DE4206769A1 - Analysing host cell shut=off caused by Picorna viruses - by comparing translation prods. of viral 2C gene from RNA with and without cap structure, used e.g. to identify inhibitors of viral 2A protease - Google Patents

Abstract

Analysis comprises (1) in vitro transcription of a rhinoviral 2C gene (provided with translation stop and start codons) to produce RNA having a cap structure, (2) adding this to a translation mixt. which has been pretreated with a picorna virus 2A protease, (3) subjecting this mixt. to in vitro translation, and (4) comparing the translation products with those generated in a comparative test using RNA wthout the cap structure. Also new are the plasmids pHRV2/5'UTR-2C, phink-2C, phink-2BC, pSVL-2BC, pSVL-2C and pbeta-2C. Pref. the human rhinovirus (HRV)2 2C-gene is transcribed, and HRV2 2A protease is used in step (2). The translation mixt. includes reticulocyte lysate (or a fraction of it). USE/ADVANTAGE - The method is used (1) to detect cellular portions of the 'host cell shut off' (HCSO) generated proteins, (2) to isolate inhibitors of HCSO, partic. of cellular and viral proteins involved in the process, specifically rhinoviral 2A protease.

Description

Infection by most representatives of the Picorna viruses very soon lead to a drastic one Reduction of cellular protein biosynthesis despite Presence of intact, endogenous messenger RNA (mRNA). This phenomenon known as "host cell shut-off" enables the virus to efficiently synthesize the virus genomic RNA and viral proteins (Rueckert R. R (1990) "Picornaviridae and their Replication", in Fields, B. N (ed.): "Virology", 2nd edition, Vol. 1-2, 507-548).

The molecular mechanism leading to the "host cell shut-off "is not yet known exactly The course of the infection will be a splitting of the protein p220 observed, a component of the eukaryotic Initiation factor eIF-4F, which is necessary for the formation of a Cap-dependent initiation complex appears necessary (Sonenberg, N. (1987) Adv. Virus Res. 33, 175-204). There the cellular mRNAs a characteristic Having a cap structure will translate them prevented. Picornavirus, however, do not contain a cap, but have a functionally preserved Region in the 5 ′ untranslated region, the one Cap-independent translation enables (Pelletier, J. and Sonenberg, N. (1988) Nature 334, 320-325; Bold et al. (1990), J. Virol. 64, 4625-4631).  

The cleavage of p220 is in entero- and rhinoviruses possibly indirectly through viral protease 2A and in the case of aphtoviruses by the leader protease pair L / L ′ caused (Kräusslich, H.-G., Nicklin, M. J. H., Toyoda, H., Etchinson, D. and Wimmer, E. (1987) J. Virol. 61, 2711-2718; Lloyd, R.E., Toyoda, H., Etchinson, E., Wimmer, E. and Ehrenfeld, E. (1986) Virology 150, 299-303; Belsham, G.J. and Brangwyn, J.K. (1990) J. Virol. 64, 5389-5395; Devaney et al. (1989) J. Virol. 62, 4407-4409). A row experimental results suggest that by these highly specific viral proteases endogenous latent in the infected cell Protease is activated proteolytically, which in turn cleavage of p220 causes (Kräusslich et al., loc. cit .; Lloyd et al., Loc. cit .; Hellen, C. U. T., Krausslich, H.-G. and Wimmer, E. (1989) Biochem. 28, 9881-9890). However, this activation is not yet proved that the to identify cellular protease. However, it could are shown to be the eukaryotic initiation factor eIF-3 is a necessary part of this p220 Fission activity is (Wyckoff, E., Hershey, J.W.B. and Ehrenfeld, E. (1990), Proc. Natl. Acad. Sci. U.S.A., 87, 9529-9523).

However, observations from other working groups show no temporal causal connection between the Cleavage of p220 and the appearance of the "host cell shut-offs ", which is its relevance as a triggering cause this phenomenon still seem controversial (Urzainqui and Carrasco. (1989) J. Virol. 63, 4729-4735).  

An analysis of the in vitro translation of rhinoviral cDNA fragments of serotype 2 (HRV2) shows that unexpected differences in the properties of the 5 ′ untranslated regions (5′UTR) of HRV2 occur. Only certain deletions in the 5 ′ UTR range ( FIGS. 1-5 and 7) lead to a translation of subgenomic HRV2 transcripts produced in vitro (example 1). FIG. 7B shows the translation products of the transcripts which have been synthesized in vitro by the plasmids GR5, GR6, GR7, GR8, GR9 and GR10 after electrophoretic separation. Translation products are only formed from the RNAs of the plasmids GR8 (-448) and GR10 (-610), whereby in the case of GR8 the AUG at position 449 was surprisingly used. Initiation of translation at this AUG provides a protein with a molecular mass of 35 kD. In the case of GR10, a translation product with a molecular mass of 30 kD occurs; this product corresponds to an initiation of translation at the AUG at position 611, the start codon, which is also used in vivo. In both cases, only a single nucleotide of HRV2 5'UTR is present.

These results are in contrast to those with HRV14 were obtained. Here is the initiation of the Translation observed only in one AUG (position 625) and only if nucleotides 1-546 are deleted. In contrast, there is no translation after deletion of nucleotides 1-620 instead (Alsaadi, S., Hassard, S. and Stanway, G. (1989), J. Gen. Virol., 70, 2799-2804).

The present invention now provides in vitro Test system available that allows the molecular Mechanism of the "host" caused by picornaviruses cell shut-off ". The test system can also for the identification of inhibitors of this Mechanism can be used. For example the influence of inhibitors on the "host cell shut-off "involved viral and cellular proteins to be examined.

The test system is based on a comparison of Translation products by transcription and Translation of the rhinoviral 2C gene can be obtained. It includes the following steps:

• - One with a translation start and one Translational stop codon labeled rhinoviral 2C Gene, in particular the HRV2 2C gene in the form of the Plasmid pLink-2C is transcribed in vitro and provided with a cap structure. The RNA obtained becomes a translation mixture given that with a picornaviral proteinase, which triggers the "host cell shut-off" is.
• - The proteins thus translated in vitro can then depending on the question, then trans tion products from comparative experiments be compared. Appropriate proteins can, for example, with Help can be obtained from transcripts using and have been synthesized without a cap structure and with a translation mixture in vitro be translated without using a proteinase is pre-incubated. This is the opportunity to Examination of the mode of action of the corresponding Given proteinase.

To identify cellular proteins that "host cell shut-off" can be involved purified cellular proteins or Protein extracts using standard methods can be manufactured for in vitro Translation can be added. In addition, the Translation mixture or the one used Rabbit reticulocyte lysate, by known Processes are fractionated to the effect of individual proteins or protein mixture on the Check translation reaction.

• - In addition to identification and characterization cellular proteins that are "host cell shut-off" are involved, the mode of operation of the picornaviral proteins which form the "host cell trigger shut-off "to be examined.
• - With the help of this test system it is also possible Inhibitors for everyone at the "host cell shut-off" substances or proteins involved (cellular how to isolate viral). For example, the Activity of the viral proteinase can be inhibited. An inhibition can on the changed Translation patterns can be read.

A. Can be used to analyze the translation products Variety of methods are used, all of which come from are known in the art, e.g. B. Installation radioactive amino acids, electrophoretic Separation and fluorography or detection of the separated translation products by immunological Methods, e.g. B. by detection of the 2C protein or of its fragments by monoclonal directed against 2C or polyclonal antibodies according to known  Methods can be produced (Sambrook, J., Fritsch, E. F., Maniatis, T. (1989): "Molecular cloning: a laboratory manual ", Cold Spring Harbor Laboratory Press) Such genes can expediently be used for the test system be used, the multiple internal translations owns starts, preferably the HRV 2C gene.

The 2C gene should have a start and stop codon be provided. The sequence before Start AUG advantageously be chosen so that it the highest possible match with the consensus sequence of Kozak (Kozak, M. 1989, J. Cell Biol. 108, 229-241). The methods necessary for this are known from the prior art (Sambrook et al. (1989), loc. cit.).

The preparation of a suitable 2C gene using Standard methods are used in Example 2 using the HRV2 2C Gene described.

The in vitro transcription of the corresponding RNA takes place according to standard methods, such as B. Duechler et al. 1989, Virology 168, 159-161, Sambrook et al. (1989), loc. cit., Krieg, P.A. and Melton, D.A. (1987), Meth. Enzymol., 155, 397-415 or Bujard, H. et al. (1987), Meth. Enzymol., 155, 416-433 described.

After the transcription reaction, the DNA is through Addition of DNase I (RNAse-free, Pharmacia) digested and RNA by phenol extraction and ethanol precipitation cleaned. For synthesis with and without a cap structure provided RNA becomes the transcription reaction 7 MeGpppG and GTP added. For example, that of Duechler et al. (1989), loc. cit., described Reaction mixture 1 mM 7MeGpppG and 250 µm GTP contain.  

The in vitro translation is carried out according to known methods (Sambrook et al. (1989), loc. Cit., Example 2). As shown in Example 2, 1 pg of the RNAs are used per reaction mixture. The reaction mixture also contains 20 µl 90 mM KCI, 0.3 mM MgCl ₂ , 10 mM creatine phosphate, 2 µl ³⁵ S-Trans-Label (ICN; 10 µCi / µpl, 1000 Ci / mmol) and 11.5 µl rabbit reticulocyte lysate, that had been treated with Micrococcal Nuclease (Sigma). The rabbit reticulocyte lysate can e.g. B. by the method of Jackson, R. and Hunt, T. (Meth. Enzymol. (1983), 96, 50-74) by treatment with micrococcal nuclease. After 1 hour at 30 ° C., the reaction is stopped by adding 1 μl of 200 mM methionine and an aliquot (1 μl) is applied to an SDS polyacrylamide gel. The gel is fluorographed after the run is complete.

The picornaviral 2A proteinase is expressed according to standard methods (Sambrook et al. (1989), loc. cit.). For example, for the expression of HRV2 2A the path given in Example 3 can be followed.

For further characterization of the "host cell shut-offs "can in addition to the plasmids described also the plasmids from Examples 1-3, especially pHRV2 / 5′UTR-2C, pβ-2C and pLink-2BC too Comparative experiments can be used.

In addition to the applications described the method also for isolating inhibitors of the "host cell shut offs" can be used. Example 3 shows that the addition of Elastatinal for inhibition which leads to 2A proteinase. The following is the Invention using the example of translation picornaviraler Transcripts, especially of the rhinoviral 2C  Transcripts, and the influence of 2A proteinase on the Translation explained in detail.

Basis for an effective translation of a 2C gene is the installation of a start and stop codon. In addition, the sequence should start AUG Similarities to Kozak's consensus sequence (Kozak, M. (1989), J. Cell. Biol. 108, 229-241). All necessary methods are up to date known in the art (Sambrook et al. (1989), loc. cit., and described in detail in Examples 2-3.

The plasmid pLink-2C (FIGS . 12 and 13, example 2), which, after linearization with Bam H1 and after in vitro transcription, provides an RNA which comprises 46 nucleotides from the start of transcription to the AUG codon, followed by nucleotides 3872 to 5219 the HRV2 sequence. The plasmid pLink-BC (FIGS . 12 and 13, example 2) can serve as a control, whereby after linearization with Bam H1 in the in vitro transcription an RNA is formed which comprises 48 nucleotides from the start of transcription to the start of translation of the HRV2 sequence, followed by nucleotides 3587 to 5219 of the HRV2 sequence. For further comparison, the 2C gene can be cloned behind the 5 ′ UTR of HRV2 and behind the 5 ′ UTR region of the rabbit globin gene. The result is the plasmids pHRV2 / 5'UTR-2C or pβ-2C ( Fig. 13, Example 2).

FIG. 15A shows the translation products of the RNAs obtained by in vitro transcription from plasmids pHRV2 / 5'UTR 2C, pβ-2C-2C and pLink pLink-2BC. Surprisingly, several proteins are translated from each RNA.

A band of 37 kD occurs among the products of the RNAs of the plasmids pLink-2C and pβ-2C; this band corresponds to the size of the mature 2C protein. This product is obviously the result of initiation on the first AUG of the two constructions (indicated by black arrows in FIG. 13). The other bands (33 kD, 28 kD and 20 kD) are probably products of initiation on the AUGs marked with open arrows. Surprisingly, when the RNA of the construction pHRV2 / 5′UTR-2C is translated, no 37 kD band occurs; it follows, therefore, that no initiation has occurred at AUG 611. The identity of the 37 kD band with the mature 2C protein is confirmed by the fact that it is not present in the translation products of the RNA of the plasmid pLink-2BC. In addition to the 33 kD, 28 kD and 20 kD bands, two further bands of 47 kD and 42 kD can be found here. The 37 kD band is missing because it comes from the synthetically introduced AUG in front of the 2C gene. The 47 kD band comes from the initiation on the first AUG of the 2BC gene (marked black in FIG. 13) and the 42 kD band of the initiation on an AUG in the 2BC gene. Table 1 shows the sequences in the region of those AUGs which are responsible for the internal initiations.

Table 1

The oligonucleotide used for the construction of pLink-2C is underlined. For all internal translation products are two AUG start codons possible - except for the 20 kD product.

Nucleotide sequences in the vicinity of the AUGs of the 2C and 2BC constructions

AUGs within the HRV2 2C and 2B sequence that could serve as translation start codons

About this unexpected phenomenon of internal initiation to further characterize RNAs from the Plasmids transcribed under conditions leading to Presence of a 5 ′ cap structure. Under Cap structure is understood to be the nucleotides 7MeGpppG that form the 5 'end of cellular RNAs.

In Fig. 15B are the translation products of the RNAs provided with a cap structure that are transcribed from plasmids pHRV2 / 5'UTR 2C-2C and pLink pLink-2BC, as shown by an electrophoretic separation. The RNAs of the plasmids pLink-2C and pLink-2BC were initiated here almost exclusively at those AUGs which are marked with black arrows in FIG. 13; the 37 kD or the 47 kD band can be recognized almost exclusively. The presence of the cap structure led to the initiation being pushed back. There are hardly any differences between the translation products of the capped structure RNA of the construction pHRV2 / 5′UTR and those with the uncapped RNA. The weak bands at 37 kD and 43 kD indicate initiation at AUG codons 449 and 611. The 5′UTR- of HRV2, therefore, even when capped, cannot suppress the property of the 2C RNA to initiate an internal translation.

In order to investigate the influence of the rhinoviral 2A proteinase on the translation, the translation experiments were carried out with the addition of the HRV2 2A proteinase ( FIG. 15B).

In Fig. 15B, lane 7 are shown the translation products. Although an RNA with a cap structure was used, the translation pattern of an RNA without a cap structure is observed; the presence of 2A obviously changes the properties of the rabbit reticulocyte lysate. That the 2A proteinase is responsible for this change is supported by the result in FIG. 15B, lane 8. In this experiment (Example 3), the 2A preparation was first spiked with Elastatinal (an inhibitor of 2A activity). In this case, the translation of an RNA without a cap structure takes place normally in rabbit reticulocytes; inhibition by 2A prevents the translation properties from changing.

Due to the unexpected occurrence of different Protein pattern depending on the cap structure and the Addition of a viral proteinase results simple method for analysis of picornaviruses caused "host cell shut-offs".

The procedure is detailed using the following examples described.

example 1 In vitro translation of rhinovirals cDNA fragments, the deletions in the 5 'UTR exhibit

To delete parts of the 5′UTR from HRV2, that was Plasmid GR6 used; it contains the HRV2 nucleotides 19-1400 in plasmid pGEM2 (Promega). The nucleotide 19 of the HRV2 sequence is around 30 base pairs dated Removal of transcription for T7 RNA polymerase removed. This creates an RNA after in vitro transcription, which corresponds to the orientation of the genomic RNA.

A) Production of GR6

Three were used to prepare the GR6 plasmid Plasmids used, namely p773 (HRV2 nucleotides 562-1012; Skern, T. et al. (1985), Nucl. Acids Res., 13, 2111-2126), p19 (HRV2 nucleotides 19-5100) (Duechler et al. (1989), loc. Cit.) And p860 (Skern et al. (1985), loc. cit.). p860 contains the HRV2 Nucleotides 660 to 1400 in the PstI site from pUC9; the orientation is such that nucleotide 1400 next to the EcoRI interface of the polylinker.

The plasmid pGEM2 was used with the restriction enzymes HindIII and BamHI cut and with alkaline Calf intestine phosphatase dephosphorylated; Likewise with HindIII and BamHI the plasmid p773 cut. The HRV2 insert released by this was eluted from an agarose gel and with the HindIII / BamHI linearized pGEM2 vector ligated. To It became a transformation into competent E. coli cells resulting plasmid (GR1) isolated. GR1 was then with the restriction enzymes HindIII and NcoI and with treated with alkaline phosphatase; also with The plasmid p19 was cut with HindIII and NcoI.  

This creates a 361 bp HindIII / NcoI fragment with nucleotides 248 to 609 of the HRV2 gene map. This fragment was eluted from an agarose gel and ligated with the GR1 plasmid treated as described above. The resulting plasmid (GR2) was cut with the restriction enzyme EcoRI and treated with calf intestine alkaline phosphatase; plasmid p860 was also cut with EcoRI. 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. After transformation into competent E. coli cells, the corresponding plasmid (GR5) was isolated. The plasmid GR5 in turn was cut with the restriction enzyme HindIII and dephosphorylated with calf intestinal alkaline phosphatase; plasmid p19 was also treated with HindIII. The 240 bp fragment formed from the HindIII site in the polylinker to the HindIII site at nucleotide 248 of the HRV2 gene map (ie nucleotide 19 of the HRV2 gene map to 248) was ligated with the linearized GR5 vector. After transformation into competent E. coli cells, a plasmid was isolated, the structure of which is shown in FIG. 1 (GR6). After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 28 nucleotides from the transcription site to the EcoRI site, the beginning of the HRV2 sequence, followed by nucleotides 19 to 1400 of the HRV2 sequence. The already described plasmid GR5 served as deletion 1-248. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 10 nucleotides from the transcription site to the HindIII site, the beginning of the HRV2 sequence, followed by nucleotides 248 to 1400 of the HRV2 sequence.

B) Production of GR7

The plasmid GR6 was cut with the restriction enzymes EcoRV and BamHI and dephosphorylated with calf intestinal alkaline phosphatase. In a second approach, the plasmid GR6 was cut with the restriction enzymes BanII and BamHI; the 988 bp fragment was eluted from an agarose gel and ligated with the GR6 DNA preparation cut with EcoRV and BamHI. After transformation into competent E. coli cells, a plasmid was isolated (GR7), the structure of which is shown in FIG. 2. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 19 nucleotides from the transcription site to the EcoRV site, the beginning of the HRV2 sequence, followed by nucleotides 412 to 1400 of the HRV2 sequence.

C) Production of GR8

The plasmid GR6 was cut with the restriction enzymes EcoRV and PstI and dephosphorylated with calf intestinal alkaline phosphatase. The 3.0 kb fragment was isolated from an agarose gel and ligated with the 5 ′ phosphorylated UKCC 1660/1661 oligonucleotides ( FIG. 6; the oligonucleotides were brought together briefly to 65 ° C. and then slowly cooled to 4 ° C.). After transformation, a plasmid (GR8a) was isolated since the 928 bp PstI / PstI fragment is lost on first digestion with EcoRV and PstI. To bring this fragment back into GR8a, this plasmid was cut with the restriction enzyme PstI and dephosphorylated with calf intestine using alkaline phosphatase. In a second approach, the plasmid dR6 was also cut with the restriction enzyme PstI; the 928 bp fragment was eluted from an agarose gel and ligated with the GR8a DNA preparation cut with PstI. After transformation into competent E. coli cells, a plasmid (GR8) was isolated, the structure of which is shown in FIG. 3. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 19 nucleotides from the transcription site to the EcoRV site, the beginning of the HRV2 sequence, followed by nucleotides 448 to 1400 of the HRV2 sequence.

D) Production of GR9

The plasmid GR6 was cut with the restriction enzymes EcoRV and BamHI and dephosphorylated with calf intestinal alkaline phosphatase. In a second approach, the plasmid GR6 was cut with the restriction enzymes AvaII and BamHI; the 874 bp fragment was eluted from an agarose gel and ligated with the GR6 DNA preparation cut with EcoRV and BamHI. After transformation into competent E. coli cells, a plasmid (GR9) was isolated, the structure of which is shown in FIG. 4. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 19 nucleotides from the transcription site to the EcoRV site, the beginning of the HRV2 sequence, followed by nucleotides 525 to 1400 of the HRV2 sequence.

E) Production of GR10

The plasmid GR6 was cut with the restriction enzymes EcoRV and Nco I and the overlapping Ncol end was filled in with the Klenow fragment of E. coli DNA polymerase I. The 3.8 kb DNA fragment was isolated from an agarose gel and ligated to itself. After transformation into competent E. coli cells, a plasmid (GR10) was isolated, the structure of which is shown in FIG. 5. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 19 nucleotides from the transcription site to the EcoRV site, the beginning of the HRV2 sequence, followed by nucleotides 610 to 1400 of the HRV2 sequence.

F) Transcription and translation

In vitro transcription of these plasmids after linearization with BamHI using T7 RNA polymerase was carried out as described (Duechler et al. (1989), loc. cit.). At the end of the reaction time, the DNA was digested by adding DNase I (RNAse-free, Pharmacia), the proteins were removed by phenol extraction, and the RNA was precipitated with ethanol after adding a third volume of 8 M ammonium acetate. After reprecipitation with ethanol, the RNA was then taken up in water and an aliquot was checked for quality by agarose gel electrophoresis. The running buffer contained 1% SDS. 1 µg of these RNAs were used for in vitro translation. The reaction contained 20 µl 90 mM KCl, 0.3 mM MgCl ₂₁ 10 mM creatine phosphate, 2 µl ³⁵ S-Trans-Label (ICN; 10 µCi / µl, 1000 Ci / mmol) and 11.5 µl rabbit reticulocyte lysate containing the Micrococcal Nuclease (Sigma) had been treated. The rabbit reticulocyte lysate was prepared by the method of Jackson, R. and Hunt, T. (1983) Meth. Enzymol., 96, 50-74) by treatment with micrococcal nuclease. After 1 hour at 30 ° C., the reaction was stopped by adding 1 μl of 200 mM methionine and an aliquot (1 μl) was applied to an SDS polyacrylamide gel. The gel was fluorographed after the run was completed.

FIG. 7B shows the translation products of the RNAs which have been transcribed from the plasmids GR5, GR6, GR7, GR8, GR9 and GR10 after electrophoretic separation. Translation products are only found from the RNAs of the plasmids GR8 (-448) and GR10 (-610), whereby in the case of GR8 the AUG at position 449 was surprisingly used. Initiation of translation in this AUG results in a protein with a molecular mass of 35 kD. In the case of GR6, a translation product with a molecular mass of 30 kD was evident; this product corresponds to initiation of translation at AUG at position 611, the start codon used in vivo. Interestingly, however, only a single nucleotide of HRV2 5′UTR was present in both cases. This result is in contrast to that obtained in similar experiments with HRV14. Firstly, the initiation of translation was only observed in one AUG (position 625). Second, a translation product was observed only when nucleotides 1-546 had been deleted. In contrast, translation was prevented by deleting nucleotides 1-620. Obviously there are unexpected differences in the properties of the non-translated regions of these HRV serotypes.

Example 2 In vitro translation derived from RNAs of rhinoviral cDNA fragments of the 2C Gene. A) Installation of the stop codons

The plasmid p15 (Duechler et al. (1989), loc. Cit.), Which contains the cDNA of HRV2 from nucleotide 2125 to 7102, was cut with Acc I and then treated with mung bean nuclease to the overhanging 5 'ends to remove the resulting fragments. Then it was digested further with Pvu II. The 1.7 kb fragment (nucleotide 3522 to 5219; Fig. 8), which is part of the 2A sequence (designated 2A '), the entire sequence of 2B, 2C, 3A, 3B and part of 3C (referred to as 3C') was cloned into the Sma I site of pUC18. The resulting plasmid (pUC18-2C3A) was subjected to double digestion with Ssp I (interface at nucleotide 4335) and Bam HI (in the polylinker). The 0.89 kb fragment containing the viral sequences was cloned into the Eco RV and BamHI site of the Bluescript (+) vector. After transfection of E. coli 5K, single-stranded DNA from Blue (+) - 2C3A was isolated using an M13 helper phase (VCS-M13, Stratagene). This DNA was used to insert a stop codon after 2C (GGG → TAG), as shown in Fig. 7. The oligonucleotide 5'-ATCAATTGGCTATTGGAATATA-3 'were used using standard techniques (Taylor et al., 1985, Nucl. Acids Res. 13, 8765-8785).

A clone with the built-in stop codons (Blue (+) - 2C * 3A) were isolated after transfection and his Accuracy through direct DNA sequencing mutated region checked (Tabor, S. and Richardson, C.C., 1987, Proc. Natl. Acad. Sci. USA 84, 4767-4771).

The mutated DNA region was isolated from Blue (+) - 2C * 3A and reintroduced into the wild-type environment in pUC18-2C3A (right side of FIG. 8). Blue (+) - 2C * 3A was partially digested with BspMI and the 312 bp fragment containing the mutation was isolated. pUC18-2C3A was digested completely with the same enzyme and the 365 bp fragment was also isolated. An aliquot of the same digest was dephosphorylated with alkaline phosphatase.

A ligation of these two fragments with digested and dephosphorylated pUC18-2C3A resulted after transformation into competent E. coli cells for pUC18-2C * 3A. Because BspMI one interrupted palindrome recognizes are the overlapping Ends always different; this makes it possible to have three Fragments controlled in this way to ligate with each other.

B) Installation of the start codons in front of the 2BC and 2C genes

The plasmid pSVL (Pharmacia; Fig. 10) was cut with the restriction enzymes XbaI and BamHI and treated with calf intestine alkaline phosphatase. pUC18-2C * 3A ( FIG. 10) was cut on the one hand with the restriction enzymes NdeI and BamHI and the 1.6 kb fragment was eluted from an agarose gel and ligated with the 5′-phosphorylated oligonucleotides EBI 1102/1094 ( FIG. 11). The oligonucleotide pair contained an AUG codon before the first codon of the 2BC gene and the codons to be supplemented up to the NdeI interface. The sequence before the AUG was chosen so that it was as close as possible to the consensus sequence of Kozak (Kozak, M., loc. Cit.). A plasmid was obtained called pSVL-2BC; the integrity of the sequence at the 5 ′ end of the 2BC gene was demonstrated by DNA sequencing.

The procedure was analogous to construct the plasmid pSVL-2C: The plasmid pSVL (Pharmacia: FIG. 10) was cut with the restriction enzymes XbaI and BamHI and treated with alkaline phosphatase from calf intestine. On the other hand, pUC18-2C * 3A ( FIG. 10) was cut with the restriction enzymes SphI and BamHI and the 1.3 kb fragment was eluted from an agarose gel and ligated with the 5′-phosphorylated oligonucleotides EBI 1099/1092 ( FIG. 11). The pair of oligonucleotides contained an AUG before the first codon of the 2C gene and the missing codons up to the SphI interface. The sequence before the AUG codon was chosen so that it was as close as possible to the consensus sequence of Kozak (Kozak, M., loc. Cit.). A plasmid called pSVL-2C was obtained; the integrity of the sequence at the 5 'end of the 2C gene was demonstrated by DNA sequencing.

For in vitro transcription by T7 RNA polymerase the XbaI / BamHI fragment of pSVL-2C with pGEM2, cut with XbaI and BamHI and treated with alkaline phosphatase, ligated. After transformation into competent E. coli cells a plasmid (pLink-2C) was isolated, its structure is shown in Figure 12. After linearization with the restriction enzyme BamHI is produced in vitro Transcription with T7 RNA Polymerase is an RNA developed by the transcription site up to the AUG codon, the beginning the HRV2 sequence, comprising 46 nucleotides, followed by the Nucleotides 3872 to 5219 of the HRV2 sequence.

Analogously, the XbaI / BamHI fragment of pSVL-2BC was ligated into pBluescript (Stratagene), which was cut with XbaI and BamHI and treated with alkaline phosphatase. After transformation into competent E. coli cells, a plasmid called pLink-2BC was isolated, the structure of which is shown in FIG. 12. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 48 nucleotides from the transcription site to the AUG codon, the beginning of the HRV2 sequence, followed by nucleotides 3587 to 5219 of the HRV2 sequence.

In order to put the 2C gene behind the 5'UTR of HRV2, the plasmid pHRV2 / 5'UTR-2C ( Fig. 13) was prepared as follows. The plasmid GR6 was cut with the restriction enzymes NcoI and BamHI and dephosphorylated with calf intestinal alkaline phosphatase. The plasmid pLink-2C was cut with the restriction enzymes SphI and BamHI; the 1.3 kb fragment was eluted from an agarose gel and ligated with the GR6 DNA preparation cut with NcoI and BamHI and with the 5 ′ phosphorylated oligonucleotides EBI 1631 and 1634 ( FIG. 14; the oligonucleotides were brought together briefly to 65 ° C. and can be slow cooled to 4 ° C). These oligonucleotides code for the amino acids that lie between the AUG codon of pLink-2C and the SphI interface. After transformation into competent E. coli cells, a plasmid was isolated (pHRV2 / 5'UTR-2C), the structure of which is shown in FIG. 13. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which comprises 19 nucleotides from the transcription site to the EcoRV site, the beginning of the HRV2 sequence, followed by nucleotides 19 to 613 and 3872 to 5219 HRV2 sequence.

In order to place the 2C gene behind the 5′UTR of the rabbit β-globin gene, the plasmid pβ-2C ( FIG. 13) was produced as follows: The plasmid GR6 was cut with the restriction enzymes EcoRV and BamHI, the 3.0 kb fragment from an agarose gel isolated and dephosphorylated with alkaline phosphatase from calf intestine. The plasmid pLink-2C was cut with the restriction enzymes SphI and BamHI; the 1.3 kb fragment was eluted from an agarose gel and ligated with the GR6 DNA preparation cut with NcoI and BamHI and with the 5 ′ phosphorylated pairs of oligonucleotides EBI 1250/1264 and EBI 1631/1634 ( FIG. 14; the oligonucleotides were together briefly at 65 ° C and then slowly cooled to 4 ° C). The oligonucleotide pair EBI 1250/1264 codes for the 5′UTR of the rabbit β-globin gene and the oligonucleotide pair EBI 1631/1634 for the amino acids which lie between the AUG codon of pLink-2C and the SphI interface. After transformation into competent E. coli cells, a plasmid was isolated (pβ-2C), the structure of which is shown in FIG. 13. After linearization with the restriction enzyme BamHI, an in vitro transcription with T7 RNA polymerase produces an RNA which, from the transcription site to the EcoRV site, the start of the β-globin 5′UTR, which comprises 19 nucleotides, followed by the 52 nucleotides, the entire β-globin 5′UTR and nucleotides 3872 to 5219 of the HRV2 sequence.

C) In vitro transcription and translation

In vitro transcription of these plasmids after linearization with BamHI using T7 RNA polymerase was carried out as described (Duechler et al. (1989), loc. Cit.). At the end of the reaction time, the DNA was digested by adding DNase I (RNAse-free, Pharmacia), the proteins were removed by phenol extraction, and the RNA was precipitated with ethanol after adding a third volume of 8 M ammonium acetate. After repeated precipitation with ethanol, the RNA was then taken up in water and an aliquot was checked for quality by agarose gel electrophoresis. The running buffer contained 1% SDS. 1 µg of these RNAs were used for in vitro translation. The reaction contained in 20 ul 90 mM KCl, 0.3 mM MgCl ₂ , 10 mM creatine phosphate, 2 ul ³⁵ S-Trans label (ICN; 10 ul Ci / ul, 1000 Ci / mmol) and 11.5 ul rabbit reticulocyte lysate, which with micrococcal Nuclease (Sigma) had been treated. The rabbit reticulocyte lysate was prepared by the method of Jackson, R. and Hunt, T. (Meth. Enzymol., (1983), 96, 50-74) by treatment with micrococcal nuclease. After 1 hour at 30 ° C., the reaction was stopped by adding 1 μl of 200 mM methionine and an aliquot (1 μl) was applied to an SDS polyacrylamide gel. The gel was fluorographed after the run was completed.

FIG. 15A shows the translation products of the RNAs transcribed by the plasmids pHRV2 / 5'UTR-2C, pβ-2C, pLink-2C and pLink-2BC after electrophoretic separation. Surprisingly, several proteins were translated from each RNA.

The unexpected phenomenon of internal initiation To further examine RNAs from the plasmids transcribed under conditions conducive to presence lead a 5 ′ cap structure. Under cap structure one understands the nucleotides 7MeGpppG, which the 5 ′ end form the RNA. The transcription was therefore as above described, only that the reaction mixture 1 mM 7MeGpppG and 250 µM GTP contained. The translation was carried out as described above.

In Fig. 15B are the translation products of the RNAs provided with a Cap-structure, which had been transcribed from the plasmids pHRV2 / 5'UTR 2C-2C and pLink pLink-2BC, as shown by an electrophoretic separation. The RNAs of the plasmids pLink-2C and pLink-2BC were initiated here almost exclusively at those AUGs which are marked with black arrows in FIG. 13; the 37 kD or the 47 kD band can be recognized almost exclusively. The presence of the cap structure led to the initiation being pushed back. There are hardly any differences between the translation products of the capped structure RNA of the construction pHRV2 / 5′UTR and those with the uncapped RNA. The weak bands at 37 kD and 43 kD indicate initiation at AUG codons 449 and 611. The 5′UTR HRV2 can therefore, even if capped, not suppress the ability of the 2C RNA to initiate internally.

Example 3

Development of an in vitro system for Detection of the influence of a picornaviral proteinase on the Translation cap-provided RNAs and the Evidence of the inhibitory effect of Elastatinal to the HRV2 2A proteinase.

It has long been known that HRV2 and poliovirus Infections to inhibit translation of capped RNAs during a viral infection.

A) Expression of HRV2 2A proteinase

To produce the 2A proteins in E. coli, the plasmid pET3b (Studier, F. et al. (1990), Meth. Enzymol. 185, 60-89; Novagen, Madison, USA) was cut with BamHI and treated with alkaline phosphatase. Double-stranded DNA of the M13 phage M13 / 2A (Liebig et al. (1991), Proc. Natl. Acad Sci., 88, 5979-5983;) was cut with MluI and HindIII, the 448 bp fragment (HRV2 nucleotides 3138 to 3586 ) isolated from an agarose gel and ligated with the BamHI cut pET3b preparation. After transformation into competent E. coli cells, a plasmid called pET / 2A was isolated, the structure of which is shown in FIG. 16. This plasmid has the promoter for the T7 RNA polymerase, the translation signals and the first 14 amino acids of the gene 10 protein of the T7 phage followed by the last 7 amino acids of the capsid protein VP1 of HRV2, all 142 amino acids of the 2A proteinase of HRV2 and 2 stop codons. After transformation of this plasmid into the E. coli strain BL21 (DE3) LysS (Novagen, Madison, USA) and addition of IPTG (isopropylthiogalactoside) this expression block is expressed because this strain controls the gene for the T7 RNA polymerase on the chromosome of the lac operator / promoter. 2A proteinase can split off from the growing polypeptide chain; a mature, active 2A molecule with a molecular weight of 15 kD is therefore formed.

For this, a culture (50 ml) of E. coli BL21 (DE3) LysS with the plasmid pET / 2A is grown to an OD ₅₉₀ of 0.6; the expression block is induced with 0.2 mM IPTG and the culture is incubated for a further 2 hours at 37 ° C. The bacteria are then centrifuged, taken up in 0.7 ml sonication buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM DTT and 1 mM EDTA) and sonicated as described by Sommergruber et al. (1989, Virology, 169, 68-77). The cell debris is centrifuged off; the proteins in the supernatant are precipitated with 30% ammonium sulfate and centrifuged. The 30% ammonium sulfate precipitate is taken up in 250 µl sonication buffer and stored at 4 ° C.

B = translation with the addition of HRV2 2A proteinase

In Fig. 15B, the effect of the addition is a preparation 2A shown on the translation of a capped RNA of the plasmid pLink-2C. 3 μl of such an extract were added to a rabbit reticulocyte extract before adding the RNA and incubated at 30 ° C. for 10 min. In Fig. 15B, lane 7 are seen the translation products with a pre-treated rabbit reticulocyte lysate. Although a capped RNA was used, a translation pattern of an uncapped RNA is observed; the presence of 2A obviously changes the properties of rabbit reticulocyte lysate.

C) Inhibition of HRV2 2A proteinase

That the 2A proteinase is responsible for this change is supported by the result in FIG. 15B, lane 8. In this experiment, the 2A preparation was first spiked with 0.5 mM elastatinal; it could be shown that Elastatinal has an IC50 for 2A of 0.05 mM (Sommergruber et al., manuscript submitted and part of the unpublished German patent application P 41 36 433.0) and is therefore a very good inhibitor of 2A activity. The translation of a capped RNA is normal in rabbit reticulocytes; the inhibition of 2A prevents the translation properties from changing.

LEGENDS TO THE FIGURES

Fig. 1 shows a schematic representation of the HRV2 genome and the construction GR6. The closed arrows show the ATG codons at 449 and 611. The black block represents the coding region of HRV2. S shows the promoter of the T7 RNA polymerase. The following restriction sites are given: A, AccI; Av, AvaII; B, BamHI; Bn, BanII; H, HincII; H3, Hind III; N, NcoI; P, PstI; RI, Eco RI; RV, EcoRV; Sa, SalI; Sm, SmaI.

Fig. 2 shows a schematic representation of the HRV2 genome and the construction GR7. The symbols are identical to those in Fig. 1. A circle at a restriction site indicates that the restriction site has inevitably been lost during manufacture.

Fig. 3 shows a schematic representation of the HRV2 genome and the construction GR8. The symbols are identical to those in FIGS. 1 and 2.

Fig. 4 shows a schematic representation of the HRV2 genome and the construction GR9. The symbols are identical to those in FIGS. 1 and 2.

Fig. 5 shows a schematic representation of the HRV2 genome and the construction GR10. The symbols are identical to those in FIGS. 1 and 2.

Fig. 6 shows the oligonucleotides used for the construction of GR8.

Fig. 7A shows a compilation of the plasmids which carry deletions in the 5'UTR of HRV2. They are: p (193-1400) (GR6); p (248-1400) (GR5); p (412-1400) (GR7); p (448-1400) (GR8); p (525-1400) (GR9); p (610-1400) (GR10); the extent of the respective deletion is given in brackets. The symbols are identical to those in FIGS. 1 and 2.

FIG. 7B shows the fluorogram of a 12.5% polyacrylamide gel with translation products of RNAs from the plasmids shown in FIG. 5B. The limits of the respective deletions are given above the tracks.

In track C the result of a translation is without RNA addition shown. The right numbers (Unit: kilodalton) indicate selected molecular Size marker. The open arrow points to the 35 kD Translation product of the RNA from p (448-1400; GR8) and the closed arrow on the 30 kD translations product of the RNA of p (610-1400; GR10).

Fig. 8 shows the production route of the plasmid pUC18-2C * 3A.

Fig. 9 shows the oligonucleotides used for the introduction of a stop codon behind the 2C gene. The sequences from this area before and after the mutagenesis are also shown; the modified codon is highlighted in bold.

Fig. 10 shows the production route of the plasmids pSVL-2BC and pSVL-2C.

Figure 11 shows the oligonucleotides used to make pSVL-2BC and pSVL-2C; the AUGs are highlighted in bold.

Figure 12 shows the plasmids pLink-2BC and pLink-2C. The black arrow marks the synthetic AUG that was introduced before the two genes. The open arrows mark AUGs where an internal initiation is likely to take place. S shows the promoter of T7 RNA polymerase. The closed circle shows the position of the stop codon introduced by targeted mutagenesis. Abbreviation: Ba, BamHI.

Fig. 13 shows the constructions pHRV2 / 5'UTR-2C, pβ-2C, pLink-2C and pLink-2BC. The open rhombus represents the 5′UTR of the rabbit β-globin gene. The other symbols are identical to those in FIG. 12.

Figure 14 shows the oligonucleotides used to make the constructions of pHRV2 / 5'UTR-2C and pβ-2C; the AUG is highlighted in bold.

FIG. 15A shows the fluorogram of a 12.5% Proteingeles with translation products from ungecappten RNAs from the four plasmids pHRV2 / 2C 5 'UTR (lane 1), pβ-2C (lane 2), pLink-2C (lane 3) and pLink-2BC (lane 4). Track C shows the result of a translation without the addition of RNA. The numbers on the left (unit: kilodalton) indicate the translation products from the RNAs from the plasmids pHRV2 / 5′UTR-2C, pβ-2C, and pLink-2C. The numbers on the right (unit: kilodalton) indicate the additional translation products from the RNA of the plasmid pLink-2BC. Track C shows the result of a translation without the addition of RNA.

FIG. 15B shows the fluorogram of an 12.5% with Proteingeles translation products of RNAs from the following plasmids:
Lane 1: RNA from plasmid pHRV2 / 5′UTR-2C, without cap structure.
Lane 2: RNA from plasmid pHRV2 / 5′UTR-2C, with cap structure.
Lane 3: RNA from plasmid pLink-2BC, without cap structure.
Lane 4: RNA from plasmid pLink-2BC, with cap structure.
Lane 5: RNA from plasmid pLink-2C, with cap structure.
Lane 6: RNA from plasmid pLink-2C, without cap structure.
Lane 7: RNA from plasmid pLink-2C, with cap structure.
Lane 8: RNA from plasmid pLink-2C, with cap structure.

In lane 7 the rabbit reticulocyte extract was included a 2A proteinase preparation pretreated as in Example 3 is described. In lane 8 the Rabbit reticulocyte extract as in lane 7 above acts; in this case it was a 2A preparation used with 0.5 mM Elastatinal was.

The numbers (unit: kilodalton) indicate the respective translation products from the different RNAs.  

Fig. 16 shows the construction of pET / 2A. The block filled with horizontal lines represents the 14 amino acids of the T7 Gen10 protein, the open block the last 7 amino acids of the HRV2 VP1 gene and the straight, black block the 142 amino acids of the HRV2 2A gene. The binding site for the PC20 antiserum is represented by the square filled with a circle. S shows the T7 RNA polymerase promoter. The following restriction sites are given: B, BamHI; H, Hind III; M, MluI. A circle at a restriction site indicates that the restriction site was lost during manufacture.

Claims (11)

1. A method for analyzing the "host cell shut offs" caused by picornaviruses, characterized in that

• a) the rhinoviral 2C gene provided with a translation start and a translation stop codon is transcribed in vitro and the RNA provided with a cap structure
• b) is added to a translation mixture which is preincubated with a picornaviral 2A proteinase, is translated in vitro and
• c) the translated proteins are compared in comparison with translation products of the RNA with and without a cap structure.

2. The method according to claim 1, characterized in that that for in vitro transcription the HRV2 2C gene is used. 3. The method according to claim 1, characterized in that the plasmids for in vitro transcription pHRV2 / 5′UTR-2C, pβ-2C, pLink-2C and pLink-2BC be used. 4. The method according to claim 1, characterized in that for the pre-incubation of the translation mixture rhinoviral 2A proteinase, especially HRV2 2A Proteinase is used.   5. The method according to claim 1, characterized in that the translation mixture reticulocyte lysate or fractional portions of a Contains reticulocyte lysates. 6. The method according to claim 1 for analyzing the "host cell shut-offs" caused by rhinoviruses, characterized in that

• a) the HRV2 2C gene cloned in pLink-2C is transcribed in vitro and the RNA provided with a cap structure
• b) added to a translation mixture which is preincubated with an HRV2 2A proteinase and translated in vitro and
• c) the translated proteins are compared in comparison with translation products of the RNA with and without cap structure by means of gel electrophoresis and radioactive detection of the proteins.

7. Use of the method according to claim 1 for Detection of the cellular part of the "host cell proteins that cause shut-off. 8. Use of the method according to claim 1 for Isolation of inhibitors of the "host cell shut offs ". 9. Use of the method according to claim 1 for Isolation of inhibitors against the host cell shut-off "involved cellular and viral Proteins.   10. Use of the method according to claim 1 for Isolation of inhibitors against the rhinoviral 2A Proteinase. 11. Plasmids pHRV2 / 5′UTR-2C, pLink-2C, pLink-2BC, pSVL-2BC, pSVC-2C and pβ-2C.