WO2012080541A1 - Novel reverse transcriptases of human immunodeficiency virus type 2 group o - Google Patents

Abstract

The invention relates to reverse transcriptases isolated from human immunodeficiency virus type 1 (HIV-1), group O, and altered at position 65 and/or position 75, which have a high copying fidelity and thermostability, as well as to use thereof for performing the reverse transcription, amplification or sequencing of a template nucleic acid. The invention also relates to a method for obtaining said reverse transcriptases. The invention further relates to a reverse transcription method, an amplification method, a sequencing method using the reverse transcriptases of the invention and to a kit comprising the elements necessary for carrying out said methods.

Description

 New type 1 group O human immunodeficiency virus retrotranscriptases

The present invention relates to retrotranscriptases isolated from a human immunodeficiency virus type 1 (HIV-1) of group O and modified at positions 65 and / or 75, which have high copy fidelity and maintain high thermostability. (similar to that of the non-mutated retrotranscriptase), as well as its use to carry out the retrotranscription, amplification or sequencing of a template nucleic acid.

STATE OF THE PREVIOUS TECHNIQUE

Human immunodeficiency viruses (HIV-1 and HIV-2) are the etiological agents of AIDS in man. They belong to the genus Lentivirus within the family Retroviridae (reirovirus) and are characterized by an enormous genetic diversity. HIV-1 has been classified into four groups: M, N, O and P. 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). 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). Currently, information on the complete genome of variants of HIV-1 group O can be obtained from nucleotide sequence databases such as GenBank (for example, in this database strain MVP5180 / 91 has accession number L20571).

In retroviruses, retrotranscriptase (RT) (or reverse transcriptase) is the enzyme responsible for viral genome replication. RT converts single stranded genomic ribonucleic acid (RNA) into double stranded deoxyribonucleic acid (DNA) capable of integrating into the host cell genome (Herschhorn and Hizi. Cell Mol Life Sci 2010; 67: 2717-2747). RTs are DNA polymerases capable of using both RNA and DNA as templates. The HIV-1 RT is a heterodimer consisting of two subunits, called p66 and p51. In HIV-1 isolates of group M - subtype B, it is known that the p51 subunit has 440 amino acids while the p66 subunit has 560. Although p51 and p66 share the same primary structure in their first 440 residues, the different sub-domains 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 mold-primer complex interacts with either RNA / DNA or DNA / DNA, in addition to the active center involved in 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. On the contrary, the p51 subunit has a mainly structural function. In vitro studies have shown that p66 / p66 homodimers and p66 / p51 RT heterodimers have DNA polymerase activity on RNA / DNA and DNA / DNA complexes, while p51 (monomeric or homodimeric) has very little or no such activity (Herschhorn and Hizí. Cell Mol Life Sci 2010; 67: 2717-2747). 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 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 the viral protease This enzyme makes an endoproteolytic cut, between amino acids 440 and 441 of p66 of HIV-1 group M subtype B, in one of the two chains that form the homodimer.

RTs are enzymes that lack error-correcting exonuclease activity and therefore have a high error rate (around 10 ^"4 ), which would partly explain the enormous genetic variability observed in some retroviruses such as human immunodeficiency virus type 1 (HIV-1) (Menéndez-Arias. Viruses 2009; 1: 1137-1 165). Despite this, RTs are widely used in recombinant DNA technology, such as for the synthesis of copy DNA from messenger RNA (mRNA), as is the case of Moloney virus leukemia RT mouse (MLV) and RT of myeloblastosis bird virus (AMV) (Gerard et al. Nucleic Acids Res 2002; 30: 31 18-3129; Yasukawa et al. J Biochem (Tokyo) 2008; 143: 261-268 ). In relation to HIV-1 RTs, it has been seen that in complementary DNA synthesis reactions, group M HIV-1 RT - subtype B shows an efficacy similar to that of MLV or AMV RTs at 37 and 42 ° C, but retains an activity greater than 50 ° C. On the other hand, it has been shown that the "wild-type" RT (wild or WT) of group O HIV-1 has a higher thermostability compared to the MLV RT and a "wild-type" HIV- 1 group M - subtype B (WO20101130864).

The RTs of the HIV-1 group O are characterized by having about 20% of different amino acids when compared with prototype "wildtype" sequences of subtype B (Quiñones-Mateu et al. Virology 1997; 236: 364-373) . In addition, the RTs of HIV-1 group O are naturally resistant to nevirapine and other non-nucleoside-like inhibitors and have greater stability in the presence of urea than subtype B RTs (Menéndez-Arias et al. J Biol Chem 2001; 276: 27470-27479). V75I and V75I / E478Q mutants of the HIV-1 group O RT provide higher yields than those obtained with the "wild-type" RT of HIV-1 subtype B in RT-PCR amplification reactions, when retrotranscription is carried carried out at 57-69 ° C (Álvarez et al. J Mol Biol 2009; 392: 872-884). Despite their high thermostability, the wild-type RTs of HIV-1, both those of group M - subtype B, and those of group O, are approximately 7 to 15 times less faithful than RTs of MLV and AMV, in genetic assays based on DNA synthesis using the template as a template lacZ gene present in phage M13mp2 (Roberts et al. Science 1988; 242: 1 171-1 173; Roberts et al. Mol Cell Biol 1989; 9: 469-476; Menéndez-Arias. Viruses 2009; 1: 1 137- 1 165; WO20101 130864). It has been observed that some amino acid changes may produce an increase in fidelity in some RTs, such as mutations K65A, K65R, V75I, D76V, R78A, V148I and Q151 N, in the context of HIV-1 RT sequence of subtype B (Menéndez-Arias. Viruses, 2009; 1: 1 137-1 165; Garforth et al. J Mol Biol 2010; 401: 33-44). However, it is not possible at first to know if the same mutation exerts the same effect on a phylogenetic variant of HIV-1 away from subtype B as is the case for group O variants. In fact, the same change may have different effects. according to the sequence context. For example, the E89G substitution in the HIV-1 RT of group M - subtype B produces an increase in fidelity in trials of erroneous incorporation and in extension trials of missing ends (Drosopoulos and Prasad. J Virol 1996; 70: 4834- 4838; Hamburgh et al. Nucleic Acids Res 1998; 26: 4389-4394; Rubinek et al. Eur J Biochem 1997; 247: 238-247). However, in the context of HIV-2 RT sequence, E89G has a minimal effect on copy fidelity, measured with the same type of trials (Taube eí al. Eur J Biochem 1997; 250: 106-1 14 ). Another example is the L74V mutation, which in the HIV-1 RT of group M subtype B produces an increase in fidelity measured in erroneous incorporation trials and in extension trials of missing ends (Rubinek et al. Eur J Biochem 1997; 247 : 238-247), although these effects were not observed when the mutation was introduced in the HIV-2 RT (Taube et al. Eur J Biochem 1997; 250: 106-114).

EXPLANATION OF THE INVENTION

The technical problem solved by the invention is to provide retrovirus retrotranscriptases (RTs) with greater copy fidelity and therefore both lower error rate and that they are also stable at elevated temperatures, greater than 54 ° C.

The present invention relates to RTs isolated from a human immunodeficiency virus type 1 (HIV-1) of group O and modified in one or more positions, which have high fidelity of copy and thermostability, as well as their use to carry out the retrotranscription, amplification or sequencing of a template nucleic acid. In order to obtain new modified RTs of group O HIV-1 mutants have been generated from an RT isolated from a group O HIV-1 (group O HIV-1 RT) whose p66 subunit has been modified by substitution of lysine 65 by arginine (K65R mutation); or by replacing lysine 65 and valine 75 with arginine and isoleucine, respectively (mutations K65R and V75I). These new RTs show an increase in their copy fidelity with respect to that of the unmodified RT, without decreasing their thermostability. In addition, the RTs of the invention have been compared with other RTs already known in the state of the art. The RTs of HIV-1 of group O carrying the K65R mutation or of the mutations K65R and V75I have higher copy fidelity and thermostability than the RTs of isolates or strains of HIV-1 of subtype B group (HIV-1 RT of subtype B), and that the MLV RT, used for the same purpose.

A first aspect of the present invention relates to an RT isolated from a group O HIV-1 and modified to comprise an amino acid sequence in which the residue corresponding to position 65 of SEQ ID NO: 1 is substituted by an arginine or by another amino acid that supposes a conservative substitution with respect to arginine, where said modified RT has an increased copy fidelity in relation to the corresponding unmodified RT, while maintaining the high temperature stability of the enzyme not modified Since p66 can be proteolyzed, preferably, the amino acid sequence comprised by the RT of the invention has an identity of at least 70%, more preferably, of at least 80% and, even more preferably, at least 90% with SEQ ID NO: 1.

A preferred embodiment of this aspect of the invention relates to a modified group O HIV-1 RT comprising SEQ ID NO: 2. A preferred embodiment of this aspect of the invention relates to an HIV-1 RT of modified group O, which comprises an amino acid sequence, mutated as described above, and which also has another substitution of the residue corresponding to position 75 of SEQ ID NO: 1 with an isoleucine or another amino acid that involves a substitution conservative with respect to isoleucine, and where said modified RT has an increased copy fidelity in relation to the unmodified RT and retains its stability at elevated temperatures.

Another preferred embodiment of this aspect of the invention relates to a modified group O HIV-1 RT comprising SEQ ID NO: 3.

From now on we will use the expression "RT of the invention" to refer to a modified RT according to any of the characteristics described above in the present description.

The term "retrotranscriptase isolated from human immunodeficiency virus type 1" (or "human immunodeficiency virus type 1 reverse transcriptase", or "HIV-1 RT") refers to a protein retrotranscriptase with DNA and RNA-dependent DNA polymerase activity , which 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 polymerase chain reaction (PCR) of the nucleotide sequence encoding the amino acid sequence of the p66 subunit of the RT from the viral RNA obtained from an HIV-1 isolate, and subsequent cloning into a expression vector.

The term "group O HIV-1 RT", as used herein, refers to a retrotranscriptase isolated from a group O human type 1 immunodeficiency virus. The term "HIV-RT Subtype B 1 "refers to a retrotranscriptase isolated from a human immunodeficiency virus type 1 group M subtype B.

The terms "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. The term "residue" corresponds to an amino acid.

The term "conservative substitution" refers to a substitution that is made with amino acids that would be considered by a person skilled in the art as similar, for example in physicochemical properties of hydrophobicity, polarity, charge or volume of their side chain, and that will lead to an activity similar to that described for the mutants of the present invention. In the present invention "similar amino acids" refers to amino acids similar to arginine in the case of the substitution in the residue of position 65 or similar to isoleucine in the case of the substitution in the residue of position 75.

The term "identity", as used herein, 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. The term "copy fidelity" refers to the accuracy of polymerization, or the ability of the RT to discriminate between correct and incorrect substrates (eg, 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 cellular RNA polymerase II, 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. proviral 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, and thus obtain mutation frequencies.

In the biochemical tests, purified RT is used for the determination of kinetic constants on an RNA or DNA template, under certain conditions (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 a stationary and in a stationary state.

Biochemical tests of erroneous incorporation in the steady state are based on the determination of 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 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 [and ³² PJATP), in the presence of different dNTP concentrations, once the complex has formed RT binary / mold-primer. The resulting products are analyzed by electrophoresis in polyacrylamide gels. The data obtained fit the Michaelís-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 _inc ) is defined as the relationship between the catalytic efficiency (kcat / Km) obtained for the incorrect nucleotide and the catalytic efficiency obtained for the correct nucleotide. Thus, greater fidelity of the RT implies a lower efficiency of erroneous incorporation.

For the fixing of an error in the nascent DNA, the incorporation of an incorrect nucleotide is not enough, the RT must also be able to extend the missing end that is generated as a result of this erroneous incorporation. This measure of fidelity is carried 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 primer-complexing complex: the complex with the 3 'end properly matched and the same complex with the 3' end missing. The efficiency of extension of missing ends (f _ex t) is defined as the ratio between k _cat IK _m obtained for the extension of the missing end and that 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 in the order of milliseconds). In this way, it is possible to calculate the affinity constant (Kd) for the interaction between the dNTP and the binary complex RT / template-primer, and the polymerization constant {k _po¡ ). The efficiency of erroneous incorporation and extension of missing ends is determined from the values of k _po and IK _d obtained for the incorporation of correct or incorrect nucleotides, or those obtained for the incorporation of correct nucleotides on template-primer complexes containing one end 3ΌΗ paired or missing.

The most commonly used genetic assays, called "forward mutation assays" are usually carried out using as a primer-primer of the DNA synthesis reaction, the double stranded phage DNA M 13mp2, from which the region corresponding to the lacZ gene of One of his threads. The DNA synthesis reaction is performed in the presence of RT and high concentrations of dNTPs. After bacterial transformation with the reaction product, the mutants are identified as blue / white plates in culture medium containing X-Gal (5-bromo-4-chloro-3-indole-galactopyranoside) and IPTG (ísopropyl- β-thio-galactopyranoside). Thus, if there is no error in the DNA synthesis reaction, the result is a dark blue plate. On the contrary, the introduction of one or several errors implies the partial or total loss of the a- 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 RT.

Another type of genetic assays are reversal assays. These assays are based on the use of templates containing an inactivating mutation (typically, termination codons or insertions of a nucleotide). Reversal of the mutation results in the correction of the error and therefore in the restoration of the activity of the marker gene.

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. For example, in biochemical tests of nucleotide incorporation, 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. For example, in genetic tests ("forward mutation assays") is considered increased fidelity when there is a significant increase (applying statistical criteria) in the mutant frequency obtained by the modified RT, typically 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. The temperature at which the polylase activity (RNA or DNA dependent) of a RT is maximum is called the optimum temperature. Above this temperature, the increase in reaction speed due to temperature is counteracted by the loss of catalytic activity due to thermal denaturation of RT, so that its polymerase activity (dependent on DNA or RNA) decreases. The term "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 ° C, preferably at least 50 ° C, more preferably of at least 55 ° C and even more preferably of at least 60 ° C.

The 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 activity of DNA polymerase at different elevated temperatures, using a template-primer complex, analyzing the residual RT activity after subjecting the enzyme to an incubation at elevated temperature for different time intervals, analyzing the half-life of the RT or measuring the quantity and / or 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. For example, the average life of the RT activity refers to the time in which the polymerase activity (RNA or DNA dependent) is reduced by half when the synthesis reaction (RNA or DNA dependent) is carried out at a high temperature. Other parameters indicative of the thermostability of an RT are the quantity and / or length of the product synthesized by the RT when the synthesis reaction is carried out at a high temperature. For example, an estimate of the stability of the RT can be obtained by analyzing the amount of the product obtained through the RT in an RT-PCR. For this, a retrotranscription reaction can first be carried out using total RNA at a high temperature as a template nucleic acid, and then the complementary DNA obtained from the retrotranscription can be amplified the reaction product by PCR. The amount of product obtained in these reactions constitutes a measure of the stability of the RT, at the temperature at which the back transcription reaction was carried out.

A "high temperature synthesis reaction", as used herein, refers to a reaction and, more preferably, a back transcription (or reverse transcription) reaction, which is performed at a temperature of at least , 45 ° C, preferably at least 55 ° C, more preferably at least 60 ° C and even more preferably at least 65 ° C. In some circumstances, 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. An RT with "increased" or "increased" thermostability is defined as an RT that has a significant increase or increase (applying statistical criteria) in 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 of the HIV-1 "wild-type" group O (SEQ ID NO: 1 ). For example, when thermostability is estimated by the quantity of the product obtained by means of RT in an RT-PCR, the RT is considered to have an increased thermostability when the amount of product obtained by RT-PCR has a significant increase or increase (applying statistical criteria), typically of at least 1, 5 times, preferably at least 3 times, and even more preferably at least 4 times, with respect to the RT of HIV-1 "wild-type" group O (SEQ ID NO: 1).

As used herein, the term "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 one-letter code 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 herein as XiNX ₂ , in which Xi is the amino acid in the non-mutated protein sequence, X2 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 a suitable codon change in the polynucleotide encoding the RT. More preferably, the mutants of the present invention can be obtained through the oligogonucleotide-directed mutagenesis technique. This technique consists in the ringing of a complementary oligonucleotide (except for the presence of one or more missing nucleotides) with the nucleotide sequence of the group O-1 HIV-RT. The partially missing oligonucleotide is then extended by a DNA polymerase, generating a double stranded DNA molecule that contains the desired change in the sequence of one of the chains. The changes introduced in the sequence result in the exchange of one amino acid for another. The double stranded polynucleotide can then be inserted into an appropriate expression vector and the mutant polypeptide produced. Oligonucleotide-directed mutagenesis can be carried out, for example, by PCR.

A second aspect of the present invention relates to a polynucleotide encoding the amino acid sequence comprised in the RT of the invention.

The terms "polynucleotide", "nucleotide sequence", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably herein, and refer to a polymeric form of nucleotides of any length, which may be, or no, chemically or biochemically modified.

A third aspect of the present invention relates to a vector comprising the polynucleotide encoding the amino acid sequence comprised in the RT of the invention.

The vector can be, for example a cloning vector or an expression vector. Preferably, said vector is an appropriate vector for the expression and purification of the RT of the invention. The term "cloning vector", as used in the present description, refers to a DNA molecule in which another DNA fragment can be integrated, without losing the ability to self-replicate. Examples of expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes. The term "expression vector", as used herein, refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a cell, called a host cell. Said nucleic acid is generally operatively linked to control sequences.

The term "expression" refers to the process by which a polypeptide is synthesized from a polynucleotide. It includes transcription of the polynucleotide into a messenger RNA (mRNA) and the translation of said mRNA into a protein or a polypeptide.

The term "cell" or "host cell", as used herein, refers to any prokaryotic or eukaryotic organism that is the recipient of an expression vector, cloning or any other DNA molecule.

The term "control sequence", as used herein, refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked. The term "control sequences" is intended to include, 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.

The term "operably linked", as used in the present description, 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 "operably linked" to a polynucleotide is linked in such a way that the expression of the sequence Encoder is achieved under conditions compatible with the control sequences.

As used herein, the term "promoter" refers to a region of DNA, generally "upstream" or "upstream" of the transcription start point, 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. Examples of 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 α-amylase gene of B. subtilis For the expression of a nucleic acid in a prokaryotic cell, the presence of an upstream ribosomal binding site of the coding sequence is also necessary. Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.

Using well known techniques, one skilled in the art would be able to obtain a suitable vector for the expression of the amino acid sequence comprising the RT of the invention in any prokaryotic or eukaryotic cell. A preferred embodiment of this aspect of the invention relates to a vector comprising a polynucleotide encoding the amino acid sequence comprised in the RT of the invention, wherein said polynucleotide is operably linked to at least one control sequence in the 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 relates to a vector comprising a polynucleotide encoding 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 tag. of purification.

The term "purification tag" or "affinity tag", as used herein, 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 protein to be purified, 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 . Preferably, the purification tag consists of at least 6 histidine residues.

From now on we will use the expression "first vector of the invention" to refer to a vector comprising a polynucleotide encoding the sequence comprised in the RT of the invention, wherein said vector has any of the characteristics described above in the present description.

Another preferred embodiment of this aspect of the invention relates to a vector comprising a polynucleotide that encodes the amino acid sequence comprised by the RT of the invention and which further comprises a polynucleotide encoding 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. From now on we will use the expression "second vector of the invention" to refer to a vector comprising a polynucleotide encoding the sequence comprised in the RT of the invention, wherein said vector has any of the characteristics of the first vector of the invention, and which further comprises a polynucleotide that encodes for 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.

In a preferred embodiment, the protease capable of performing an endoproteolytic cut in the amino acid sequence comprised in the RT of the invention between 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. The term "HIV-1 protease," as used herein, refers to a protease isolated from a human immunodeficiency virus type 1. The term "protease isolated from human immunodeficiency virus type. 1 "refers to a protease capable of performing an endoproteolytic cut in the amino acid sequence comprised in the RT of the invention between residues corresponding to positions 440 and 441 of SEQ ID NO: 1, or in nearby positions, and which It is substantially or completely free of the components that normally accompany it or interact with it in its natural form. It can be obtained, for example, by amplification by the polymerase chain reaction (PCR) of the nucleotide sequence encoding the amino acid sequence of the protease from the viral RNA obtained from an isolate of HIV-1, and subsequent cloning into an expression vector. Preferably, the HIV-1 protease has the amino acid sequence SEQ ID NO: 4. SEQ ID NO: 4 corresponds to the HIV-1 protease sequence isolated from the prototypic strain of group M-subtype B (strain NL4 -3). In the virus, said protease forms homodimers by non-covalent binding of its subunits.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the HIV-1 protease. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.

As used herein, 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%.

The term "fragment", as used herein, refers to a portion of the HIV-1 protease or a variant thereof.

The term "functionally equivalent", as used herein, means that the protein or fragment of the protein in question maintains the ability to perform an endoproteolytic cut at or around the amino acid sequence comprised in the RT of the invention between residues corresponding to positions 440 and 441 of SEQ ID NO: 1. A fourth 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.

A preferred embodiment of this aspect of the invention relates to a cell comprising the second vector of the invention. From now on we will use the expression "second cell of the invention" to refer to a cell comprising the second vector of the invention.

Another preferred embodiment of this aspect of the invention refers to a cell that comprises the first vector of the invention and which, furthermore, comprises a vector comprising a polynucleotide encoding 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, or between residues close to them. From now on we will use the expression "third cell of the invention" to refer to a cell with said characteristics. From now on we will use the expression "cell of the invention" to refer to the first cell, the second cell or the third cell of the invention. The cell of the invention can be a prokaryotic or eukaryotic cell. Preferably, the cell of the invention is a prokaryotic cell. A fifth aspect of the present invention relates to a method for producing the amino acid sequence comprised in the RT of the invention comprising: a) culturing the cell of the invention, and

b) isolate the amino acid sequence comprised in the RT expressed step (a) by said cell. A sixth aspect of the present invention relates to a method for producing active RT of the invention comprising: a) culturing the cell of the invention, and

 b) isolate the RT expressed in step (a) by said cell and preserve it under conditions that allow the RT to be active.

The conditions that would allow the activity of the isolated RT in the method described in the sixth aspect of the invention are those applicable to any protein and known to any person skilled in the art, for example conditions that allow the protein to fold correctly and be functionally active (for example, that possesses catalytic activity, in the case of enzymes). Examples of factors to be taken into account to maintain protein functionality include, but are not limited to, adequate pH conditions, adequate concentration of electrolytes and salts, presence of reducing agents such as dithiothreitol at appropriate concentrations, or the addition of stabilizing agents. A seventh 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.

An eighth 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.

A ninth 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.

A tenth aspect of the present invention relates to a method of retrotranscription of a template nucleic acid, preferably mRNA, comprising: a) mixing said template nucleic acid with the RT of the invention, eb) incubating the mixture of step (a) under conditions that allow the synthesis of DNA complementary to the template nucleic acid .

An eleventh aspect of the present invention relates to a method of amplifying a template nucleic acid, preferably mRNA, comprising: a) mixing said nucleic acid with the RT of the invention and with at least one DNA-dependent DNA polymerase e

 b) incubate the mixture from step (a) under conditions that allow amplification of DNA complementary to the template nucleic acid.

A twelfth aspect of the present invention relates to a method of sequencing a nucleic acid, preferably mRNA, comprising: a) contacting said nucleic acid with the RT of the invention, b) incubating said mixture under conditions that allow the synthesis of a population of DNA molecules complementary to the template nucleic acid, and

 c) separating said population of complementary DNA molecules to determine the nucleotide sequence.

The term "retrotranscription" or "reverse transcription", as used herein, refers to the synthesis of a DNA complementary to an RNA.

The term "amplification", as used in the present description, is refers to the increase in the number of copies of a template nucleic acid. In a preferred embodiment, the amplification takes place by PCR.

The term "sequencing", as used herein, refers to the determination of the nucleotide order of a template nucleic acid.

The term "template nucleic acid" or "template" as used herein refers to a single or double stranded nucleic acid molecule that is to be retrotranscribed, amplified or sequenced.

The term "conditions that allow the synthesis of complementary DNA" refers to the conditions under which the incorporation of nucleotides into nascent DNA can take place by complementing bases with the template nucleic acid.

Generally the conditions under which DNA synthesis takes place include: (a) contacting said template nucleic acid with the RT of the invention in a mixture which further 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 nucleotide incorporation into the primer by complementing bases with the template nucleic acid, and Place a population of complementary DNA molecules of different sizes. The separation of said population from complementary DNA molecules makes it possible to determine the nucleotide sequence of the template nucleic acid.

The incorporation of mismatched nucleotides during complementary DNA synthesis may result in one or more mismatched bases. Therefore, the synthesized DNA chain may not be exactly complementary to the template nucleic acid. The term "conditions that allow the synthesis of a population of DNA molecules complementary to the template nucleic acid" refers to the conditions under which sequencing is performed, and which generally include (a) contacting said template nucleic acid with the RT of the invention in a mixture which further 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 ios nucleotides to the primer by base complementarity with the template nucleic acid, and then to a population of complementary DNA molecules of different sizes. The separation of said population from complementary DNA molecules, generally, by electrophoresis, allows the nucleotide sequence to be determined.

The term "primer", as used herein, refers to an oligonucleotide capable of acting as the starting point of DNA synthesis when hybridized with the template nucleic acid. Preferably, 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).

The term "specific primer", as used herein, 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. The term "arbitrary primer" refers to a primer whose sequence is synthesized at random and that is used to initiate DNA synthesis at random positions of the template nucleic acid that is to be re-transcribed, amplified or sequenced. A population of different arbitrary primers is often used. The term "arbitrary primers" refers to a set of primers whose sequence is synthesized at random and that is used to initiate DNA synthesis at random positions of the template nucleic acid that is intended to be re-transcribed, amplified or sequenced.

The term "hybridization," as used herein, refers to the pairing of two complementary single stranded nucleic acid (DNA and / or RNA) molecules to give a double stranded molecule. Preferably, 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. However, those with normal experience in the field will recognize that two nucleic acid molecules that possess a region with complementarity less than 100% can also hybridize.

The term "nucleotide", as used herein, refers to an organic molecule formed by the covalent bond of a pentose, a nitrogenous base and a phosphate group. The term "nucleotide" includes deoxyribonucleoside triphosphates, such as, but not limited to, dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. The term nucleotide also includes dideoxypyribonucleoside triphosphates (ddNTPs), such as ddATP, ddCTP, ddGTP, ddITP, ddTTP or derivatives thereof. According to the present invention a "nucleotide" or a "primer" can be labeled or labeled by techniques well known in the state of the technique. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels or enzymatic labels. The term "DNA-dependent DNA polymerase," as used herein, refers to a DNA polymerase capable of catalyzing the polymerization of deoxyribonucleotides using DNA as the template nucleic acid. Examples of DNA-dependent DNA polymerase that can be employed in the amplification method of the following invention are, but are not limited to, the DNA polymerases of Thermus thermophilus (Tth), Thermus aquaticus (Taq), Neapolitan Thermotoga (Tne), Maritime Thermotoga (Tma), Thermococcus litoralis (Tii or Vent ™), Pyrococcus furiosis (Pfu), Pyrococcus specíes GB-D (Deep Vent ™), Pyrococcus woosii (Pwo), Bacillus stearo-thermophilus (Bst), Bacillus caldophilus (Bca), Sulfolobus acidocaldarius (Sac), Thermoplasma acidophilum (Tac), Thermus flavus (Tfl / Tub), Thermus ruber (Tru), Thermus brockianus (DyNAzyme ™), Methanobacterium thermoautotrophicum (Mth) or Mycobacterium sp. (Mtb, Mlep).

A thirteenth 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 relates to a kit for carrying out any of the methods described above in the present description, which comprises: a) the RT of the invention, and

 b) at least one element of the list comprising: a buffer,

a primer, iii) a DNA-dependent DNA polymerase, and iv) a nucieotide.

A fourteenth aspect of the invention relates to the use of the thirteenth aspect of the invention kit for retrotranscription, amplification or sequencing of a template nucleic acid, where preferably this is messenger RNA.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Electroforesls of the RTs analyzed. It shows the purity of the different RTs obtained. Polyacrylamide and SDS gel electrophoresis shows: 1, molecular weight markers (top to bottom: 97.4 kDa, 66.2 kDa, 45 kDa and 31 kDa); 2, RT of the HIV-1 group "wíld-type" (WT); 3, RT of HIV-1 group O carrying the K65R mutation; 4, RT of the HIV-1 group O carrying the K65R and V75I mutations; 5, RT of the HIV-1 group O carrying the R78A mutation; wells 6 and 7: bovine serum albumin (66.2 kDa) (1 and 2 pg respectively).

Figure 2. Thermostability of the RTs analyzed. It shows the thermal stability of variants of the RT of HIV-1 group O compared to the RT of HIV-1 group M - subtype B (BH10_WT). For each enzyme, the specific RNA-dependent DNA polymerase residual activity, determined after preincubating the enzyme with the mold-initiator for 5 min at the indicated temperature, and relative to that obtained when the preincubation is performed at 37 ° C. The polymerization reactions are carried out with poly (rA) / oligo (dT) and ₆ as a template-initiator and [ ³ H] dTTP as a substrate. The nucleotide incorporation rates obtained for the different enzymes at 37 ° C (reference values) were: 0.72 ± 0.28 s ¹ for RTO_WT (HIV-1 RT group O "wild-type"), 0.92 ± 0.25 s ^"1 for RTO_K65R (RT of HIV-1 group O carrying the K65R mutation), 0.61 ± 0.26 s ¹ for RTO_K65R / V75l (RT of HIV-1 group O carrying mutations K65R and V75I), 0.45 ± 0.06 s ^" for RTO_R78A (RT of the HIV-1 group O carrying the R78A mutation, where the arginine at position 78 was replaced by an alanine), 0.25 ± 0.06 s ¹ for RTO_V75l / R78A (RT of the HIV-1 group O carrying the V75I mutations and R78A), 0.57 ± 0.06 s ^"1 for RTO_V75l (HIV-1 RT group carrying the V75I mutation) and 1.17 ± 0.4 s for BH10_WT (HIV-1 group M RT - subtype B BH10).

Figure 3. RNA-dependent DNA polymerase activity of the RTs analyzed. It shows the amplification by RT-PCR of fragments of RNA coding for actin of 900 base pairs (A) and for tubulin of 1200 base pairs (B), from total RNA of mouse liver. The amplification was carried out with the following RTs: well 2, HIV-1 RT group O carrying the K65R mutation (RTO_K65R); well 3, RT of HIV-1 group O carrying mutations K65R and V75I (RTO_K65R / V75l); well 4, RT of the HIV-1 group O carrying the R78A mutation (RTO_R78A); well 5, RT of the HIV-1 group O carrying mutations V75I and R78A (RTO_V75l / R78A); well 6. RT of HIV-1 group O "wild-type"(RTO_WT); well 7. V75I in the context of 0_WT RT (RTO_V75l); well 8. RT of HIV-1 subtype B BH10 (RT BH10_WT); and well 9, Moloney virus RT from mouse leukemia (RT MLV) (well 8). In wells 1 and C molecular weight markers (phage DNA digested Φ29, carried out with Hindlll) and a negative control performed in the absence of complementary DNA, respectively, are shown. The indicated temperatures refer to the DNA synthesis reaction copy. Figure 4. Copy fidelity of the RTs analyzed. It shows the efficiency of incorporation of incorrect nucleotides (A) and the extension efficiency of missing ends (B), obtained with two of the three characterized mutant RTs (RTO_K65R and RTO_K65R / V75l), in comparison with those of HIV RT- 1 group "wild-type" (RTO_WT) and the RT carrier of the V75I mutation (RTO_V75l).

EXAMPLES OF EMBODIMENT OF THE INVENTION

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1: Generation, expression and purification of variants of group 1 HIV RT.

The expression and purification of the RTs was carried out with a modified version of plasmid p66RTB using the molecular tools described in the literature, which we briefly describe (Boretto et al. Anal Biochem 2001; 292: 139-147; Álvarez eí al J Mol Biol 2009; 392: 872-884; WO20101130864). The expression and purification of the RTs was carried out with a modified version of plasmid p66RTB containing the ampicillin resistance gene and in which the coding region of the p66 subunit of the RT of an isolate of HIV-1 was cloned group O. The nucleotide sequence comprising the region encoding the p66 subunit of HIV-1 (group O) in the expression plasmid is shown in SEQ ID NO: 5, while the amino acid sequence of the obtained RT with said Plasmid is indicated in SEQ ID NO: 6. Using this construct, the p66 subunit was produced, modified at the N-terminal (amino terminal) end by the presence of three amino acids: Met-Asn-Ser, and at the C- end terminal (carboxyl terminal) by the presence of a 9 amino acid tail (Glu-Ser-Thr-His-His-Hís-His-His), which contained the six histidine residues, which facilitate its purification. The p51 subunit was generated by the proteolytic processing of p66, by the co-expressed HIV-1 protease through the use of the plasmid pATprotease (Boretto et al. Anal Biochem 2001; 292: 139-147).

Plasmids for the expression of the mutant RTs RTO_K65R and RTO_K65R / V75l were obtained by directed mutagenesis using Stratagene's "Quik-Change Site-Directed Mutagenesis" kit, following the manufacturer's instructions. As mutagenic olygonucleotides, SEQ ID NO: 7 and SEQ ID NO: 8 were used to introduce the K65R mutation. As a template for the introduction of K65R, the plasmid carrying the coding sequence for HIV-1 (group O) ("wild-type") p66, described above, was used. The K65R mutation was also introduced into the V75I carrier plasmid, to obtain the double mutant K65R / V75I. After mutagenesis, it was verified by sequencing that the coding region of p66 in said plasmids was correct and that it contained only the mutations introduced. The nucleotide sequences of the inserts carrying the K65R and K65R / V75I mutations are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. SEQ ID NO: 9 and SEQ ID NO: 10 contain nucleotides at the 5 ^' and 3 ^' ends that code for additional amino acids of the N-terminal (Met-Asn-Ser) and the C-terminal (Glu-Ser-Thr) -His-His-His-His-His-His) of the p66 subunit of the RT. The amino acid sequences of the RTs that were obtained from the expression of the mulant plasmids are indicated in SEQ ID NO: 1 1 (K65R) and SEQ ID NO: 12 (K65R / V75I). These sequences contain the additional amino acids of the N- and C-terminal ends indicated above. The p66 subunit (with its modified ends) was co-expressed 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 (standard Luria-Broth medium with 100 g / ml ampicillin and 50 pg / ml kanamycin) of E. coli bearing the expression plasmids of group O RT ("wild-type" or the corresponding mutants) and pATprotease, in exponential phase of growth, and RT expression was induced with isopropyl-D-thiogalactopyranoside (IPTG) for 20-24 hours.

After that time the bacteria were collected and processed following the procedure described by Boretto et al. Anal Biochem 2001; 292: 139-147, which includes a stage of bacterial lysis and homogenization, followed by ion exchange chromatographs [in phosphocellulose P1 1 (Whatman)] and affinity [in Ní ²⁺ -nitriloacetic-agarose columns (ProBond ™, Invítrogen)]. In phosphocellulose chromatography, the RT was eluted with a gradient of NaCl (0 to 2 M) in 50 mM sodium phosphate buffer (pH 6.8). In affinity chromatography, RT was eluted with a gradient of imidazole (0 to 500 mM) in 50 mM sodium phosphate (pH 6.0). The fractions containing the RT were pooled and dialyzed three times against 50 mM Tris-HCI buffer (pH 7.0), containing 25 mM NaCl, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid (EDTA) and 10% glycerol and subsequently concentrated using Centriprep-30 and Centricon-30 devices (Amícon, Millipore).

The yield obtained from 3 liters of culture (approximately 10 g of cells), estimated from the molar extinction coefficient of the RT at 280 nm (£ 280 = 260 450 M ^"1 cm ^{" 1} ), was greater than 10 mg for RT with the K65R mutation and greater than 20 mg for the double mutant K65R / V75I. The purity of the RTs obtained was greater than 95% according to estimates carried out with polyacrylamide gels and SDS (Figure 1). EXAMPLE 2: Effect of the K65R mutation alone or in combination with V75I (mutant K65R / V75I) on the thermostability and efficiency of the retrotranscription reaction collected with PCR amplification.

The thermal stability of the RTs was determined by measuring the residual RNA-dependent DNA polymerase activity, obtained after pre-incubating the enzymes for 5 minutes at different temperatures in the range of 37 to 58 ° C, in the presence of the template-initiator complex. Then, polymerization reactions were carried out at 37 ° C, in the presence of poly (rA) / oligo (dT) and ₆ 1 μ 1 and [ ³ HjdTTP 50 μΜ, in 50 mM Tris-HCI buffer (pH 8.0) which It contained 20 mM NaCI, 10 mM ₂ gCI and 8 mM dithiothreitol. The reactions were initiated by the addition of 0.2-1 pmol of enzyme (in 30 μΙ of reaction volume) and incubated for 0-10 min, to calculate the rate of incorporation of dTTP. The reactions were stopped by adding 20 μΙ of 0.5 M EDTA (Quiñones-Mateu eí al. Virology 1997; 236: 364-373; Álvarez eí al. J Mol Biol 2009; 392: 872-884).

The thermal stability tests showed that all the RTs of HIV-1 group O (like the wild-type RT of HIV-1 subtype B) retained more than 60% of their activity after being incubated at 46 ° C during 5 min (Figure 2). When preincubation is performed at 50 ° C, all of them, except the R78A mutant, retained more than 40% of the initial activity. The comparative study of the different enzymes reflected important differences at 54 ° C. When preincubation was done at this temperature, only four variants of the RT of HIV-1 group O were retained significant activity (25-35%): the wild-type enzyme and the K65R, V75I and K65R / V75I mutants. Under these conditions, the "wild-type" RT of HIV-1 subtype B retains about 5%, while the R78A and V75I / R78A mutants do not show significant activity. To analyze the effects of the mutations on the efficiency of the back-transcription reaction coupled with PCR amplification, back-transcription reactions were carried out at different temperatures, and subsequently the reaction products (complementary DNA, cDNA) were amplified by PCR under standard conditions. (Álvarez eí al. J Mol Biol 2009; 392: 872-884). Typically, the back transcription reaction was carried out in a volume of 20 μΙ [4 μΙ of 250 mM Tris-HCI buffer (pH 8.3 at 25 ° C) containing 375 mM KCI, 15 mM MgCl ₂ and 50 mM dithiothreitol ; 1 μΙ of total RNA isolated from mouse liver (1 pg / μΙ) (Stratagene); 4 μΙ of a mixture of the 4 dNTPs (at 2.5 mM each); 1 μΙ of oligo-dT (100 μΜ); 0.5 μΙ of RNase inhibitor (40 units / μΙ) (RNasin® Plus, Promega); RT at an approximate concentration of 150 nM and the rest up to 20 μΙ of water]. Initially, the mouse liver RNA and oligo-dT were incubated at 68 ° C for 3 min. Then the other reaction components (including RT) were added and incubated for 1 hour at the desired temperature to see thermostability. Finally, the reaction was stopped by incubating 10 min at 92 ° C, to obtain the cDNA. The cDNA was amplified by PCR under standard conditions, using Taq polymerase or other similar enzymes (eg, Expand High Fidelity DNA polymerase, Roche). The efficiency of the retrotranscription reaction at different temperatures was determined after PCR amplification of the obtained copy DNA. The wild-type RT of HIV-1 group O and the K65R, V75I and K65R V75I mutants were found to be effective in amplifying an approximately 900 base pair RNA fragment derived from the actin gene, from reactions of retrotranscription carried out at different temperatures (Figure 3A). Similar results were obtained in the amplification of a fragment of approximately 1200 base pairs of an RNA encoding tubulin (Figure 3B). The primers used for the amplification of actin (ACT1 and ACT3) or tubulin (TUB1 and TUB2) copy DNA have been previously described (Álvarez eí al. J Mol Biol 2009; 392: 872-884). The effectiveness of Four enzymes were similar to those obtained with the "wild-type" RT of HIV-1 group M - subtype B (strain BH 0), and superior to that of the MLV RT.

EXAMPLE 3: Effect of the K65R mutation alone or in combination with V75l (mutant K65R V75I) on the copy fidelity of the group O RT.

The copy fidelity of the RTs was determined by genetic tests using the plasmid M13mp2 lacZa (Bebenek and Kunkel. Methods Enzymol 1995; 262: 217-232). In addition, kinetics of nucleotide incorporation and kinetic tests of extension of missing ends were carried out, under pre-stationary conditions, in order to determine the ability of the different enzymes to discriminate between correct and incorrect nucleotides or template-initiator complexes. correctly or incorrectly paired. For this purpose, the 31T / 21 P mold-initiator complex, or derivatives thereof, was used in which the 3ΌΗ end of the 21 P primer could be missing.

The copy fidelity of the different RTs was measured in complementation tests in which M13mp2 phage derivatives carrying the lacZ gene were used. With these tests, the frequency with which mutants were obtained was determined when the synthesis process was carried out with the different recombinant RTs (Table 1). An increase in copy fidelity is reflected in the appearance in the assay of a smaller number of mutant plates. The RTs carrying the K65R and K65R / V75I mutations improved the copy fidelity of the wild-type RT of the HIV-1 group O by 10.8 and 9.3 times, respectively. Its fidelity is also superior in 1, 5 to 1, 8 times to that obtained with the MLV RT.

Table 1. Loyalty of the RT of HIV-1 group O "wild-type" (RTO_WT) compared to those of HIV-1 group M - subtype B ("wild-type" BH10) (BH10_WT) and LV (RT_MLV) and different mutants of the HIV-1 RT group O: K65R (RTO_K65R), K65R / V75I (RTO_K65RA 75l), V75I (RTO_V75l), R78A (RTO_R78A) and E478Q (RTO_E478Q (RTO_E478Q) in which the glutamic acid residue of position 478 was replaced by glutamine), estimated by means of genetic complementation tests (M13mp2 lacZa "forward mutation assay").

 392: 872-884.

 Data for BH10_WT taken from Matamoros et al. J Mol Biol 2008; 375: 1234-1248.

Nucleotide incorporation assays (biochemical tests based on the use of gels) showed that the variants of the group O RT K65R and K65R / V75I (i.e., RTO_K65R and RTO_K65R / V75l) exhibited similar or even slightly catalytic efficacy. higher (in the case of K65R) than that of the "wild-type" enzyme (RTO_WT). Nucleotide misincorporation assays demonstrated that the K65R mutant was 3.9 to 8 times more faithful than the wild-type enzyme for incorporation into the DNA of C [cytidine (monophosphate)], G [guanosine (monophosphate) ] or A [adenosine (monophosphate)] instead of T [thymidine (monophosphate)]; while the K65R / V75I mutant showed even greater fidelity in trials of incorporation of C or A instead of T (Table 2). Both enzymes improved the fidelity of the V75I mutant for the incorporation of C or A instead of T.

In missing end extension assays, the two mutants (K65R and K65R / V75I) were more faithful than the wild-type enzyme in missing end extension reactions, although the differences with the V75I mutant were small (Table 3) . In any case, the double mutant K65R / V75I exhibited a fidelity 5.2 times greater than that of the ia RT "wild-type" for the extension of ends G: A, 9.5 times higher for the extension of G: T and 22.8 times higher for the extension of G: C. The differences between the four RTs are reflected graphically in Figure 4.

Table 2. Kinetic parameters of nucleotide misincorporation on the 31T / 21 P template-initiator complex for the HIV-1 RT group "wild-type" (RTO_WT) and ios mutants K65R (RTO_K65R), K65R / V75I (RTO_K65R / V75l) and V75I (RTO_V75l), determined in the pre-stationary state.

Efficiency

Nucleotide Enzyme Incorporation

(μΜ) (MivrV)

wrong {f _in ) ^b

RTO_WT ³ dTTP 14.7 ± 1, 0 11, 8 ± 2.9 1, 25 ± 0.32

dCTP 0.72 0.10 7652 ± 2083 (9.5 ± 2.9) x 10 * 7.57 x 10 * dGTP 0.15 ± 0.03 11730 ± 3750 (1, 3 ± 0.5) 10 * 1, 03 x 10 * dATP (7.3 ± 0.2) 10 ^"J 1485 ± 150 (4.9 ± 2.2) x 10 ° 3.94 x 10 ^" °

RTO_V75l ^at dTTP 13.9 ± 1, 2 14.6 ± 4.3 0.96 ± 0.30

dCTP 0.27 ± 0.05 11023 ± 3792 (2.4 ± 0.9) 10 * 2.54 x 10 ^' ° (3.0) dGTP (6.3 ± 0.9) x 10 ^"¿ 7481 ± 2307 (8.4 ± 2.9) x 10 * 8.76 x 10 ^" ° (1, 2) dATP (2.2 ± 0.2) x 10 ^{" J} 2308 ± 720 (9.5 ± 3.1 ) x 10 ^' ' 9.98 10 ^" '(3.9)

RTO_K65R dTTP 10.7 ± 0.9 5.8 ± 1, 9 1, 82 ± 0.60

dCTP 0.17 ± 0.02 9559 ± 1908 (1, 7 ± 0.4) 10 * 9.51 x 10 * (8.0) dGTP (3.1 ± 1, 9) x 10 ^"¿ 6452 ± 857 (4.8 ± 0.7) x 10 * 2.66 x 10 * (3.9) dATP (4.8 ± 0.1) x 10 ^"J 3680 ± 178 (1, 3 ± 0.1) x 10 * 7.09 10 ^" '(5.6)

a Datos para RTO_WT y RTO_V75l tomados de Álvarez et al. J Mol Biol 2009; 392: 872-884. RTO_ dTTP 12.7 ± 1, 3 13.3 ± 3.7 0.95 ± 0.28

a Data for RTO_WT and RTO_V75l taken from Álvarez et al. J Mol Biol 2009; 392: 872-884.

b The wrong incorporation efficiency value (/ ¡ _nc ) is defined as:

Zinc = [/ (poi (incorrect) / Kd (incorrect) / / _p0 i (correct) / d (correct) 3, where the incorrect nucleotides were dCTP, dGTP or dATP, while the correct nucleotide was dTTP. The numbers between parentheses represent the relative increase in fidelity obtained for each incorrect nucleotide, from the expression: f _ínc (RTO_WT) / / j _nc (mutant RT).

Table 3. Kinetic parameters of extension of missing ends on the 31T / 21P mold-initiator complex for the RT of HIV-1 "wild-type" group O (RTO_WT) and mutants K65R (RTO_K65R), K65R / V75I (RTO_K65R / V75l) and V75I (RTO_V75l), determined in the pre-stationary state.

Efficiency

Pair

extension base _{κ ύ} kpoJKá

Enzyme of extremes in position (μΜ) (MM ^' V)

missing 3 ^'

( ^b

RTO_WT ³ G: C 14.7 ± 1.0 11.8 ± 2.9 1.25 ± 0.32

 G: T 7.6 ± 0.9 2638 ± 785 (2.9 ± 0.9) x 10 "2.30 x 10"

G: G 0.56 ± 0.07 1132 ± 316 (4.9 ± 1.5) 10 ^"4 3.95 x 10 ^'4

G: A (2.1 ± 0.3) x 10 ^"¿ 7817 ± 2334 (2.7 ± 0.9) x 10 ^" ° 2.17 x 10 ^" °

RTO_V75l ^at G: C 13.9 ± 1.2 14.6 ± 4.3 0.96 ± 0.30

G: T 2.8 ± 0.2 4400 ± 789 (6.3 ± 1.2) x 10 ^'4 6.56 x 10 ^"4 (3.5)

G: G 0.32 ± 0.01 2426 ± 178 (1.3 ± 0.1) 10 ^{~ 4} 1.39 10 ^{~ 4} (2.8)

G: A (6.8 ± 0.8) x 10 "12110 ± 2536 (5.6 ± 1.3) x 10" 5.86 x 10 "(3.7)

RTO_K65R G: C 10.7 ± 0.9 5.8 ± 1.9 1.82 ± 0.60

G: T 3.1 ± 0.5 2214 ± 1058 (1.4 ± 0.7) x 10 "7.80 x 10 ^{" 4} (2.9)

G: G 0.19 ± 0.02 1416 ± 328.4 (1.3 ± 0.3) 10 ^{~ 4} 7.25 10 ^{~ 3} (5.4)

G: A (1.3 ± 0.2) x 10 15180 ± 4239 (8.7 ± 2.9) x 10 "4.78 x 10" (4.5)

RTO_ G: C 12.7 ± 1.3 13.3 ± 3.7 0.95 ± 0.28

a Data for RTO_WT and RTO_V75l taken from Álvarez et al. J Mol Biol 2009; 392: 872-884.

b The extension efficiency value of missing ends (f _ex t) is defined as:

fext = [/ fpoi (missing) / K _d (missing) / fp ₀ i (paired) / c¡ (paired)], where the missing base pairs were G: T, G: G and G: A, while the correctly matched pair was G: C. The numbers in brackets represent the relative increase in fidelity obtained for each base pair, from the expression: f _ex t (RTO_WT) / f _and xt (mutant RT).

Claims

1. Retrotranscriptase isolated from a human immunodeficiency virus type 1 of group O and modified to comprise an amino acid sequence in which the residue corresponding to position 65 of SEQ ID NO: 1 is replaced by a arginine or by another amino acid that supposes a conservative substitution with respect to arginine. 2. Retrotranscriptase according to claim 1, wherein the amino acid sequence has an identity of at least 70% with SEQ ID NO: 1. 3. Retrotranscriptase according to claim 2, wherein the amino acid sequence has an identity of at least 80% with SEQ ID NO: 1. 4. Retrotranscriptase according to claim 3, wherein the amino acid sequence has an identity of at least 90% with SEQ ID NO: 1. 5. Retrotranscriptase according to claims 1 to 4, wherein the amino acid sequence is SEQ ID NO: 2. 6. Retrotranscriptase according to any one of claims 1 to 5, wherein the amino acid sequence also has another substitution of the residue corresponding to position 75 of SEQ ID NO: 1 with an isoieucin or another amino acid that supposes a conservative substitution with respect to Isoieucina 7. Retrotranscriptase according to claim 6, wherein the amino acid sequence is SEQ ID NO: 3. 8. Polynucleotide encoding the amino acid sequence of a retrotranscriptase according to any one of claims 1 to 7. 9. Vector comprising the polynucleotide according to claim 8. 10. Vector according to claim 9, wherein the polynucleotide is operatively linked to at least one control sequence of the list comprising:  a) a promoter,  b) a transcription initiation signal,  c) a transcription termination signal,  d) a polyadenylation signal, or  e) a transcriptional activator. 11. Vector according to any of claims 9 or 10 wherein the polynucleotide is linked in reading phase to a sequence encoding a purification tag. 12. Vector according to claim 11 wherein the purification tag consists of a tail of histidine residues. 13. Vector according to any of claims 9 to 12, further comprising a polynucleotide encoding a protease capable of performing an endoproteolytic cut in the amino acid sequence of a retrotranscriptase according to any one of claims 1 to 7 between, or around, waste corresponding to positions 440 and 441 of SEQ ID NO: 1. 14. A cell comprising a vector according to any of claims 9 to 12. 15. A cell according to claim 14, further comprising another vector comprising a polynucleotide encoding a protease capable of making an endoproteolytic cut in the amino acid sequence of the retrotranscriptase according to any one of claims 1 to 7 between or around the residues which correspond to positions 440 and 441 of SEQ ID NO: 1. 16. Cell comprising the vector according to claim 13. 17. Method for producing the amino acid sequence of a retrotranscriptase according to any one of claims 1 to 7, comprising: a) culturing the cell according to any of claims 14, 15 or 16, and  b) isolating the amino acid sequence of the retrotranscriptase expressed in step (a) by said cell. 18. A method of producing retrotranscriptase according to any one of claims 1 to 7, comprising: a) culturing the cell according to any of claims 14, 15 or 16, and  b) isolating the retrotranscriptase expressed in step (a) by said cell. 19. Use of the retrotranscriptase according to any of claims 1 to 7 for the retrotranscription of a template nucleic acid. 20. Use of the retrotranscriptase according to any of claims 1 to 7 for the amplification of a template nucleic acid. 21. Use of the retrotranscnptase according to any of claims 1 to 7 for the sequencing of a template nucleic acid. 22. Use of the retrotranscriptase according to any of claims 19 to 21 wherein the template nucleic acid is mRNA. 23. A method of retrotranscription of a template nucleic acid comprising: a) mixing said template nucleic acid with the retrotranscriptase according to any one of claims 1 to 7, and  b) incubate the mixture from step (a) under conditions that allow the synthesis of DNA complementary to the template nucleic acid. 24. A method of amplifying a template nucleic acid comprising: a) mixing said nucleic acid with the retrotranscriptase according to any one of claims 1 to 7 and with at least one DNA-dependent DNA polymerase, and  b) incubate the mixture from step (a) under conditions that allow amplification of DNA complementary to the template nucleic acid. 25. A sequencing method of a template nucleic acid comprising: a) contacting said nucleic acid with the retrotranscriptase according to any one of claims 1 to 7,  b) incubating said mixture under conditions that allow the synthesis of a population of DNA molecules complementary to the template nucleic acid, and c) separating said population of complementary DNA molecules to determine the nucleotide sequence. 26. Method according to any of claims 23 to 25 wherein the template nucleic acid is mRNA. 27. Kit comprising: a) the retrotranscriptase according to any one of claims 1 to 7, and  b) at least one element of the list comprising:  i) a buffer,  ii) a primer,  iii) a DNA-dependent DNA polymerase, and  iv) a nucleotide. 28. Use of a kit according to claim 27, for retrotranscription, amplification or sequencing of a template nucleic acid. 29. Use of a kit according to claim 28 wherein the nucleic acid is messenger RNA.