RU2234535C2 - Substrate for determination of human immunodeficiency virus integrase activity - Google Patents

Abstract

FIELD: biochemistry, virology, medicine.

SUBSTANCE: substrate is designated for determination of activity of integrase of human immunodeficiency virus in vitro. Substrate is prepared by synthesis of oligonucleotide primers that are complementary to conservative region with the most similarity of genomes of different strains of human immunodeficiency virus and amplification of DNA sequence that is complementary to covalently closed ring forms of DNA of immunodeficiency virus, and by polymerase chain reaction. Then DNA product is prepared by repeated amplification with pair of nest primers and DNA product consists of two parallel oriented intact LTR-sequences with length 1200-1300 nucleotide pairs. Then prepared DNA product is labeled with Klenow's fragment and [α-³²P]-TTP for determination of activity of integrase in the 3'-processing reaction and using Klenow's fragment and a set of [α-³²P]-TTP for determination of activity of integrase in the insertion reaction.. The labeled DNA-product is used for determination of activity of integrase in vitro and the following analysis of products reaction in polyacrylamide gel and radioautography. The proposed substrate elevates the rate of determination of integrase activity in vitro in retention of specificity and identity of constructed substrate with natural substrate for integrase of human immunodeficiency virus.

EFFECT: improved assay method, valuable properties of substrate.

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Description

The invention relates to molecular biology, namely to enzymology, and can be used in virology to identify potential inhibitors of human immunodeficiency virus integrase.

The human immunodeficiency virus, a member of the family of retroviruses, causes a chronic infection of sensitive cells, using a replication strategy that sets it apart from other animal viruses. After the virus binds to the target cell, the viral genome in the form of single-stranded RNA is released into the cytoplasm of the host cell. During replication in a cell, viral RNA goes through the reverse transcription stage, which is carried out by a virus specific enzyme - reverse transcriptase, with the formation of double-stranded DNA with long terminal repeats (LTR) [1]. In the process of reproduction of the human immunodeficiency virus, at least three forms of provirus DNA are formed: a linear copy of the viral genome, which is a substrate for integration [2] and two ring forms with one or two LTRs, the functional role of which is still unclear [3]. The insertion of a linear form of provirus DNA into the genome of a host cell is carried out using a viral enzyme, integrase.

Viral integrase (IN) in several stages implements the insertion of provirus DNA into the genome of the host cell. In the first stage, two nucleotides are removed from the 3 ′ ends of the LTR of the newly synthesized proviral DNA. This site-specific endonuclease activity is called a 3’-processing reaction. The subsequent stages of integration occur after the complex penetrates from the cytoplasm into the nucleus. The nuclear integration step involves the formation of single-stranded cuts in the host DNA and ligation with the processed 3’-OH LTR ends — an insertion reaction.

To date, various radioactive double-stranded duplexes of 20-26 nucleotides in length are used to determine the in vitro integrase activity of retroviruses.

Known radioactive duplex used in the method for determining the activity of integrase [4].

Duplex is obtained by the synthesis of complementary deoxy oligonucleotides, the construction of hybridized complementary oligonucleotides, and radiolabeling. The resulting radiolabeled duplex is incubated with a purified integrase preparation and the resulting products are analyzed on a polyacrylamide gel.

Known fluorescently labeled duplex from the method of measuring the activity of retroviral integrase [5]. Measurement of the activity of retroviral integrase in the reaction of 3’-processing occurs by increasing the fluorescence intensity upon cleavage of the GT dinucleotide.

The disadvantages of these substrates include the fact that in an inhibitor analysis the results obtained using the test system for evaluating the in vitro integrase activity based on oligonucleotide duplexes do not correlate with the results obtained in the culture of HIV-infected cells [6]. This raises the question of an adequate interpretation of the current data on the functional role of integrase. As a result of this, today a search is underway for a test system for evaluating integrase activity based on the use of an extended DNA substrate flanked by LTR fragments that are recognized by the integrase of the human immunodeficiency virus.

Closest to the proposed invention is a substrate obtained by constructing a plasmid containing terminal sequences of proviral DNA, known from the method for determining the activity of HIV integrase [7, prototype]. The substrate is prepared as follows: the ends of the proviral DNA are amplified by polymerase chain reaction (PCR), in the presence of two pairs of HIV-specific primers, cloned into the vector plasmid pBKRSV (Stratagene, Canada), the resulting plasmid construct is generated, and the plasmid is linearized using restriction enzyme , a 5'-protruding end is obtained by means of an ExoIII nucleus and subsequently completed with T7 DNA polymerase using radioactively labeled nucleotides. The resulting radiolabeled substrate is used to determine the activity of integrase in the incorporation reaction, followed by separation of the reaction products in a polyacrylamide gel and radioautography. To determine the activity of integrase in the reaction of 3’-processing, the resulting plasmid construct is restricted to obtain a 107 bp fragment. followed by the introduction of a radioactive label.

The main disadvantage of this substrate is that it turns out to be different in length and composition, which affects the results of determining the activity of integrase in the reactions of 3’-processing and embedding. The use of this system for screening integrase inhibitors for the selection of potential anti-HIV drugs is difficult due to the multi-stage design of the substrate, the use of expensive inaccessible enzymes, as well as due to the additional stage of plasmid production in the bacterial system of E. coli. Also, due to the complexity of obtaining the substrate, the use of this method makes it difficult to construct substrates homologous to various strains of the human immunodeficiency virus to study the effect of natural nucleotide substitutions in the substrate on the functional activity of integrase.

An object of the invention is the construction of a substrate identical to the natural substrate of HIV integrase, which makes it possible to increase the speed of in vitro measurement of integrase activity and makes it possible to conduct a comprehensive study of the functional activity of integrase in several reactions (3’-processing and incorporation) on one substrate.

The technical task of the invention is solved by the selection and synthesis of oligonucleotide primers, so that only DNA fragments homologous to the circular forms of human immunodeficiency virus cDNA are amplified.

The substrate for determining the activity of integrase consists of a fragment of 1200-1300 bp complementary to intact parallel oriented long terminal repeat (LTR) HIV. The substrate is a non-infectious mini-copy of the viral genome, which allows you to study the integration process in conditions as close to natural as possible.

Long terminal repeats (size 600-650 bp) are located at the 5 ’and 3’ ends of the proviral DNA, and the orientation relative to each other is parallel. 5 ’the LTR region is the promoter-enhancer region of the virus and consists of many functional elements that carry out the basal and regulated expression of viral genes. HIV LTR is divided into three regions: U3, R and U5, which contain different regions for binding of cellular transcription factors.

The primary LTR sequence plays an important role in the integration process. Both 3’-OH end LTRs play an important role in the integration process. Both 3’-OH ends of the LTR carry a conserved CA dinucleotide at position 3,4, to which integrase specifically binds. After binding to the substrate, integrase cleaves two terminal GT dinucleotides from the 3’-OH end. The resulting DNA product with hydrolyzed 3’-OH ends of integrase integrates into the genome of the host cell. When using the proposed substrate as DNA for embedding, an excess of plasmid DNA is used.

Primers are selected in the conservative region of the genomes of different strains of human immunodeficiency virus so that they have 100% homology between different strains of human immunodeficiency virus, which allows amplification of an integrase substrate homologous to different strains of the virus using the same primers. The first pair of primers amplifies a sequence consisting of two parallel oriented LTR sequences from the 5 ’end of the env gene portion and from the 3’ end of the gag gene. The second pair of primers on the inner region of the obtained fragment directly amplifies the integrase substrate containing the dinucleotide CA at the ends, at positions 3, 4 of the 3’-OH end, which is specifically recognized by integrase. Next, the DNA fragment obtained is radioactively labeled with a Klenov fragment and is used to determine the activity of integrase in 3’-processing and embedding reactions. A positive reaction result was judged by the appearance of a product detected by polyacrylamide gel electrophoresis.

The substrate is prepared as follows.

At the first stage, two pairs of oligonucleotide primers are selected and synthesized, complementary to the conservative region of the greatest similarity of the genomes of various strains of human immunodeficiency virus type 1. Moreover, the orientation of the primers should be such that only the fragment complementary to the circular forms of DNA is amplified (Fig. 1). For example: S1-5'-GGGGTGGGAGCAGCATCTCG-3 '(at the end of the env gene) and S2-55-TCTTGCCGTGCGCGCTTCAG-3' (at the beginning of the gag gene). Then by re-amplification with a pair of nested primers directly at the ends of the LTR: N1-5'-ACTGGAAGGGCTAATTCACTCC-3 'and N2-5'-ACTGCTAGAGATTTTCCACACTG-3'; get a DNA product that contains a site - CA-dinucleotide at the 3'-OH end for specific recognition of integrase and consists of two parallel oriented intact LTR sequences of 1200-1300 bp long The resulting DNA product is labeled by means of a Klenov fragment and [α-p ³² ] TTP to determine the integrase activity in the 3'-processing reaction and by means of a Klenov fragment and a set of [α-p ³² ] dNTP to determine the integrase activity in the incorporation reaction.

A significant difference between the claimed substrate from the known prototype:

An extended 1200–1300 bp is used as a substrate. a DNA sequence homologous to human immunodeficiency virus cDNA, consisting of only long terminal repeats, which makes it possible to study the functional activity of integrase in vitro using a non-infectious copy of the HIV mini-genome.

Therefore, we can consider the claimed technical solution meets the criterion of "inventive step".

GRAPHIC MATERIALS

Figure 1 Schematic illustration of the construction of LTR-containing DNA substrate.

Figure 2 The dependence of the accumulation of the product in the reaction 3’-processing, catalyzed by integrase, from time to time: 1 - Control; 2-5 minutes; 3-15 minutes; 5-45 min; 6-90 minutes

Figure 3 The activity of recombinant HIV integrase in the insertion reaction using LTE-containing DNA substrate. Radio autograph with 4% polyacrylamide gel.

The essence of the technical solution is disclosed in specific examples of the method.

Example 1. The construction of an LTR-containing substrate to determine the activity of integrase based on the circular forms of DNA of the virus.

Operation 1. Isolation of proviral DNA of human immunodeficiency virus from peripheral blood lymphocytes of HIV-infected patients.

The peripheral blood sampling of patients to prevent the formation of a blood clot is carried out in test tubes containing 0.5 M EDTA pH 8.0, the final concentration of EDTA is 4 mm. Isolation of lymphocytes is carried out by separation of the patient’s blood in a ficoll-verographin gradient. 60 ml of a 15% solution of ficoll in deionized water are prepared and, with stirring, 20 ml of a 76% solution of verographin (Spofa, Czechoslovakia) are poured onto it. From this solution under the control of a hydrometer, solutions are prepared with a density of 1.097 g / ml and 1.075 g / ml. The prepared solutions are autoclaved at 121 ° C for 30 minutes. A gradient composed of 5 ml of a solution with a density of 1.097 g / ml and 5 ml of a solution with a density of 1.075 g / ml is layered with 10 ml of diluted Hanks solution in a 1: 2 ratio of blood. Centrifuged for 40 min at 400 g and a temperature of 22 ° C. A light fraction is taken that contains lymphocytes and monocytes, washed by centrifuging (2-3 min at 400 g) with Hanks solution.

Cells are resuspended in lysis buffer (10 mM Tris pH 8.3; 1 mM EDTA; 0.5% Tween 20; 0.5% NP-40) supplemented with Proteinase K (concentration 8 mg / ml) and incubated in an incubator at 56 ° C for 3 hours. Then phenol extraction and DNA reprecipitation with ethanol are carried out.

Operation 2. Isolation of total cellular DNA from a cell culture suspension.

The study uses a MT-4 cell culture highly sensitive to human immunodeficiency virus infected with HIV-1 / GKV4046, HIV-1 / Bru strains with an infectious titer of 10 ⁴ -10 ⁶ infectious units / ml and a concentration of 0.5 × 10 ⁶ cells / ml

Cells pre-washed with Hanks solution or DMEM (M) are resuspended in lysis buffer (10 mM Tris pH 8.3; 1 mM EDTA; 0.5% Tween 20; 0.5% NP-40) supplemented with proteinase K (concentration 8 mg / ml) and incubated in an incubator at 56 ° C for 3 hours. Then, phenol extraction and DNA reprecipitation with ethanol are carried out.

Operation 3. Polymerase chain reaction.

PCR is carried out in 50 μl of the reaction mixture containing 1.6 mm (NH ₄ ) ₂ SO ₄ , 6.7 mm Tris Hcl (pH 8.8), 1.5 mm MgCl ₂ , 0.01% tween-20, 0.1 mM dATP, dGTP, dCTP, dTTP, 2 units of Tag polymerase, 2 μM primers S1 and S2, and 5 μl of the extracted DNA from the infectious material are added. On top of the reaction mixture layered 30 μl of liquid paraffin. PCR consists of 30 cycles: 95 ° C for 1 min, 55 ° C for 1.2 min, 72 ° C for 1.5 min. At the end of the reaction, the final synthesis is carried out at 72 ° C for 5 minutes. After completion of the PCR, the aqueous phase is taken and mixed with an equal volume of a solution containing phenol: chloroform: isoamyl alcohol (25: 25: 1). After centrifugation at 14000 rpm for 30 s, the aqueous phase was collected and DNA was precipitated with ethyl alcohol and sodium acetate. The precipitate was dissolved in 20 μl of water. PCR is carried out under the same conditions with N1 and N2 nested primers, adding 5 μl of the solution obtained by amplification with S1 and S2 primers.

Step 4. Obtaining a radioactively labeled DNA substrate to measure integrase activity in a 3’-processing reaction.

A blunt-ended DNA fragment is labeled with a Klenov fragment by substituting a dTTP nucleotide at the 3’-hydroxyl end.

The substitution reaction is carried out in 50 μl of the reaction mixture containing 10 mm Tris Hcl (pH 7.5), 5 mm MgCl ₂ , 7.5 mm DTT, 33 μm [ ³² p] TTR (50 μCi), 1.2 units of the Big ( Maples) of a DNA polymerase fragment I and 1-3 μg of DNA substrate synthesized in a polymerase chain reaction. The mixture is incubated for 1 hour at 37 ° C. Upon completion of the reaction, add 5 μl of a solution containing 0.4 M EDTA, 60% glycerol, 0.1% bromphenol blue. The radiolabeled substrate was purified by preparative electrophoresis on a 4% polyacrylamide gel under native conditions, followed by radiography.

Step 5. Obtaining a radioactively labeled substrate DNA to measure integrase activity in the insertion reaction.

The DNA fragment is subjected to partial hydrolysis from the 3’-OH ends due to the 3’-5 ’exonuclease activity of the Maple fragment of E. coli DNA polymerase I. Then, after dNTP is added, the protruding 5’-ends are completed due to the 5’-3 ’polymerase activity of the Klenov fragment.

The reaction mixture contains 10 mM Tris HCl (pH 7.5), 5 mM MgCl ₂ , 7.5 mM DTT, 1.2 units of the Large (Maples) DNA polymerase I fragment, and 1-3 μg DNA substrate synthesized in the polymerase chain reactions. The mixture was incubated for 30 min at 37 ° C, then 33 μM dATP, dGTP, dCTP and [ ³² p] TTP (50 μCi) were added. Incubate the mixture for 60 minutes at 37 ° C. The radiolabeled substrate was purified by preparative electrophoresis on a 4% polyacrylamide gel under native conditions, followed by radiography.

Step 6. Elution of the radiolabeled DNA substrate from the polyacrylamide gel.

The strip corresponding to the DNA substrate according to radioautography, is cut out from the gel and 1 volume of an elution buffer containing 0.5 M NH ₄ CH ₃ CO ₂ , 1 mM EDTA (pH 8.0) is added to it. Incubated during rotation (100 rpm) for 12 hours at 37 ° C. Centrifuge for 10 min at 10,000 g. Collect the supernatant and precipitate the DNA with ethanol. The precipitate was dissolved in 50 μl of 50 mM Tris-Hcl buffer (pH 8.0), 2 mM EDTA (pH 8.0). The concentration of the obtained substrate DNA is measured spectrophotometrically.

Example 2. Determination of the reaction rate of 3’-processing catalyzed by HIV IN.

Integrase activity is measured by cleavage of the radiolabeled GT * dinucleotide from the 3 'end of both DNA substrate chains. The reaction mixture (45-50 μl) contains: 10 mM HEPES, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl ₂ , 80 mM NaCl, 10% PEG 6000, 70 ng recombinant HIV integrase and 2 nM radiolabeled substrate.

The reaction mixture was placed on ice for 10 minutes and then incubated for 60 minutes at 37 ° C. During the incubation, 10 μl aliquots are taken every 10-20 minutes. The reaction products are analyzed electrophoretically using 20% PAAG under denaturing conditions (7.5 M urea), followed by radioautography (Fig.2). To quantify the reaction product, gel fragments corresponding to the product on a radio autograph are cut out and radioactivity is measured on a counter (Mark-III).

Example 3. Determination of the kinetic parameters of the reaction of 3’-processing, catalyzed by integrase.

When determining the kinetic parameters, the reaction mixture (45-50 μl) for the 3'-processing reaction contains: 10 mM HEPES, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl ₂ , 80 mM NaCl, 10% PEG 6000, 70 ng recombinant HIV integrase and variable concentrations of radioactively labeled substrate (10 ^-11 -10 ^-8 M).

The reaction mixture was placed on ice for 10 minutes and then incubated for 60 minutes at 37 ° C. During the incubation, 10 μl aliquots are taken every 10-20 minutes. The reaction products are analyzed as in example 2.

Km reactions of 3 'processing are found according to the Michaelis-Menten equation using the Origin Professional 5.0 program (Micrococal inc.). All measurements are performed on linear sections of the dependences of the accumulation of products on time and enzyme concentration. The error in determining constants does not exceed 10-30%. For the recombinant HIV integrase used in the work, the Km value in the 3'-processing reaction for the radiolabeled substrate is (1.2 ± 0.3) · 10 ^-10 M. All measurements are carried out on linear sections of the dependence of product accumulation on time and enzyme concentration. The error in determining constants does not exceed 10-30%.

Example 4. Inhibition of recombinant HIV integrase by aurintricarboxylic acid in a 3’-processing reaction using DNA substrate.

When determining the concentration of aurin tricarboxylic acid at which the relative activity of the enzyme is 50% ([I] ₅₀ ); the reaction mixture (45-50 μl) contains: 10 mM HEPES, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl ₂ , 80 mM NaCl, 10% PEG 6000, 70 ng recombinant HIV integrase, 2 nM radiolabeled DNA substrate and aurintricarboxylic acid in various concentrations (10 ^-7 -10 ^-3 M)

The reaction mixture was placed on ice for 10 minutes and then incubated for 60 minutes at 37 ° C. During the incubation, 10 μl aliquots are taken every 10-20 minutes. The reaction products are analyzed as in Example 2.

[I] ₅₀ aurintricarboxylic acids are found graphically by constructing experimental data in the coordinates of the concentration of ATK on the relative reaction rate of 3'-processing. [I] ₅₀ aurintricarboxylic acid in the reaction of 3'-processing, catalyzed by integrase, for this DNA substrate is (7.2 ± 0.7) · 10 ^-7 M.

Example 5. Measurement of HIV integrase activity in the insertion reaction.

The integrase activity in the embedding reaction is evaluated by the amount of incorporation of the radioactively labeled substrate DNA into plasmid pUC19 (FIG. 3). The reaction mixture (45-50 μl) contains: 10 mM HEPES, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl ₂ , 80 mM NaCl, 10% PEG 6000, 0.5 μg pUC19, 70 ng integrase and 1 nM radioactive labeled substrate. The reaction is carried out at 37 ° C for 30-90 minutes. The reaction products are analyzed electrophoretically using 4% PAG under native conditions, followed by radioautography. To quantify the reaction product, gel fragments corresponding to the product on a radio autograph are cut out and radioactivity is measured on a counter (Mark-III).

LIST OF REFERENCES

1. Fausi A.S. // Science. 1988. V.239. P.617-622; Weiss R.A. // Science. 1993. V.260. P.1273-1279; De Clercq E. // Clinical Microbiology Reviews V. 8, No. 12, P.200-239.

2. Hindmarsh P., Leis J. // Microbiology and Molecular Biology Reviews, Dec. 1999, p. 836-843.

3. Gashnikova N.M. and others // DAN, No. 4, 2001

4. Chow S.A. (1997) In vitro assays for activities of retroviral integrase // Methods, 12, 306-317.

5. US patent No. 5763181.

6. O’Brien C. // Science. 1994. V.266. R. 1946; Sourgen F. et all // Eur. J. Biochem. V.240. P.765-73.

7. Activity of recombinant HIV-1 integrase on mini-HIV DNA. Cherepanov P., Surratt D., Toelen J., Pluymers W., Griffith J., De Clercq E. and Debyser Z. (1999) Nucleic Acids Res., 27, 2202-2210.

Claims (3)

1. The substrate for determining the activity of integrase of the human immunodeficiency virus, containing the end sequences of proviral DNA and labeled with a radioactive label, characterized in that as a substrate using a fragment of proviral DNA HIV-1, consisting of two parallel oriented intact LTR sequences with a length of 1200-1300 p .n. 2. The substrate according to claim 1, characterized in that the long terminal repeats (size 600-650 bp) are located at the 5 ’and 3’ ends of the proviral DNA. 3. The substrate according to claim 1, characterized in that an excess of plasmid DNA is used as DNA for embedding.

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