG. 20.
  • mutant M13-2A (Phel30: Leu) * no "second-site" revertants could be found either; all blue plaques examined were reverse mutations or uncut M13-2A vector DNA.
  • mutant (M13-I-18521Q-MluI / SalI (42/43), which has Arg: Glu (AGA: CAA) at amino acid position 134, deletions were detected by sequence analysis in 2 blue progeny, which increased by reading frame shift Stop codons in the anterior part of the VPl / 2A gene resulted in ⁇ complementation, and a further mutation Glul34: Arg (CAA: CGA) was detected in 10 other blue offspring.
  • Example 11 In vitro translation of rhinoviral cDNA fragments to check the activity of viral proteinases
  • VP1 / ⁇ 2A gene fragments of HRV2 and HRV14 are expressed in rabbit reticulocyte lysates; the resulting protein (sulfur, 35S) is then labeled with bacterial
  • Extracts containing the IPTG-induced proteins from various M13-2A or pBR2A clones are incubated. If the cleavage site is processed, the labeled, in vitro translated protein is cleaved; the result can be on a
  • the plasmid GR6 (FIG. 23) served as the cloning vector for the 5 'UTR of the rabbit ⁇ -globin; it contains the HRV2 nucleotides 19-1400 in the plasmid pGEM2 (Promega). Nucleotide 19 of the HRV2 sequence is approximately 30 base pairs away from the start of transcription for the T7 RNA polymerase. After in vitro transcription, this results in an RNA that corresponds to the orientation of the genomic RNA.
  • p773 HRV2 nucleotides 562-1012; Skern, T. et al., 1985, loc. Cit.
  • Pl9 HRV2 nucleotides 19-5100
  • p860 Skern, T. et al., 1985, loc. cit.
  • p860 contains the HRV2 nucleotides 660 to 1400 in the PstI site of pUC9; the orientation is such that nucleotide 1400 is adjacent to the EcoRI site of the polylinker.
  • the plasmid ⁇ GEM2 was cut with the restriction enzymes Hindlll and BamHI and dephosphorylated with calf intestine using alkaline phosphatase; plasmid p773 was also cut with HindIII and BamHI.
  • the HRV2 insert released by this was eluted from an agarose gel and ligated with the pGEM2 vector linearized with HindIII / BamHI.
  • GR1 was isolated, the structure of which is shown in FIG. 23.
  • GR1 was then treated with the Hindlll and Ncol restriction enzymes and with alkaline phosphatase; the plasmid pl9 was also cut with HindIII and Ncol.
  • the resulting 680 bp fragment from the EcoRI site at nucleotide 743 of the HRV2 gene map to the EcoRI site of the pUC9 polylinker (ie to nucleotide 1400 of the HRV2 gene map) was isolated and ligated with the GR2 vector cut with EcoRI and treated with alkaline phosphatase.
  • a plasmid, GR5 was isolated, the structure of which is shown in FIG. 23.
  • the plasmid GR5 in turn was cut with the restriction enzyme Hindlll and dephosphorylated with alkaline phosphatase from calf intestine; plasmid pl9 was also treated with HindIII.
  • the 240 bp fragment formed from the HindIII interface in the polylinker to the HindIII interface at nucleotide 248 of the HRV2 gene map was ligated with the linearized GR5 vector.
  • a plasmid was isolated, the structure of which is shown in FIG. 23 (called GR6).
  • the interfaces for the restriction enzymes EcoRV and EcoRI in the plasmid pl9 between the HindIII site and the nucleotide 19 of the HRV2 gene map can naturally also be found in the plasmid GR6.
  • the plasmid GR6 was digested with EcoRV and Ncol, the larger fragment was eluted from an agarose gel and with alkaline Treated phosphatase from calf intestine.
  • EBI 1264 and 1250 FIGG. 24; the oligonucleotides were brought together briefly to 65 ° C. and then slowly cooled to 4 ° C.
  • RNA is formed which comprises 18 nucleotides from the transcription site to the EcoRV site, the beginning of the ß-globin 5 * UTR, followed by the 52 nucleotides of the entire ß-globin 5'UTR and Nucleotides 611 to 1400 of the HRV2 sequence.
  • a plasmid ( Figure 25) was constructed which has the ⁇ -globin 5 'UTR in the reverse orientation.
  • the plasmid GR6 was again digested with Ncol. After the overlapping ends had been filled in with the Klenow fragment of E. coli DNA polymerase I, EcoRV was digested, the larger fragment was eluted from an agarose gel and ligated with the 5 'phosphorylated oligonucleotides EBI 1264 and 2497 (FIG. 24; the oligonucleotides were brought together briefly to 65 ° C and then slowly cooled to 4 ° C). After transformation into competent E. coli cells, a plasmid was isolated, the structure of which is shown in FIG. 25.
  • RNA After in vitro transcription with T7 RNA polymerase, an RNA is formed which comprises 18 nucleotides from the transcription site to the EcoRV interface, followed by the 52 nucleotides of the inverse 5 'UTR of the ⁇ -globin gene and the nucleotides 611-1400 from HRV2.
  • plasmid which contains the 5 'UTR of the ⁇ -globin gene before the ATG at nucleotide 449 of the HRV2 sequence was carried out as follows: First, plasmid GR6 was digested with EcoRV and PstI and treated with alkaline phosphatase. This DNA was then analyzed with the 5 'phosphorylated oligonucleotide pairs EBI 1264/2497 (which corresponds to the sequence of the 5' UTR of the ⁇ -globin gene (FIG. 24)) and UKCC 1660/1661 (which corresponds to the HRV2 sequence 448-471 (FIG.
  • plasmid p20 a plasmid was isolated in which the cloned 1st okb PstI fragment had the same orientation to the T7 RNA promoter has as in plasmid GR6 (in Figure 27a); it was called pß + 449.
  • an RNA is formed which comprises 18 nucleotides from the transcription initiation site to the EcoRV site, followed by the 52 nucleotides of the ß-globin 5 * UTR and the nucleotides 448-1400 of the HRV2 sequence.
  • RNA is formed, which in turn comprises 18 nucleotides from the transcription initiation site to the EcoRV site, 52 nucleotides of the inverse ⁇ -globin 5 'UTR and nucleotides 449-1400 from HRV2.
  • RNA polymerase In vitro transcription of these plasmids after linearization with BamHI using T7 RNA polymerase was carried out as described (Düchler et al, 1989; EPA 0 356 695). At the end of the reaction time, the DNA was digested by adding DNase I (RNAse-free, Pharmacia) and the RNA was precipitated with ethanol after adding a third volume of 8M ammonium acetate. After reprecipitation with ethanol, the RNA was then taken up in water and its quality was checked by agarose gel electrophoresis. The running buffer contained 1% SDS. 1 ⁇ g of these RNAs were used for in vitro translation.
  • DNase I RNAse-free, Pharmacia
  • reaction was stopped by adding 1 .mu.l 200 mM methionine and is applied, an aliquot (1 .mu.l) on a SDS-polyacrylamide gel. The gel was fluorographed after the run was completed.
  • Figure 27 shows the translation products of the RNAs transcribed from the four plasmids (pß + 449, pß-449, pß + 611 and pß-611) after electrophoretic separation.
  • Translation products are only found when the ⁇ -globin 5'UTR is in the positive orientation (pß + 449 and pß + 611), surprisingly the translation of the ATG 449 (probably not used in vivo) is stronger than the translation of ATG 611, which is believed to be used in vivo.
  • the translational products behave in their gel electrophoretic properties in accordance with the molecular masses predicted from the amino acid sequence.
  • the translation product obtained with the RNA from pB611 + could be precipitated with the antibody 8F5. This antibody recognizes an amino acid sequence of the VP2 protein. (Skern, T., et al. (1987) J. Gen. Virol., Loc. Cit). This result supports the assumption that translation actually starts at the ATG 611.
  • the plasmid p20 is very suitable for such purposes because it has a number of interfaces which are suitable for the Insertion of fragments can be used.
  • the three enzymes AccI, Hindll and Sall recognize the sequence 5 'GTCGAC 3'; the position of the enzymatic cleavage is different for these restriction enzymes.
  • Sall cuts after the first G, AccI after the T and HindII after the first C. Therefore, after appropriate manipulation, each fragment can be brought into the correct reading frame.
  • the other interfaces can be used to linearize the plasmid prior to in vitro transcription.
  • the plasmid p20 was cut with HindII and each with cDNA fragments of HRV2 and an HRV14 acid-resistant mutant, HRV14-as3 (Skern, T., Torgersen, H., Auer, H., Kuechler, E. and Blaas, D. (1991 ), Virology, 183, 757-763).
  • HRV2 an SspI / XmnI fragment comprising nucleotides 2368-3752 was used; in the case of the acid-resistant mutant of HR14, an Hpal fragment with the nucleotides 1810-3560 was used.
  • the SspI / XmnI fragment of HRV2 was excised from plasmid pl9 (Duechler, M., et al. 1989, loc. Cit.); the Hpal fragment of the acid-resistant mutant of HRV14 was excised from the plasmid pHRV14-as3 (Skern, T. et al., 1991, Virology, loc. cit.). After transformation in E. coli 5K cells, plasmids were isolated in both cases, in which the orientation of the HRV insert enables translation of the HRV amino acid sequences. The plasmids were named pßHRV2 / sub and pßHRV14 / s * ub.
  • the plasmid pßHRV2 / sub was cut with Apal so that only up to amino acid glycine 99 was transcribed.
  • the plasmid pHRV14 / sub was with BamHI (the interface is in the polylinker of p20) cut; in this case, the HRV14 insert was transcribed until the end, ie up to the amino acid leucine 122. As a result, both 2As are inactive in the cis cleavage.
  • the translation products of the RNAs transcribed in vitro by these plasmids are shown in FIG. 28.
  • the 40 kD product corresponds to a translation of the VP1 / ⁇ 2A region of HRV2, which is contained in the plasmid pßHRV2 / sub;
  • the 66 kD product corresponds to a translation of the ⁇ VP3 / VP1 / ⁇ 2A region of HRV14, which is contained in the plasmid pßHRVl4 / sub.
  • the pßHRV2 / sub translation product was then incubated with E. coli extracts containing native 2A expressed either from an M13 genome or from a plasmid. This is to check to what extent the ⁇ VP1 / ⁇ 2A region can be cleaved by the HRV2 2A proteinase expressed in bacteria. For this purpose, the proteins were induced with IPTG as described above and the cells were worked up.
  • the cells of 1.5 ml culture for plasmid constructions or 3 ml for M13 constructions were taken up in 500 ⁇ i washing solution, disrupted with ultrasound and 15 ⁇ l (for M13 constructions) or 10 ⁇ l (for plasmid constructions) from the supernatant Incubate 1 ⁇ l of the translation product of pßHRV2 / sub for 3 hours at 30 ° C.
  • the result of the incubations is shown in Figure 29A; after incubation with extracts of M13-2A or pBR2A / l and pBR2A / 2, two further bands (32 kD and 30 kD) can be seen.
  • Extracts from M13-2A ⁇ 3Gly or pBR2A ⁇ no further bands to be seen since no active 2A was expressed.
  • the presence of two fission products allows two explanations; either the 2A protease cleaves the translation product of pßHRV2 / sub twice or the correct cleavage product is additionally processed by an E. coli protease.
  • bacterial extracts of M13-2A and pBR2A / l and pBR2A / 2 were partially purified by ammonium sulphate precipitation. The extracts were mixed with ammonium sulphate to a final concentration of 30%.

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Abstract

Un système d'essai par coloration in-vivo permet de contrôler l'activité de protéases virales.
EP19910914980 1990-08-28 1991-08-22 Systeme d'essai permettant de controler l'activite de proteinases virales Withdrawn EP0497949A1 (fr)

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DE4027153 1990-08-28
DE19904027153 DE4027153A1 (de) 1990-08-28 1990-08-28 Testsystem zur aktivitaetsueberpruefung viraler proteinasen
DE19914112952 DE4112952A1 (de) 1991-04-20 1991-04-20 Testsystem zur aktivitaetsueberpruefung viraler proteinasen
DE4112952 1991-04-20

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US5965124A (en) * 1991-12-06 1999-10-12 Whitehead Institute For Biomedical Research Replication-competent recombinant viral vaccines and method of producing same
MX9206981A (es) * 1991-12-06 1994-08-31 Whitehead Biomedical Inst Vacunas de recombinantes y metodo para producirlas.
AU1837897A (en) * 1996-01-26 1997-08-20 Boehringer Mannheim Corporation Bis-maleimido cross-linking agents
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
WO1999006072A1 (fr) * 1997-07-30 1999-02-11 Boehringer Mannheim Corporation Promedicaments cyclises
WO2000039348A1 (fr) * 1998-12-24 2000-07-06 Small Molecule Therapeutics, Inc. Methodes et compositions pour identifier des modulateurs de protease
EP1994178A4 (fr) 2006-03-13 2009-11-11 Univ Leland Stanford Junior Détection d'interactions moléculaire en utilisant un système rapporteur de complémentation enzymatique à affinité réduite
US8101373B2 (en) 2007-10-12 2012-01-24 Discoverx Corporation β-galactosidase donor fragments
WO2012178079A1 (fr) 2011-06-23 2012-12-27 Discoverx Corporation Suivi du trafic des protéines par complémentation d'un fragment rapporteur de bêta-galactosidase

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DE2712615A1 (de) * 1977-03-18 1978-09-21 Max Planck Gesellschaft Verfahren zur herstellung von filamentoesen phagen als vektor fuer synthetische rekombinate
ES2059482T3 (es) * 1987-12-23 1994-11-16 Boehringer Ingelheim Int Expresion de la proteasa p2a de hrv2 codificada por virus.
DE3819846A1 (de) * 1988-06-10 1989-12-14 Hans Prof Dr Dr Wolf Testsystem fuer die entwicklung von inhibitoren der protease von hiv
FI901459A7 (fi) * 1988-07-25 1990-03-23 Boehringer Ingelheim Int Tarttuvan RNA:n in vitro-synteesi

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