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WO2010130864A1 - Transcriptase inverse du vih-1 de groupe o modifiée - Google Patents

Transcriptase inverse du vih-1 de groupe o modifiée Download PDF

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Publication number
WO2010130864A1
WO2010130864A1 PCT/ES2010/070320 ES2010070320W WO2010130864A1 WO 2010130864 A1 WO2010130864 A1 WO 2010130864A1 ES 2010070320 W ES2010070320 W ES 2010070320W WO 2010130864 A1 WO2010130864 A1 WO 2010130864A1
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retrotranscriptase
amino acid
nucleic acid
acid sequence
seq
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Luis MENÉNDEZ ARIAS
Tania Matamoros Grande
Mª del Mar ÁLVAREZ GARCÍA
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Consejo Superior de Investigaciones Cientificas CSIC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses

Definitions

  • the present invention falls within the field of biotechnology. More specifically, it refers to isolated retrotranscriptases of a human immunodeficiency virus type 1 (HIV-1) of group O and modified that have high fidelity of copy and thermostability, as well as its use to carry out the back transcription, amplification or sequencing of a template nucleic acid.
  • the invention also relates to a method for obtaining said retrotranscriptases.
  • RT retrotranscriptase
  • reverse transcriptase The enzyme responsible for the replication of the genome of retroviruses.
  • This enzyme converts the single stranded genomic RNA into double stranded DNA capable of integrating into the genome of the host cell [reviewed by Telesnitsky and Goff. In Retroviruses. Coffin et al. (ed.), p. 121-160, CoId Spring Harbor Lab. Press, Plainview USA, 1997]. It is a polymerase capable of synthesizing DNA, using RNA or DNA as a template, interchangeably.
  • RT has endonuclease activity (RNase H), which allows the RNA template to be degraded during the RNA-dependent DNA synthesis process.
  • retrovirus RTs are useful enzymes for obtaining complementary DNA (cDNA) from messenger RNA, which once amplified by conventional techniques [polymerase chain reaction (PCR), for example] can be used to detect gene expression in organisms or tissues. From cDNA libraries it is possible to select gene transcripts that once amplified by PCR can be cloned into plasmids or viral vectors. For these applications it is interesting to have RTs that have greater thermal stability, that is, to retain polymerase activity at relatively high temperatures, since under these conditions the secondary structure levels in the RNA and It would improve the amplification process.
  • PCR polymerase chain reaction
  • RTs are enzymes that lack exonuclease activity and therefore, are polymerases with a relatively high error rate, so an increase in their fidelity would be desirable if what is intended is to clone products derived from the generated cDNA during the retrotranscription.
  • RTs from various retroviruses have been isolated and purified.
  • AMV bird myeloblastosis virus
  • MMV Moloney virus of the mouse leukemia
  • Virus Res 2008; 134: 186-202 of the equine infectious anemia virus (EIAV), of the feline immunodeficiency virus (IVF), of the bovine immunodeficiency virus (BIV), of the mouse mammary tumor virus (MMTV), of the bovine leukemia virus (BLV), and of foam-retrovirus (reviewed in Hizi and Herschhorn. Virus Res 2008; 134: 203-220); in addition to the RTs of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) and simian immunodeficiency (SIV).
  • HMV-1 human immunodeficiency virus type 1
  • HV-2 type 2
  • SIV simian immunodeficiency
  • the most commercially used RTs in amplification reactions are those of MLV, AMV and HIV-1.
  • the reference enzyme in this type of analysis is a "wild-type" variant, which is phylogenetically classified as belonging to subtype B (group M).
  • the HIV-1 RT (subtype B) is more stable than the MLV RT at temperatures above 45 or V "and.
  • the furthest are those belonging to the O group, which are characterized by having about 20% different amino acids when compared with prototype "wild-type" sequences of subtype B (Qui ⁇ ones-Mateu et al.
  • Group O "wild-type" RTs are naturally resistant to nevirapine and other non-nucleoside-like inhibitors and present greater stability in the presence of urea than subtype B RTs (Menéndez-Arias et al. J. Biol. Chem. 2001; 276: 27470-27479). However, its expression and purification is difficult due to the poor performance of the procedures described to date.
  • RTs obtained by directed mutagenesis have allowed us to study the role of different amino acid changes on copy fidelity, as well as the inactivation of the RNase H activity of the HIV-1 RT (subtype B).
  • the context of the sequence has proven to be of great importance (Taube et al. Eur. J. Biochem. 1997; 250: 106-114), so it seems unlikely that a mutation analyzed in the RT of HIV- 1 subtype B can exert the same effect in a variant of HIV-1 phylogenetically remote as the RT of HIV-1 of group O.
  • the present invention relates to retrotranscriptases (RTs) isolated from a virus of human immunodeficiency type 1 (HIV-1) of group O and modified that have a high fidelity of copy and thermostability, as well as its use to carry out Ia retrotranscription, amplification or sequencing of a template nucleic acid.
  • RTs retrotranscriptases isolated from a virus of human immunodeficiency type 1 (HIV-1) of group O and modified that have a high fidelity of copy and thermostability, as well as its use to carry out Ia retrotranscription, amplification or sequencing of a template nucleic acid.
  • HIV-1 human immunodeficiency type 1
  • the invention also relates to a method for obtaining said RTs.
  • Retrovirus RTs are useful enzymes, for example, for obtaining complementary DNA from messenger RNA, which once amplified can be cloned into a vector. It is desirable that the RTs used for this purpose have high thermal stability, since under these conditions the levels of secondary structure in the RNA would decrease and the amplification process would improve. However, RTs are enzymes that lack error-correcting exonuclease activity and therefore have a relatively high error rate, so an increase in their fidelity would be desirable in order to be used for this purpose.
  • mutants of an isolated RT of a group O HIV-1 (group O HIV-1 RT) whose p66 subunit has been modified by replacing the valine 75 with isoleucine (mutation V75I) are described ), so that an increase in its fidelity of copy is observed with respect to that of the unmodified RT.
  • group O HIV-1 RTs carrying this mutation have higher copy fidelity and thermostability than the RTs present in isolates or strains of group M HIV-1 subtype B (HIV-1 RT subtype B), currently employed for the same purpose.
  • the V75I mutation in the RT of group O HIV-1 leads to a loss of thermostability with respect to the unmodified RT.
  • a second amino acid change (mutation) consisting in the replacement of glutamic acid of position 478 of the p66 subunit by glutamine allows to increase its thermostability with respect to the RT that only presents The V75I mutation. Therefore, the present invention provides RTs with high thermostability and fidelity of copy, appropriate to carry out the retrotranscription, amplification or sequencing of a template nucleic acid.
  • a first aspect of the present invention relates to a modified group O HIV-1 RT comprising an amino acid sequence that has a mutation that modifies said amino acid sequence in the residue corresponding to position 75 of SEQ ID NO : 1, in which the modified RT has an increased copy fidelity, in relation to the corresponding unmodified RT.
  • said RT comprises an amino acid sequence that has a mutation so that the residue corresponding to position 75 of SEQ ID NO: 1 of this amino acid sequence is isoleucine.
  • a preferred embodiment of this aspect of the invention refers to an RT of the modified group O HIV-1, which comprises an amino acid sequence, mutated as described above, and which also has another mutation that modifies said amino acid sequence in the residue corresponding to position 478 of SEQ ID NO: 1, in which the modified RT has an increased thermostability in relation to the modified RT in the residue corresponding to position 75 of SEQ ID NO: 1.
  • said RT comprises an amino acid sequence that has a mutation so that the residue corresponding to position 478 of SEQ ID NO: 1 of this amino acid sequence is glutamine.
  • RT of the invention refers to a modified RT according to any of the characteristics described above in this description.
  • the amino acid sequence comprised by the RT of the invention has an identity of at least 90%, more preferably, of at least 95% and, even more preferably, of at least 98% with SEQ ID NO: 1.
  • a preferred embodiment of this aspect of the invention refers to a modified group O HIV-1 RT comprising SEQ ID NO: 2.
  • Another preferred embodiment of this aspect of the invention refers to a modified group O HIV-1 RT comprising SEQ ID NO: 3.
  • HIV-1 and HIV-2 Human immunodeficiency viruses
  • retroviridae Retrovirus
  • HIV-1 has been classified into three groups: M, O and N.
  • At least 10 AK subtypes are known from the M group, in addition to multiple recombinants of these subtypes (Buonaguro et al. J. Virol 2007; 81: 10209-10219; Ram ⁇ rez et al. Virus Res. 2008; 134: 64-73).
  • HIV-1 group O The first isolate of HIV-1 group O was obtained from infected patients in 1987, and its nucleotide sequence was published three years later (De Leys et al. J. Virol. 1990; 64: 1207-1216).
  • variants of HIV-1 group O for example, strain MVP5180 / 91
  • NIH AIDS Research & Reference Reagent Program http://www.aidsreagent.org
  • the HIV-1 RT is a multifunctional enzyme that exhibits RNA and DNA-dependent DNA polymerase activities and endonuclease or ribonuclease H (RNase H) activity. It is a heterodimer consisting of two subunits, called p66 and p51.
  • the p51 subunit has 440 amino acids while the p66 has 560.
  • p51 and p66 share the same primary structure in their first 440 residues, the different subdomains contained in their respective sequences are oriented differently in both subunits.
  • the p66 subunit participates in an important way in the formation of the groove in which the complexer-primer interacts either RNA / DNA or DNA / DNA, in addition to the active center involved in the catalysis that leads to the formation of phosphodiester bonds between deoxyribonucleotides 5 'triphosphate (dNTP) and the free 5' end of the DNA strand being synthesized.
  • dNTP deoxyribonucleotides 5 'triphosphate
  • the p51 subunit has a mainly structural function.
  • the p51 and p66 subunits are generated from the processing of the Gag-Pol polyprotein. Although it is not known exactly which are the intermediaries in the Gag-Pol processing that lead to the formation of the functional p66 / p51 heterodimer, it is known that in vitro, p66 / p66 homodimers can be converted into p66 / p51 heterodimers by the action of Ia viral protease
  • This enzyme makes an endoproteolytic cut, between amino acids 440 and 441, in one of the two chains that form the homodimer.
  • RT of group O HIV-1 refers to an isolated retrotranscriptase of a human type 1 immunodeficiency virus of group O.
  • HIV-1 RT of subtype B refers to an isolated retrotranscriptase of a human immunodeficiency virus type 1 of group M subtype B.
  • retrotranscriptase isolated from human immunodeficiency virus type 1 refers to a protein with retrotranscriptase activity that is substantially or completely free of the components that normally accompany or interact with it in its natural form. It can be obtained, for example, by amplification by means of the polymerase chain reaction (PCR) of the nucleotide sequence encoding the amino acid sequence of the p66 subunit of the retrotranscriptase from the viral RNA obtained from an isolate of the virus of Ia human immunodeficiency type 1, and subsequent cloning in an expression vector.
  • PCR polymerase chain reaction
  • SEQ ID NO: 1 is the amino acid sequence of the p66 subunit of the RT isolated from a strain or an isolate of HIV-1 group O.
  • the p66 subunit of the RT isolated from other strains or other isolates of HIV-1 Group O has an identity in its amino acid sequence with SEQ ID NO: 1 of up to 90%.
  • amino acid sequence or “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may or may not be chemically or biochemically modified.
  • identity refers to the proportion of identical amino acids between two amino acid sequences that are compared. The percentage of identity existing between two sequences can be easily identified by a person skilled in the art, for example, with the help of an appropriate computer program to compare sequences.
  • copy fidelity refers to the accuracy of the polymerization, or the ability of the RT to discriminate between the correct and incorrect substrates (for example, nucleotides) when it is synthesizing nucleic acid molecules that are complementary to a nucleic acid mold.
  • the fidelity of the RT of the present invention can be analyzed by different types of analysis, such as, but not limited to, fidelity assays in cell cultures or in vitro fidelity assays (genetic or biochemical).
  • Fidelity assays in cell cultures are based on the use of a retroviral vector that contains a marker gene (such as, but not limited to, lacZa, thymidine kinase or neomycin), in addition to all the elements necessary for transcription to take place. of viral proteins, their encapsidation, and the synthesis of proviral DNA.
  • This vector from which essential genes such as gag, pol, env, and / or accessory genes have been removed, is introduced into a packaging cell line that provides these genes in trans, and the virus produced is used to infect target cells. In these cells, the vector is able to complete a round of replication and integrate into the genome of the target cell to form provirus.
  • a single round of replication involves a stage of RNA transcription by the cellular RNA polymerase Il, and a retrotranscription stage that includes the RNA-dependent DNA synthesis of a proviral DNA chain, and the subsequent synthesis of the second DNA chain through the DNA-dependent DNA polymerase activity of the RT. Since the provirus is unable to express essential viral proteins, no additional replication cycles occur. By selecting an appropriate cell culture, the natural or mutant phenotypes of the marker gene can be detected, and thus obtain mutation frequencies.
  • the purified RT is used for the determination of kinetic constants on an RNA or DNA template, under conditions determined (pH, substrate concentration, etc ...) - In this way the kinetic parameters of the DNA copy fidelity (dependent on RNA or DNA) of the RT can be obtained, both in stationary and pre-stationary state.
  • the biochemical tests of erroneous incorporation in the steady state are based on the determination of the kinetic constants (k cat and K m ) for the incorporation of nucleotides at the 3 'end of a primer and provide an estimate of the selectivity of the RT by the nucleotide
  • the determination of the kinetic parameters is carried out by measuring the incorporation of nucleotides at the 3 'end of the primer, previously labeled at its 5' end with [ ⁇ 32 P] ATP, in the presence of different concentrations of dNTP, once formed the RT / mold-primer binary complex.
  • the resulting products are analyzed by electrophoresis in polyacrylamide gels.
  • the data obtained are consistent with the Michaelis-Menten equation, and the parameters k cat (incorporation rate) and K m (Michaelis-Menten constant) are determined for the correct and incorrect nucleotides.
  • the efficiency of erroneous incorporation ⁇ f ⁇ nc is defined as the relationship between the catalytic efficiency (/ Cca ⁇ / Km) obtained for the incorrect nucleotide and the catalytic efficiency obtained for the correct nucleotide.
  • greater fidelity of the RT implies a lower efficiency of erroneous incorporation.
  • the RT In order for the fixing of an error in the nascent DNA to occur, the incorporation of an incorrect nucleotide is not sufficient, the RT must also be able to elongate the missing end that is generated as a result of this erroneous incorporation.
  • This fidelity measurement is carried out by extension tests of missing ends. In these tests, the steady state kinetic constants are calculated for the incorporation of a correct nucleotide on two types of complexer-primer complex: the complex with the 3 'end properly matched and the same complex with the 3' end missing.
  • the extension efficiency of missing ends (f ext ) is defined as the ratio between k ca t / K m obtained for the extension of the missing end and the one obtained for the extension of the correctly paired end.
  • Biochemical tests in the pre-stationary state examine the ability of RT to bind and incorporate dNTP at very short times (such as, for example, in the order of milliseconds). In this way, it is possible to calculate the affinity constant (K d ) for the interaction between the dNTP and the binary complex RT / mold-primer, and the polymerization constant (/ c po / ).
  • K d affinity constant
  • the efficiency of erroneous incorporation and extension of missing ends is determined from the values of k po ⁇ / Kd obtained for the incorporation of correct or incorrect nucleotides, or those obtained for the incorporation of correct nucleotides on mold-primer complexes containing a 3'OH end paired or missing.
  • the most used genetic assays are usually carried out using as a template-primer of the DNA synthesis reaction, the double stranded DNA of phage M13mp2, from which the zone corresponding to the lacZ gene of One of the threads.
  • the DNA synthesis reaction is carried out in the presence of RT and high concentrations of dNTPs. After the bacterial transformation with the reaction product, the mutants are identified as blue / white plates in culture medium containing X-GaI (5-bromo-4-chloro-3-indole- ⁇ -galactopyranoside) and IPTG (isopropyl - ⁇ -thio-galactopyranoside).
  • the result is a dark blue plate.
  • the introduction of one or several errors implies the partial or total loss of the ⁇ -complementation, which translates into light blue or white plates.
  • the DNA recovered from these plates can be sequenced, to determine exactly the number, type and position in the genome of the mutations introduced by the RT.
  • Another type of genetic assays are reversal assays. These tests are based on the use of molds that contain an inactivating mutation (typically, termination codons or insertions of a nucleotide). The reversal of the mutation results in the correction of the error and therefore in the restoration of the activity of the marker gene.
  • an inactivating mutation typically, termination codons or insertions of a nucleotide.
  • the "increase in copy fidelity" can be measured by comparing parameters indicative of the copy fidelity of the RTs of the invention and the unmodified RT. These parameters can be analyzed, for example, but not limited, by any of the methods previously described in this document.
  • An RT with an "increased” or “increased” copy fidelity is defined as an RT that has a significant increase or increase (applying statistical criteria) in the copy fidelity with respect to the unmodified RT, typically of at least , 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times, and even more preferably at least 4 times.
  • an increase in fidelity is considered when the value obtained for the modified RT is significantly higher than that of the unmodified RT (applying statistical criteria), typically, at least, 1, 5 times, preferably, at least 2 times, more preferably at least 3 times, and even more preferably at least 4 times.
  • increased fidelity is considered when there is a significant increase (applying statistical criteria) in the frequency of mutant obtained by the modified RT, typically of at least 50% (1, 5 times), preferably, at least 2 times, more preferably at least 3 times, and even more preferably at least 4 times.
  • thermostability refers to the stability shown by an RT when it is subjected to an elevated temperature, for example, typically, a temperature of at least 45 0 C, preferably at least 55 0 C, more preferably of at least 60 0 C and even more preferably of at least 65 or Vy.
  • thermostability of the RT of the present invention can be analyzed by different types of analysis.
  • the thermostability of the RT of the present invention can be estimated, for example, but not limited, by measuring the specific DNA polymerase activity at different elevated temperatures, using a template-primer complex, analyzing the residual RT activity after subjecting the enzyme to an incubation at elevated temperature during different time intervals, analyzing the half-life of the RT or measuring the quantity and / or the length of the product synthesized by the RT at a high temperature.
  • the "half-life" of a protein is a parameter that allows measuring its thermostability.
  • the half-life of the RT activity refers to the time in which the polymerase activity (dependent on RNA or DNA) is reduced by half when the synthesis reaction (dependent on RNA or DNA) is carried out at a high temperature.
  • thermostability of a RT are the quantity and / or the length of the product synthesized by the RT when the synthesis reaction is carried out at a high temperature.
  • an estimate of the stability of the RT can be obtained, analyzing the quantity of the product obtained by means of the RT in an RT-PCR.
  • a retrotranscription reaction can be performed first using as a nucleic acid total RNA template at an elevated temperature, and then the complementary DNA obtained from the retrotranscription can be amplified the reaction product by
  • a "high temperature synthesis reaction”, as used in the present description, refers to a reaction and, more preferably, a back transcription (or reverse transcription) reaction, which is carried out at a temperature of at least , 45 0 C, preferably at least 55 0 C, more preferably at least 60 0 C and even more preferably at least 65 0 C.
  • the RT of the invention may show an increase in thermostability in the presence or absence of the template nucleic acid. It is known in the state of the art that RTs are typically more stable in the presence of the template nucleic acid.
  • the term "increased thermostability”, as used in the present description refers to the fact that the modified group O HIV-1 RT, which comprises an amino acid sequence, mutated in the residues corresponding to positions 75 and 478 of SEQ ID NO: 1, retains its activity at a higher synthesis reaction temperature, or acts longer at the same temperature, than the RT that only has a mutation in the residue corresponding to position 75 of the SEQ ID NO: 1.
  • thermostability is defined as an RT that has a significant increase or increase (applying statistical criteria) in the thermostability, typically at least 1.5 times, more preferably at least , 2 times, and even more preferably, at least 3 times, and even more preferably at least 4 times, with respect to the RT which only has a mutation in the residue corresponding to position 75 of the SEQ ID NO: 1.
  • a significant increase or increase typically at least 1.5 times, more preferably at least , 2 times, and even more preferably, at least 3 times, and even more preferably at least 4 times, with respect to the RT which only has a mutation in the residue corresponding to position 75 of the SEQ ID NO: 1.
  • the RT in an RT-PCR it is considered that the RT has a fidelity of copy increased when the quantity of product obtained by means of RT-PCR has a significant increase or increase (applying statistical criteria), typically of at least 1.5 times, preferably of at least 3 times, and even more preferably of, at least 4 times, with respect to the RT that only has a mutation in the residue that corresponds to position 75 of SEQ ID NO: 1.
  • mutation refers to a substitution of one amino acid for another different amino acid.
  • the individual amino acids in a sequence are represented here as XN, in which X is the amino acid in the sequence (designated by the code of a letter universally accepted in the amino acid nomenclature), and N is the position in the sequence.
  • Point substitution mutations in an amino acid sequence are represented here as XiNX 2 , in which Xi is the amino acid in the non-mutated protein sequence, X 2 is the amino acid in the mutated protein sequence, and N is the position in The amino acid sequence.
  • the amino acid sequence comprised by the RT of the invention can be obtained by genetic or recombinant engineering techniques well known in the state of the art. They can be obtained, for example, by mutating the RT gene by randomized or directed mutagenesis. Preferably, the mutation is introduced into the amino acid sequence by means of a suitable codon change in the polynucleotide encoding the RT. More preferably, the mutants of the present invention can be obtained by the oligonucleotide-directed mutagenesis technique. This technique consists in the ringing of a complementary oligonucleotide (except for
  • oligonucleotide-directed mutagenesis can be carried out, for example, by PCR.
  • Another aspect of the present invention refers to a polynucleotide that encodes the amino acid sequence included in the RT of the invention.
  • nucleotide sequence refers to a polymeric form of nucleotides of any length, which may be, or no, chemically or biochemically modified.
  • Another aspect of the present invention refers to a vector comprising the polynucleotide that encodes the amino acid sequence included in the RT of the invention.
  • the vector can be, for example a cloning vector or an expression vector.
  • said vector is an appropriate vector for the expression and purification of the RT of the invention.
  • cloning vector refers to a DNA molecule in which another DNA fragment can be integrated, without losing the capacity for self-replication.
  • expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes.
  • expression vector refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a cell, called host cell. Said nucleic acid is, in general, operatively linked to control sequences.
  • expression refers to the process by which a polypeptide is synthesized from a polynucleotide. It includes the transcription of the polynucleotide in a messenger RNA (mRNA) and the translation of said mRNA into a protein or a polypeptide.
  • mRNA messenger RNA
  • host cell refers to any prokaryotic or eukaryotic organism that is the recipient of an expression vector, of cloning or of any other DNA molecule.
  • control sequence refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked. It is intended that the term “control sequences” includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous. Examples of control sequences are, for example, but not limited to, promoters, transcription initiation signals, transcription termination signals, polyadenylation signals or transcriptional activators.
  • operatively linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended manner.
  • a control sequence "operatively linked" to a polynucleotide is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • promoter refers to a region of DNA, generally “upstream” or “upstream” of the starting point of Ia transcription, which is capable of initiating transcription in a cell. This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters.
  • control sequences depend on the origin of the cell in which the nucleic acid is to be expressed.
  • prokaryotic promoters include, for example, but not limited to, promoters of the trp, recA, lacZ, lacl, tet, gal, trc, or tac genes of E. coli, or the promoter of the ⁇ -amylase gene of B. subtilis
  • promoters of the trp, recA, lacZ, lacl, tet, gal, trc, or tac genes of E. coli or the promoter of the ⁇ -amylase gene of B. subtilis
  • Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.
  • a preferred embodiment of this aspect of the invention refers to a vector comprising a polynucleotide that encodes the amino acid sequence included in the RT of the invention, wherein said polynucleotide is operatively linked to at least one control sequence of the Ia list comprising:
  • a) a promoter b) a transcription initiation signal, c) a transcription termination signal, d) a polyadenylation signal, or e) a transcriptional activator.
  • a preferred embodiment of this aspect of the invention refers to a vector comprising a polynucleotide that encodes the amino acid sequence comprised by the RT of the invention, wherein said polynucleotide is linked in reading phase to a nucleotide sequence encoding a purification tag.
  • purification tag or "affinity tag”, as used in the present description refers to an amino acid sequence that has been incorporated (generally, by genetic engineering) into a protein to facilitate its purification.
  • the tag which can be another protein or a short amino acid sequence, allows the purification of the protein, for example, by affinity chromatography.
  • Some examples of purification labels known in the state of the art are, for example, but not limited to: the calmodulin-binding peptide (CBP), the glutathione-S-transferase enzyme (GST) or a tail of histidine residues .
  • the purification tag consists of at least 6 histidine residues.
  • first vector of the invention refers to a vector comprising a polynucleotide that encodes the sequence included in the RT of the invention, where said vector has any of the characteristics described above in the present description.
  • Another preferred embodiment of this aspect of the invention refers to a vector that comprises a polynucleotide that encodes the amino acid sequence comprised by the RT of the invention and that, in addition, comprises a polynucleotide that codes for a protease capable of performing an endoproteolytic cut. in the amino acid sequence included in the RT of the invention between the residues corresponding to positions 440 and 441 of SEQ ID NO: 1.
  • second vector of the invention refers to a vector comprising a polynucleotide encoding the sequence included in the RT of the invention, wherein said vector has any of the characteristics of the first vector of the invention, and also comprising a polynucleotide encoding a protease capable of making an endoproteolytic cut in the amino acid sequence comprised in the RT of the invention between the residues corresponding to positions 440 and 441 of SEQ ID NO: 1.
  • the protease capable of performing an endoproteolytic cut in the amino acid sequence included in the RT of the invention between the residues corresponding to positions 440 and 441 of SEQ ID NO: 1 is the HIV-1 protease, a variant of the HIV-1 protease or a fragment thereof, as long as said variant or said fragment is functionally equivalent.
  • HIV-1 protease refers to a protease isolated from a human immunodeficiency virus type 1.
  • protease isolated from human immunodeficiency virus type 1 refers to a protease capable of making an endoproteolytic cut in the amino acid sequence included in the RT of the invention between the residues corresponding to positions 440 and 441 of SEQ ID NO: 1, which is substantially or completely free of the components that normally accompany or interact with it in its natural form.
  • the HIV-1 protease has the amino acid sequence SEQ ID NO: 4.
  • SEQ ID NO: 4 corresponds to the sequence of the HIV-1 protease isolated from the prototypic strain of group M-subtype B NL4-3 .
  • said protease forms homodimers by means of non-covalent binding of its subunits.
  • variant refers to a protein substantially homologous to HIV-1 protease.
  • a variant includes additions, deletions or substitutions of amino acids.
  • variant also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.
  • a protein is "substantially homologous" to the HIV-1 protease when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 4, that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 4, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably of at least 95%, and even more preferably of at least 99%.
  • fragment refers to a portion of the HIV-1 protease or one of its variants.
  • Another aspect of the present invention relates to a cell comprising the first vector of the invention. From now on we will use the expression "first cell of the invention” to refer to a cell comprising the first vector of the invention.
  • the cell of the invention can be a prokaryotic or eukaryotic cell.
  • the cell of the invention is a prokaryotic cell.
  • Another aspect of the present invention relates to a method for producing the amino acid sequence comprised in the RT of the invention comprising:
  • step (a) culturing the cell of the invention, and b) isolating the amino acid sequence comprised in the RT expressed in step (a) by said cell.
  • Another aspect of the present invention relates to a method for producing the RT of the invention comprising:
  • step (a) a) cultivate the cell of the invention, and b) isolate the RT expressed in step (a) by said cell.
  • Another aspect of the present invention relates to the use of the RT of the invention for the RT of a template nucleic acid, preferably mRNA.
  • Another aspect of the present invention relates to the use of the RT of the invention for the amplification of a template nucleic acid, preferably mRNA.
  • Another aspect of the present invention relates to the use of the RT of the invention for the sequencing of a template nucleic acid, preferably mRNA.
  • Another aspect of the present invention relates to a method of RT of a template nucleic acid, preferably mRNA, comprising:
  • step (a) a) mixing said template nucleic acid with the RT of the invention, and b) incubating the mixture of step (a) under conditions that allow the synthesis of DNA complementary to the template nucleic acid.
  • Another aspect of the present invention relates to a method of amplifying a template nucleic acid, preferably mRNA, comprising:
  • step (a) mixing said nucleic acid with the RT of the invention and with at least one DNA-dependent DNA polymerase, and b) incubating the mixture of step (a) under conditions that allow the amplification of DNA complementary to the template nucleic acid.
  • Another aspect of the present invention relates to a method of sequencing a nucleic acid, preferably mRNA, comprising:
  • retrotranscription or reverse transcription
  • reverse transcription refers to the synthesis of a DNA complementary to an RNA.
  • amplification refers to the increase in the number of copies of a template nucleic acid. In a preferred embodiment, the amplification takes place by PCR.
  • sequencing refers to the determination of the order of the nucleotides of a template nucleic acid.
  • template nucleic acid refers to a single or double chain nucleic acid molecule that is going to be retrotranscribed, amplified or sequenced.
  • condition that allow the synthesis of complementary DNA refers to the conditions under which the incorporation of nucleotides into nascent DNA can take place by means of complementarity of bases with the template nucleic acid.
  • the conditions in which the DNA synthesis takes place include: (a) contacting said template nucleic acid with the RT of the invention in a mixture that also comprises a primer, a bivalent cation, for example, Mg 2+ , and nucleotides, and (b) subjecting said mixture to a temperature sufficient for a DNA polymerase, for example, the RT of the invention, to initiate the incorporation of nucleotides to the primer by means of complementarity of bases with the template nucleic acid, and of Place a population of complementary DNA molecules of different sizes.
  • the separation of said population of complementary DNA molecules allows determining the nucleotide sequence of the template nucleic acid.
  • the synthesized DNA chain may not be exactly complementary to the mold nucleic acid.
  • condition that allow the synthesis of a population of DNA molecules complementary to the template nucleic acid refers to the conditions in which the sequencing is performed, and which generally include (a) contacting said template nucleic acid with the RT of the invention in a mixture that also comprises a primer, a bivalent cation, (for example, Mg 2+ ), and nucleotides, generally, dNTPs and, at least, a ddNTP, and (b) subjecting said mixture to a temperature sufficient for a DNA polymerase, for example, the RT of the invention, to initiate the incorporation of the nucleotides to the primer by means of complementarity of bases with the template nucleic acid, and thus giving rise to a population of complementary DNA molecules of different sizes.
  • the separation of said population of complementary DNA molecules generally, by electrophoresis, allows to determine the nucleotide sequence.
  • primer refers to an oligonucleotide capable of acting as the starting point of DNA synthesis when hybridized with the template nucleic acid.
  • the primer is a deoxyribose oligonucleotide.
  • the primers can be prepared by any suitable method, including, but not limited to, cloning and restriction of appropriate sequences and direct chemical synthesis.
  • the primers can be designed to hybridize with specific nucleotide sequences in the template nucleic acid (specific primers) or can be synthesized at random (arbitrary primers).
  • primer refers to a primer whose sequence is complementary to a specific nucleotide sequence in the template nucleic acid that is intended to be re-transcribed, amplified or sequenced.
  • arbitrary primer refers to a primer whose sequence is synthesized at random and that is used to initiate the synthesis of DNA at random positions of the template nucleic acid that is intended to be re-transcribed, amplified or sequenced. A population of different arbitrary primers is often used.
  • arbitrary primers refers to a set of primers whose sequence is synthesized at random and that is used to initiate the synthesis of DNA at random positions of the template nucleic acid that is intended to be re-transcribed, amplified or sequenced.
  • hybridization refers to the pairing of two complementary single stranded nucleic acid (DNA and / or RNA) molecules to give a double stranded molecule.
  • the complementarity is 100%. That is, in the region of complementarity each nucleotide of one of the two nucleic acid molecules can form hydrogen bonds with a nucleotide present in the other nucleic acid molecule.
  • hybridize one of the two nucleic acid molecules that possess a region with complementarity less than 100% can also hybridize.
  • nucleotide refers to an organic molecule formed by the covalent junction of a pentose, a nitrogenous base and a phosphate group.
  • nucleotide includes deoxyribonucleoside triphosphates, such as, but not limited to, dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • nucleotide also includes dideoxypyribonucleoside triphosphates (ddNTPs), such as ddATP, ddCTP, ddGTP, ddITP, ddTTP or derivatives thereof.
  • a "nucleotide” or a “primer” can be labeled or labeled by techniques well known in the state of the art.
  • Detectable tags include, for example, radioactive isotopes, fluorescent tags, chemiluminescent tags, tags bioluminescent or enzymatic labels.
  • DNA dependent DNA polymerase refers to a DNA polymerase capable of catalyzing the polymerization of deoxynucleotides using DNA as a template nucleic acid.
  • DNA-dependent DNA polymerase examples include the DNA polymerases of Thermus thermophilus (Tth), Thermus aquaticus (Taq), Thermotoga neapolitana (Tne), Thermotoga maritima (Tma), Thermococcus litoralis (TIi or VENTTM), Pyrococcus furiosis (Pfu), Pyrococcus species GB-D (Deep Vent TM), Pyrococcus woosii (Pwo), Bacillus stearo-thermophilus (Bst), Bacillus caldophilus (Bca), Sulfolobus acidocaldarius (Sac), Thermoplasma acidophil
  • Tth Thermus thermophilus
  • Taq Thermus aquaticus
  • Tne Thermotog
  • Another aspect of the present invention relates to a kit comprising the elements necessary to carry out any of the methods described above in the present description.
  • a preferred embodiment of this aspect of the invention refers to a kit for carrying out any of the methods described above in the present description, which comprises:
  • Figure 1 Shows the electrophoresis in polyacrylamide gel and SDS of the RT of HIV-1 group O "wild-type" resulting from purification.
  • M molecular weight markers
  • wells 1-4 bovine serum albumin (66.2 kDa) (0.5, 1, 2 and 4 ⁇ g respectively);
  • wells 5-7 aliquots of the purified HIV-1 RT group O.
  • the purification yield was greater than 3 mg of RT per liter of processed culture.
  • Figure 2 Shows the efficiency of extension of missing ends obtained with the three mutants analyzed.
  • Figure 3 It shows the amplification by RT-PCR of fragments of RNA coding for actin, of (A) 500 and (B) 1000 base pairs, from total RNA of mouse liver. Amplification was carried out with the indicated enzymes [HIV-1 RT group "wild-type" (RTO), HIV-1 RT subtype B BH 10 (RTB), mutant V75I in the context of RTO (RTO V75I ) and the double mutant V75I / E478Q in the context of RTO (RTO V75I / E478Q)].
  • the indicated empires refer to the reaction of the DNA copying synthis.
  • Esias enzymes efficiency compared ⁇ ambién Ia Ia RT virus Moloney leukemia caren ⁇ e Ia Raion ac ⁇ ividad RNase H (MLV) (see reactions at higher ⁇ empera ⁇ uras re ⁇ ro ⁇ ranscrip
  • EXAMPLE 1 Generation, expression and purification of the RTs of type 1 "wild type" HIV virus and mutants.
  • plasmid p66RTB (Boretto et al. Anal. Biochem. 2001; 292: 139-147; Matamoros et al. J. Mol. Biol. 2005; 349: 451 -463), which contains the ampicillin resistance gene and in which we clone the coding region of the p66 subunit of the RT of an isolate of group O HIV-1 (Menéndez-Arias et al. J. Biol. Chem. 2001; 276: 27470-27479).
  • the plasmid p66RTB carrying the SS RT enzyme (described in Matamoros et al. J.
  • the p66 subunit modified at the N-terminal end by the presence of three amino acids: Met-Asn-Ser, and at the C-terminal end by the presence of a 9 amino acid tail (Glu-Ser-Thr- His-His-His-His-His-His), which contains the six histidine residues, which facilitate its purification.
  • the p51 subunit is generated by the proteolytic processing of p66, by the HIV-1 protease co-expressed by the use of the plasmid pATprotease (Boretto et al. Anal. Biochem. 2001; 292: 139-147).
  • Plasmids for the expression of the mutant RTs RTO_V75I and RTO_V75I / E478Q were obtained by directed mutagenesis using Stratagene's "Quik-Change Site-Directed Mutagenesis" kit, following the manufacturer's instructions.
  • mutagenic oligonucleotides were used: SEQ ID NO: 7 and SEQ ID NO: 8 to introduce the V75I mutation, and SEQ ID NO: 9 and SEQ ID NO: 10 for E478Q.
  • As a template for the introduction of V75I the plasmid carrying the sequence encoding p66 of HIV-1 (group O), described above, was used.
  • the E478Q mutation was introduced into the V75I carrier plasmid.
  • the p66 subunit (with its modified ends) is coexpressed in E. coli XL1 Blue with the HIV-1 protease (subtype B) using the vector pATprotease (Boretto et al. Anal. Biochem. 2001; 292: 139-147), Kanamycin resistance carrier. 3 cultures of 1 liter each were obtained (Luria-Broth standard medium with 100 ⁇ g / ml ampicillin and 50 ⁇ g / ml kanamycin) of E.
  • coli carrying the expression plasmids of RT group O ("wild-type” or the corresponding mutants) and pATprotease, in exponential phase of growth, and the expression of RT was induced with isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) for 20-24 hours.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the proportion of active enzyme obtained was equal to or greater than 40%.
  • the purity of the RT obtained was greater than 95% according to estimates carried out with polyacrylamide gels and SDS ( Figure 1). These yields are significantly higher than those obtained using previously described protocols, in which the yield did not exceed 50-100 ⁇ g for the same volume of starting culture (Qui ⁇ ones-Mateu et al. Virology 1997; 236: 364-373).
  • the copy fidelity of the RTs has been determined by kinetic tests of nucleotide incorporation in the pre-stationary state, using 31T / 21 P template-primer complexes in which the 3'OH end may be correctly or incorrectly paired (Matamoros et al. J. Mol. Biol. 2008; 375: 1234-1248), and by genetic testing using the plasmid M13mp2 lacZa (Bebenek and Kunkel. Methods Enzymol. 1995; 262: 217-232; Matamoros et al. J. Mol. Biol. 2008; 375: 1234-1248).
  • the group O RT carrying the change V75I retains catalytic activity similar to the "wild-type" RT and has a lower capacity to extend primers containing a missing 3 'end (Table 1; Figure 2).
  • Table 1 Figure 2
  • the presence of the V75I mutation causes a decrease in the efficiency of extension of missing ends of 3.5 times for the G pair : T; 2.9 times for the G: G pair and 3.7 times for the G: A pair, when compared with the HIV-1 RT group "wild-type", and 2 to 6 times when compared with the RT of HIV-1 subtype B (prototype strain BH 10).
  • fidelity was measured in complementation assays using phage derivatives M13mp2 in which the lacZ gene is present.
  • the frequency with which mutants were obtained was determined when the synthesis process is performed with the different recombinant RTs (Table 2).
  • An increase in copy fidelity is reflected in the appearance in the assay of a smaller number of mutant plates.
  • the RT of the HIV-1 group "wild-type" presented a fidelity increase of 2.5 times with respect to the RT of the HIV-1 subtype B ("wild-type" BH10), while the presence of the mutation V75I in the context of the RT of HIV-1 group O improved the copy fidelity by 4.7 times with respect to the RT of subtype B.
  • the double mutant V75I / E478Q has a copy fidelity similar to that of V75I, in complementation assays using M13mp2 phage derivatives in which the lacZ gene is present.
  • REPLACEMENT SHEET (Rule 26) group O (RTO_V75I and RTO_V75I / E478Q), estimated by genetic tests (M13mp2 lacZa "forward mutation assay").
  • the back-transcription reaction was carried out in a volume of 20 ⁇ l [4 ⁇ l of 250 mM Tris-HCI buffer (pH 8.3 at 25 0 C) containing 375 mM KCI, 15 mM MgCl 2 and 50 mM dithiothreitol ; 1 ⁇ l of total RNA isolated from mouse liver (1 ⁇ g / ⁇ l); 4 ⁇ l of a mixture of the 4 dNTPs (at 2.5 mM each); 1 ⁇ l of oligo-dT (100 ⁇ M); 0.5 ⁇ l of RNase inhibitor (40 units / ⁇ l); Ia RT at an approximate concentration of 150 nM and the rest up to 20 ⁇ l of water].
  • RNA and oligo dT incubated at 68 0 C for 3 min. Then the other components of the reaction are added and incubated for 1 hour at the desired temperature to see thermostability. Finally, the reaction is stopped by incubating 10 min at 92 0 C, to obtain cDNA.
  • This is amplified by PCR under standard conditions, using Taq polymerase and primers ACT1 and ACT2 (500 base pair fragment) or ACT1 and ACT3 (1000 base pair fragment).
  • the sequences of the primers used are: ACT1: SEQ ID NO: 15, ACT2: SEQ ID NO: 16, and ACT3: SEQ ID
  • EXAMPLE 4 Loss of the RNase H activity in the RT carriers carrying the E478Q change.
  • thermostability studies carried out with the four RTs of HIV-1 (HIV-1 group O "wild-type", HIV-1 group O mutant V75I, HIV-1 group O mutant V75I / E478Q and HIV-1 subtype B "wild-type BH10") and the Moloney virus RT of mouse leukemia (MLV) demonstrated that the three enzymes derived from HIV-1 group O were the most thermostable ( Figure 3).
  • the presence of V75I decreased the thermostability of the enzyme, while when accompanied by the change E478Q, there was an improvement in its stability that was reflected in an increase in the intensity of the amplified band. This is due to the fact that the presence of the E478Q mutation leads to the loss of RNase H activity of the enzyme ( Figure 4), which facilitates the stability of the RNA in amplification reactions.
  • the RNase H activity of the generated mutants was determined with a template-primer complex, whose structure appears in the lower part.
  • the template RNA is labeled at its 5 'end with 32 P and the reaction is carried out at 37 0 C, in the presence of template-primer RNA / DNA 50 nM and Ia RT corresponding to 100 nM, in a buffer contained: 50 mM Tris-HCI (pH 8.0), 50 mM NaCI and 5 mM MgCl 2 .
  • the reactions were started after adding 1 ⁇ l of the concentrated RT and MgCl 2 . Aliquots were taken at 0, 15, 60, 120, 240 and 480 s, and the reaction was stopped by adding 10 mM EDTA dissolved in 90% formamide.

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Abstract

La présente invention trouve une application en biotechnologie. Plus particulièrement, elle concerne des transcriptases inverses isolées d'un virus de l'immunodéficience humaine de type 1 (VIH-1) de groupe O, avec mutations V75I et E478Q, qui présentent une haute fidélité de copie et une thermostabilité élevée, ainsi que leur utilisation pour réaliser la transcription inverse, l'amplification ou le séquençage d'un acide nucléique matrice. L'invention se rapporte en outre à un procédé d'obtention de ces transcriptases inverses.
PCT/ES2010/070320 2009-05-13 2010-05-13 Transcriptase inverse du vih-1 de groupe o modifiée Ceased WO2010130864A1 (fr)

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WO2014184409A1 (fr) 2013-05-17 2014-11-20 Consejo Superior De Investigaciones Científicas (Csic) Rétrotranscriptases du vih type 1 groupe o, actives à températures élevées

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Publication number Priority date Publication date Assignee Title
WO2012080541A1 (fr) * 2010-12-16 2012-06-21 Consejo Superior De Investigaciones Científicas (Csic) Nouvelles rétrotranscriptases du virus de l'immunodéficience humaine de type 1 de groupe o
ES2386264A1 (es) * 2010-12-16 2012-08-14 Consejo Superior De Investigaciones Científicas (Csic) Nuevas retrotranscriptasas del virus de la inmunodeficiencia humana tipo 1 grupo 0
WO2014184409A1 (fr) 2013-05-17 2014-11-20 Consejo Superior De Investigaciones Científicas (Csic) Rétrotranscriptases du vih type 1 groupe o, actives à températures élevées
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US9428738B2 (en) 2013-05-17 2016-08-30 Consejo Superior De Investigaciones Científicas (Csic) HIV type 1 group O reverse transcriptases that are active at high temperatures
JP2016525878A (ja) * 2013-05-17 2016-09-01 コンセホ スペリオール デ インベスティガシオネス シエンティフィカス 高温で活性なｈｉｖタイプ１グループｏ逆転写酵素
CN105339491B (zh) * 2013-05-17 2021-02-19 西班牙高等科研理事会 在高温下具有活性的hiv-1型o组的逆转录酶

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