KR0155598B1 - Nef gene separated novel hiv-2 and protein - Google Patents

Abstract

The present invention relates to HIV-2 viruses newly isolated from HIV-2 carriers, their novel genes and proteins translated therefrom.

In the present invention, after extracting the total cell DNA from the blood of four HIV-2 carriers (OG, MB, FT, TG) and amplifying the nef gene using a primer, and then known HIV-2 ROD, ST and GH species As a result of comparing the homology between the nucleotide sequence and the amino acid sequence of the nef gene, the inventors of the present invention found that the HIV-2 TG species isolated by the present inventors were novel.

Using the novel HIV-2 TG species genes and proteins of the invention, it will be possible to prepare monoclonal antibodies, vaccines and diagnostic reagents specific for HIV-2.

Description

Nef gene isolated from novel human immunodeficiency virus-2 (HIV-2) and proteins inferred therefrom

1 is a schematic showing the location of the HIV-2 virus nef gene and oligonucleotide primers used in the PCR reaction.

FIG. 2 is an electrophoretic photograph showing the result of amplification of DNA isolated from HIV-2 infected person by PCR reaction.

Figure 3 shows the nucleotide sequence of the nef gene of the novel HIV-2 TG species of the present invention and the amino acid sequence translated therefrom.

Figure 4 compares the DNA sequence of the nef gene of the novel HIV-2 TG species of the present invention with HIV-2 ROD, ST and GH species.

Figure 5 compares the amino acid sequence of the nef gene of the novel HIV-2 TG species of the present invention with HIV-2 ROD, ST and GH species.

The present invention relates to human immunodeficiency virus-2 (HIV-2). More specifically, the present invention relates to novel HIV-2 viral genes isolated from HIV-2 carriers, proteins translated therefrom, and antibodies, vaccines and diagnostic reagents using genes or proteins of previous HIV-2.

Human immunodeficiency virus (HIV), along with another type of retrovirus T-cell leukemia virus (HTLV-1, HTLV-2), is a gene structure and expression In that the mechanism of regulation is very complicated. It is largely distinguished from other animal reverse transcription viruses. For example, HIV not only has nine genes, although their genes are as short as 9 to 10 kb, but also fuse at the time of RNA splicing between existing open reading frames. This happens to be known to produce a hybrid protein (hybrid protein).

In addition to the gag, pol and env genes that determine structural proteins, HIV has regulatory genes with relatively short lengths: tat, rev, nef, vif, vpu (for HIV-1), vpx (HIV- 2) and vpr, among which tat and rev are essential genes for virus growth, and their mechanism of action is very well known. The inventors of the present invention have reported that, due to the interaction between each of these regulatory genes, HIV is divided into two phases, namely, an early phase and a late phase. : Kim et al., J. Virol. 63: 3708 (1989).

In addition to tat and rev, an important regulatory gene known as nef (also called 3'-orf, E ', F, orfB) is a gene of about 27 kDa whose N-terminus is myristoylated. Make nef is not a gene essential for virus growth, at least in vitro culture, but it has been reported that the virus grows faster if it is defective [Luciw et al., Proc. Natl. Acad. Sci. USA, 84: 1434 (1987); Ahmad and Vankatesan, Science, 241: 1481 (1988); Niederman et al., Proc. Natl. Acad. Sci. USA, 86: 1128 (1989). According to this report, the gene has been considered to downregulate the growth of HIV, and in this respect it is called a negative factor, which is weakly named 'nef'. On the other hand, nef protein has the same biochemical properties as G-protein (Guy et al., Nature 330: 266 (1987)) and has been reported to inhibit the transcription of HIV through the HIV LTR region. and Venkatesan, Science, 241: 1481 (1988); Niederman et al., Proc. Natl. Acad. Sci. USA, 86: 1128 (1989).

However, the inventors have carefully analyzed the various HIV species in various types of host cells. As a result, at least in vitro culture nef does not have a significant effect on the growth rate of the virus. It has been found that it may play a role [Kim et al., Proc. Natl. Acad. Sci. USA, 86: 9544 (1989).

This finding was confirmed by Hammes et al., And many researchers have undertaken a re-evaluation of the nef gene. Hammes et al., Proc. Natl. Acad. Sci. USA, 86: 9549 (1989). As a result, a number of data conflicting with earlier researchers have emerged. For example, Hames et al. Reported that nef protein inhibited transcription through the LTR site, and Kaminchick et al. It is reported that it does not have the same biochemical properties as the protein (Kaminchick et al., J. Viro., 64: 3447 (1990)).

In addition, examination of the blood of a large number of asymptomatic carriers revealed that the neph protein was produced incorrectly during the incubation period faster or more than other viral gene products (eg, Figure 3b). . These findings were to deny the conventional hypothesis that nef has the effect of inhibiting virus growth and thus plays a large role in inducing latency.

The function of the nef gene is still unknown, but there is much contextual evidence that the nef protein plays an important role in viral growth and the pathogenesis of AIDS.

First, Kessler et al. Reported that in the case of simian immunodeficiency virus (SIV), the nef gene is an important factor in causing AIDS and growing viruses in monkeys. Kestler et al. al., Cell, 65: 651 (1991). These include wild type nef (hereinafter referred to as 'nef +') and variants with mutations in the nef gene (species with point mutations in the nef gene or deletions in the nef) in monkeys. When infected, only nef + species induced AIDS in monkeys and nef-deleted mutants found no disease at all. Meanwhile, another mutant, the nef-point mutation, causes AIDS with a low probability of re-collection of the virus in monkeys and found that all of these viruses reversed to nef and turned into wild type nef. These findings indicate that nef plays a very important role in AIDS pathology and viral growth in the case of SIV causing AIDS in monkeys, and further negates the conventional hypothesis that nef is a negative gene.

Second, serological findings indicate that most patients and asymptomatic carriers have large amounts of antibodies against the nef protein, which is expressed in the early stages of infection in AIDS patients and HIV carriers. Therefore, it can be seen that these viruses actively express nef gene in human body.

Third, HIV-1, HIV-2 and SIV have an open reading frame of the complete nef gene. The nef gene has between 600 and 800 bp of nucleotides depending on the type, and the existence of such a long open reading frame without being blocked by stop codons indirectly proves the importance of this gene.

Fourth, recent reports have shown that nef proteins downregulate CD4 cell surface proteins. The mechanism is not yet clear, but it is important evidence that nef is linked to the pathology of AIDS.

Thus, the evidence suggests that the nef gene plays an important role in viral growth and AIDS pathogenesis through some unknown mechanism. Until now, nef has been mainly used for HIV-1 and SIV, and little research has been carried out on the nef gene of HIV-2, which has been frequently captured in Africa and is recently spreading in the West. Therefore, the inventors of the present invention have studied the role of the nef gene in the growth and pathogenesis of HIV-2, and investigated whether the nef protein can be used as a diagnostic reagent. To this end, the present inventors collected peripheral blood mononuclear cells (PBMC) from people infected with HIV-2 to extract total DNA, and isolated DNA base by separating nef gene using targeted PCR (target PCR) technology. Sequence determination and investigation of the expected amino acid sequences revealed surprisingly the HIV-2 species reported so far (see WO 8909815: WO 8906276: EP 345559; Guyader et al., Nature, 326: 662 (1987); Zagury) et al., Proc. Natl. Acad. Sci., USA, 85: 5941 (1988); Human Retroviruses and AIDS 1993; A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Los Alamos National Laboratory, USA) The present invention was completed by concluding that it is a novel HIV-2 species which is significantly different from. On the other hand, the novel HIV-2 virus of the present invention is a pathogenic microorganism, and as of June 2, 1993, the deposit was refused by the Korean spawn association (KCCM).

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, in order to separate nef residues of HIV-2 directly from carriers of HIV-2, blood was collected from HIV-2 carriers, treated with heparin, and centrifugation gradient (Ficoll-Hypaque, density gradient). PBMC was isolated by centrifugation. Total cell DNA was extracted therefrom, and primer No. 1 was determined by primary PCR from DNA isolated on agarose gel. 5-1 and No. The nef gene was amplified using 3-1, and the primer No. 5-2 and No. Amplification using 3-2. The nef gene of amplified HIV-2 was cloned into plasmid vector pCR ^™ II (Invitrogen, USA). Comparing the nucleotide sequence and amino acid sequence of the cloned nef gene with three types of nef genes (ROD, ST and GH species) previously reported, the four carriers (OG, MB, Nef gene isolated from FT, TG) The nef gene isolated from TG patients was found to be novel, and HIV-2 containing it could be identified as a new species (hereinafter referred to as 'HIV-2 TG species').

Meanwhile, the TG virus was cultured under laboratory conditions by mixing with PBMC isolated from the above-mentioned patients and PBMC isolated from normal individuals. After culturing for 3 weeks, DNA was separated from the serova, and the sequence was determined by amplifying the nef gene by PCR. As a result, the nucleotide sequence was found to be identical to the nef gene isolated from the previous. Thus, the virus was confirmed to have a nef sequence different from other HIV-2 so far known.

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention. In particular, methods for producing monoclonal antibodies, vaccines and diagnostic reagents using the novel human immunodeficiency virus-2 (HIV-2) of the present invention and proteins expressed from the respective genes thereof are well known in the art. It can be achieved with the method, and therefore the present invention should be seen to cover all of them.

Example 1 Collection of Samples

In the present invention, blood was collected and analyzed from patients at the Medical University Hospital of the University of Lisbon, Portugal in order to directly separate the nef gene of HIV-2 from HIV-2 carriers, and the background of the analyzed 4 patients is shown in Table 1 below. As it is. Because of the possibility of variation in the nef gene according to the patient's background, patients were included to include all male / female, black / white, high risk groups, and patients suspected of heterosexual transmission.

As shown in Table 1, OG, MB, FT and TG species in the present invention means HIV-2 species isolated from each carrier with the above background.

Example 2

isolation of nef gene]

All known HIV-2 species have been amplified and isolated in laboratory conditions. Blood collected from AIDS patients and amplified by incubating in laboratory conditions mixed with normal human PBMC. Because this isolation process selects only the fastest growing virus species in the virus population, the virus isolated in this way may be different from the virus present in the patient's body. In order to minimize such artifacts due to selection pressure, the inventors chose a method of separating nef genes directly from cells of AIDS patients.

Different types of primers were used to directly separate nef genes from cells of AIDS patients. The positions and base sequences of the primers successfully used for gene amplification are shown in FIG. The nef gene of HIV-2 is located at the 3 'end of the viral gene and overlaps with the 3'-LTR as shown in FIG. 1, but there are some differences depending on the species, but mostly composed of DNA of 750 to 780bp. have.

In carriers, including AIDS patients, the number of virus-infected cells is known to be quite low, and less than 1% of PBMCs have been reported for HIV-1. Ho et al., N. Eng. J. Med., 321: 1621 (1989)]. If we assume about 1 to 5 genomes of integrated HIV per infected cell, it is believed that the genes of the virus will be present at a low number of less than 0.2 per cell based on total PBMCs. Therefore, in the present invention, in order to maximize direct cloning of the nef gene, the viral genome was amplified in two steps using nested primers as disclosed in FIG.

10-15 ml of blood was collected from HIV-2 carriers, treated with heparin, and PBMCs were isolated by concentration gradient centrifugation. The isolated PBMCs were immediately dissolved using TES buffer (pH 7.5, Tris. HCI, 10 mM EDTA, 0.7% SDS). This was treated with 400 μg / ml proteinase K for 1 hour at 50 ° C., and total cell DNA was isolated through phenol / chloroform extraction and ethanol precipitation. The isolated DNA was identified on agarose gel. .

In the primary PCR, 0.5 µg of total DNA was obtained by primer No. 5-1 and No. Using 3-1, amplification was carried out by 30 cycles under conditions of 1 minute at 92 ° C, 1 minute at 50 to 55 ° C, and 1 minute at 72 ° C, and 1/10 of the primary PCR sample in secondary PCR. Primer No. 5-2 and No. 3-2 was used to amplify by 30 revolutions under the same conditions. At this time, DNA obtained from two normal persons was used for negative regulation, and DNA of cells infected with a known ROD strain of HIV-2 was used as positive regulation.

2 is an electrophoretic photograph showing PCR results, in which lanes 1 and 14 are cleaved λ DNA with Hind III as molecular weight markers; Lanes 2 and 8 amplify the DNA of cells infected with the positive regulatory HIV-2 ROD species; Lanes 3 to 7, as a result of the first PCR, lane 3 amplified the negative regulatory DNA, lanes 4, 5, 6 and 7 amplified the nef gene of OG, MB, FT and TG, respectively: From 9 to 13, as a result of the secondary PCR, lane 9 amplified negative regulatory DNA, and lanes 10, 11, 12, and 13 were amplified nef genes of OG, MB, FT, and TG, respectively.

As shown in FIG. 2, in all four carriers, the band corresponding to nef gene DNA was not seen by the primary PCR, but the DNA band of the nef gene of 750 to 780 bp was clearly seen in the second PCR using nested primers. have. Of course, no amplified DNA was seen from the two normal subjects who were used for negative regulation.

Example 3

Cloning of nef Gene and Determination of Sequence

The nef gene of HIV-2, amplified as above, was transferred to the plasmid vector pCR. Cloned into II. This vector has one T-base at each end of the nef gene to be inserted, which facilitates the cloning of DNA fragments amplified by PCR, and also facilitates sequencing of the cloned DNA fragments since all nucleotide sequences are known. As a control for measuring errors in PCR and sequencing, the neural gene of ROD species, which was amplified and cloned in the same manner as before, was found to be consistent with known nucleotide sequences. Therefore, the nef gene analyzed by this method could be seen to represent the nef sequence of HIV-2 actually present in the patient.

To investigate whether the cloned nef gene is a novel nef gene, three types of 159 bp (ie 53 amino acids) nucleotide sequences at the N-terminus of the nef gene have been previously reported (ROD, GH and ST species). The nef genes of carriers OG and MB were about 98% similar to those of ROD, and the carrier FT was about 99% similar to that of ST. On the other hand, nef isolated from carrier TG showed about 20% difference with ROD, GH and ST. As a result, since there is a difference of about 20% in nucleotide sequences between nef genes of ROD, GH, and ST which have already been reported, HIV-2 having a nef gene of TG isolated in the present invention can be classified as a new species. there was.

Example 4

Sequence determination of nef gene of HIV-2 TG species

In order to more clearly demonstrate the novel HIV-2 TG species, the nucleotide sequence of the nef gene of TG species was determined. 3 shows the nucleotide sequence of the nef gene of HIV-2 TG species and the amino acid sequence translated therefrom. The nef gene of TG species has 762 bases except for the stop codon, which is 3 bases less than GH and ST and 6 bases less than ROD. 4 and 5 respectively compare the nucleotide sequence of the nef gene of HIV-2 TG species and the amino acid sequence translated therefrom with known ROD, GH and ST species. In FIGS. 4 and 5, (*) indicates that the nucleotide sequence of the nef gene of four kinds of viruses or the amino acid sequence translated therefrom are all identical. In addition, the results obtained by comparing the base sequence of the known ROD, GH and ST species with the nef genes of the present invention TG species and the amino acid sequences translated therefrom are shown in Tables 2 and 3 below.

As shown in Table 2, while there is a difference of about 14-18% in sequence homology between the known ROD, ST, GH, TG species can be seen that there is a difference of about 17 to 20%. In addition, as shown in Table 3, in the amino acid sequence homology, there is a difference of about 21 to 27% between ROD, ST, and GH, whereas TG species have a difference of about 29 to 33%. As a result, the results of Tables 2 and 3 show that the TG species is a novel HIV-2 species.

Example 5

Isolation of the virus

Since HIV-2 isolated from patient TG described above is significantly different from other HIV-2 species so far identified, TG virus was mixed with PBMC isolated from patients and PBMC isolated from normal individuals. Incubated in laboratory conditions. Gag antigens of HIV-2 were measured from the cell culture after 2 weeks of culture to confirm that viruses were produced. Then, one week later, DNA was separated from the cells, and the nef gene was separated again by PCR to determine the sequence, and the nucleotide sequence was found to be identical to that by direct PCR. Thus, this barrier has been confirmed to have a different nef sequence from other HIV-2 so far identified, and it is expected that there will be a big difference in other gene regions of HIV-2.

As described and demonstrated in detail above, the present invention provides novel human immunodeficiency virus-2 (HIV-2), its genes and amino acid sequences translated therefrom. With the antigen of the novel human distant deficiency virus-2 of the present invention, the application of conventional methods in the art will improve monoclonal antibodies, vaccines and diagnostic reagents against HIV-2.

Claims (2)

A nef gene having the following nucleotide sequence isolated from a novel human immunodeficiency virus-2 (HIV-2) isolated from an acquired immunodeficiency syndrome (AIDS) patient of TG species: A nef protein having the following amino acid sequence inferred from the nef gene of human immunodeficiency virus-2 (HIV-2) of claim 1: