JP2006191891A - Attenuated hiv-1 base sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To carry out the cloning of an attenuated HIV gene supposed to play an important role in the structure-function correlation of HIV gene, replication mechanism of virus and analysis of AIDS onset mechanism and to clarify the structure. <P>SOLUTION: The DNA comprises (1) a specific base sequence or (2) a base sequence obtained by the deletion, substitution and/or addition of one or more bases in the above specific base sequence and encoding an attenuated HIV virus variant. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel DNA encoding the HIV-1 genome.

  The rate of progression of HIV disease varies greatly among patients infected with HIV-1 due to complex confounding between the host's genetic and immune factors and the virulence of the virus. The viral nef gene is one of the important factors involved in disease progression, as demonstrated in animal models (Non-Patent Documents 1 to 3). The nef gene is an important factor involved in human AIDS progression, indicating that long-term non-progressive infections (LTNPs) with low viral titers despite being infected with HIV-1 for 10-14 years Some of them have large deletions in the nef gene (Non-Patent Documents 4 to 6) or hold viruses with small structural defects or mutations (Non-Patent Document 7). Strongly supported by the facts.

  Suppression of cell surface expression of CD4 and class I MHC molecules (Non-patent Documents 8 and 9), enhancement of virus replication and infectivity (Non-patent Documents 10 and 11), T cells (Non-patent Document 12) and macrophages (non-patent documents Several functions of the nef gene have been reported, including induction of cytokine and chemokine expression in Patent Literature 13) and blocking of pro-apoptotic signals in HIV-1 infected cells (Non-Patent Literatures 14 and 15). Many cell interaction partners that are important for the function of the nef gene have been identified, and their binding sites are mapped to different positions in Nef (Non-patent Document 16).

  Genetic alterations in the nef gene involved in slow disease progression have been identified in HIV-1 subtype B in Western populations (Non-Patent Documents 4-7), but these alterations are in subtype B strains. It was not clear whether or not only existed.

Du, Z., et al., 1995. Identification of a nef allele that causes lymphocyte activation and acute disease in macaque monkeys Cell. 82: 665-74 Hanna, Z., et al., 1998. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice Cell. 95: 163-75 Kestler, H.W.d., et al., 1991.Importance of the nef gene for maintenance of high virus loads and for development of AIDS Cell. 65: 651-62 Deacon, N. J., et al., 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients [see comments] Science. 270: 988-91 Kirchhoff, F., TC Greenough, DB Brettler, JL Sullivan, and RC Desrosiers 1995. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection [see comments] N Engl J Med. 332: 228-32 Salvi, R., et al., 1998. Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor J Virol. 72: 3646-57 Casartelli, N., et al., 2003. Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-progressor children Aids. 17: 1291-301 Aiken, C., et al., 1994. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain Cell. 76: 853-64 Schwartz, O., et al., 1996. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein Nat Med. 2: 338-42 Miller, M. D., et al., 1994. The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages J Exp Med. 179: 101-13 Spina, C. A., et al., 1994. The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes J Exp Med. 179: 115-23 Wang, J. K., et al., 2000. The Nef protein of HIV-1 associates with rafts and primes T cells for activation Proc Natl Acad Sci U S A. 97: 394-9 Swingler, S., et al., 1999. HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages [see comments] Nat Med. 5: 997-103 Geleziunas, R., et al., 2001. HIV-1 Nef inhibits ASK1-dependent death signaling providing a potential mechanism for protecting the infected host cell Nature.410: 834-8 Wolf, D., et al. 2001. HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals Nat Med. 7: 1217-24 Geyer, M., et al., 2001. Structure--function relationships in HIV-1 Nef EMBO Rep. 2: 580-5

  The isolation of attenuated HIV molecular clones not only plays an important role in the analysis of the structure and function of HIV genes, the mechanism of viral replication, and the mechanism of the onset of AIDS. Expression system) and immunological reagents (monoclonal antibodies, etc.), as well as vaccines and evaluation systems (animal models of AIDS, etc.). An object of the present invention is to clone an attenuated HIV gene and elucidate its structure.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a large deletion in the nef-LTR region of asymptomatic patients whose virus titer cannot be detected due to abnormally weak antibody response during the 5-year follow-up period Has succeeded in identifying attenuated mutants of HIV-1 recombinant epidemic strain (CRF) _01 (CRF01_AE). The present inventors have demonstrated for the first time an association between the slow progression of disease in a non-subtype B strain and nef deletion. The present invention has been completed based on these findings.

That is, according to the present invention, DNA having any of the following base sequences is provided.
(1) the base sequence set forth in SEQ ID NO: 1; or (2) in the base sequence set forth in SEQ ID NO: 1, wherein one to a plurality of bases are deleted, substituted and / or added, Base sequence encoding an attenuated HIV-1 virus variant:

According to another aspect of the present invention, a recombinant vector having the above-described DNA of the present invention is provided.
According to still another aspect of the present invention, a transformant having the above-described DNA or recombinant vector of the present invention is provided.
According to still another aspect of the present invention, there is provided a virus particle prepared by culturing a transformant obtained by transforming the above-described DNA or recombinant vector of the present invention into a host.
According to still another aspect of the present invention, there is provided an HIV vaccine comprising the above-described virus particles.

  The present invention provides naturally attenuated HIV-1 mutants of non-subtype B strains. The attenuated HIV-1 mutant of the present invention has not been reported so far and is a novel HIV mutant. This infectious case of a unique deletion HIV-1 strain can provide insights to understand AIDS pathology and strategies to prevent disease progression.

Hereinafter, the implementation method and embodiment of this invention are demonstrated in detail.
In the present invention, an attenuated variant of HIV-1 recombinant epidemic strain 01_AE (CRF01_AE) from an asymptomatic patient (GM43) whose virus titer cannot be detected due to an abnormally weak antibody response during a 5-year follow-up period Was identified. A large deletion was included in the nef-LTR region of HIV-1 provirus carried by GM43. Sequence analysis of the nef-LTR region at GM43 revealed that the size of the deletion gradually increased from 84 bp to over 400 bp over the past 5 years. Six years after the follow-up, a highly attenuated HIV-1 strain with a 391-bp nef-LTR deletion was isolated. A phylogenetic analysis showed that this female patient, GM43, acquired HIV-1 CRF01_AE from her husband (GM46). The nef-LTR region of GM46 was found to carry a deletion ranging from 12 to 562 bp in the HIV-1 subspecies subpopulation, but the deletion pattern in GM46 was not identical to the deletion pattern of GM43. There wasn't. The gene organization of the nef-LTR region in GM43 was surprisingly similar to that of previously reported HIV-1 subtype B infected individuals, such as the Sydney Blood Bank Cohort. HIV-1 strains have convergent evolutionary characteristics that the deletion configuration will be essentially the same, even if the strain is different, so there is a common mechanism for pathogenic attenuation, Therefore, it is suggested that the defective mutant has an uncertain advantage over the complete competent HIV-1 strain in terms of infection. There has been no report on a naturally attenuated HIV-1 mutant of non-subtype B strain, and the present invention is the first. This infectious case of a unique deletion HIV-1 strain can provide insights to understand AIDS pathology and strategies to prevent disease progression.

(A) DNA of the present invention
The DNA of the present invention has any of the following base sequences.
(1) the base sequence set forth in SEQ ID NO: 1; or (2) in the base sequence set forth in SEQ ID NO: 1, wherein one to a plurality of bases are deleted, substituted and / or added, Base sequence encoding an attenuated HIV-1 virus variant:

  In the present specification, the range of “1 to plural” in the “base sequence in which one or more bases are deleted, substituted and / or added” is not particularly limited, but for example 1 to 60, preferably Means 1 to 30, more preferably 1 to 20, further preferably 1 to 10, particularly preferably about 1 to 5.

  The DNA of the present invention can be obtained by the method described in the following examples, or an appropriate probe or primer is designed based on the information of the base sequence described in SEQ ID NO: 1 in the sequence listing of this specification. And can be obtained by screening from HIV particles.

  The DNA of the present invention is a base sequence in which one to a plurality of bases are deleted, substituted and / or added in the base sequence shown in SEQ ID NO: 1, and encodes an attenuated HIV-1 virus variant It may have a base sequence.

  The term “attenuated HIV-1 virus mutant” as used herein refers to whether the increase in HIV-1 antibody titer is abnormally slow even if the mutant is infected, and the virus titer cannot be detected over a long period of time. A variant that shows a low value even if it can be detected. Therefore, even when infected with attenuated HIV-1 virus mutants, the progression of HIV-1 pathology is very slow.

Methods for introducing a desired mutation into a given DNA sequence are known to those skilled in the art. For example, constructing DNA having mutations by appropriately using known techniques such as site-directed mutagenesis, PCR using degenerate oligonucleotides, exposure of cells containing nucleic acids to mutagens or radiation, etc. Can do. Such known techniques are, for example, Molecular Cloning:.. A laboratory Mannual, 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplement 1~38 , John Wiley & Sons (1987-1997).

(B) Recombinant vector of the present invention The DNA of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes.
Preferably, the vector used in the present invention is an expression vector. In the expression vector, the DNA of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately determined depending on the type of host.

The promoters which are operative in bacterial cells, P _R or P _L promoters of phage lambda, the E. coli lac, trp or tac promoter, Bacillus stearothermophilus maltogenic amylase gene or Bacillus licheniformis α amylase gene Examples include promoters. Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are promoters. Examples of promoters operable in yeast host cells include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, and an ADH2-4c promoter.

The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes whose complement is lacking in host cells (eg, dihydrofolate reductase (DHFR)), or drug resistance genes (eg, ampicillin, kanamycin, tetracycline, etc.).
Methods of ligating the DNA of the present invention, promoter, and optionally terminator and / or secretory signal sequence, respectively, and inserting them into an appropriate vector are well known to those skilled in the art.

(C) Transformant of the Present Invention A transformant can be prepared by introducing the DNA or recombinant vector of the present invention into a suitable host.
The host cell into which the DNA or recombinant vector of the present invention is introduced may be any cell as long as it can express the DNA of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.

The bacterium may be either a gram positive bacterium (for example, Bacillus, Streptomyces, etc.) or a gram negative bacterium (for example, Escherichia coli, etc.). Bacterial transformation can be performed by using competent cells by a protoplast method or a known method.
Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. For transformation of mammalian cells, for example, an electroporation method, a calcium phosphate method, a lipofection method, or the like can be used.

Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, for example, Saccharomyces cerevis 1ae or Saccharomyces kluyveri. Transformation of a yeast host can be performed using, for example, an electroporation method, a spheroblast method, a lithium acetate method, or the like.
In addition, insect cells can also be used as a host. In that case, after a recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain a recombinant virus in the insect cell culture supernatant, Furthermore, it is possible to infect insect cells with a recombinant virus to express a protein.

(D) Virus particle of the present invention The above transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced DNA, whereby the HIV virus particle of the present invention is cultured in the medium. Can be generated.
The method for isolating and purifying the virus particles of the present invention from the transformant culture can be carried out according to conventional methods known to those skilled in the art. Specifically, for example, the DNA of the present invention is introduced into a host (for example, mammalian cells such as HeLa cells), and the culture supernatant after culturing for 48 to 72 hours is collected, and a filter (for example, a pore size of 0.45 μm) is collected. ) To obtain a cell-free virus preparation.

(E) Utilization of DNA of the Present Invention In the present invention, attention was paid to a case where the antibody response was remarkably low over a long period of time after HIV-1 infection, and the amount of blood virus was below the detection limit, and the genomic structure was analyzed. The HIV-1 CRF01_AE strain has a deletion in the nef / LTR region. A similar attenuated virus has been known for Western subtype B, but it was the first example of a non-subtype B virus that has become a major etiology of the pandemic. The development of AIDS vaccines is currently facing great difficulties due to various factors. Among them, nef deletion attenuated type in the SIV system is the only clear protective effect that has been established. This is a live attenuated vaccine system. CRF01_AE is an epidemic strain that has an important role in the formation of epidemics in Asia, including Japan, and is expected to be a material that contributes to vaccine development targeting the Asian epidemic. That is, the DNA of the present invention is useful for developing a live attenuated vaccine.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(A) Materials and methods (1) Subject and HIV-1 serum test The patient (GM43) is a 22-year-old Thai woman who has lived in Japan for the past 8 years. This patient married a Thai husband (GM46). An HIV-1 screening test (first blood collection) was performed on January 27, 1996 at the 18th week of pregnancy, and Western blot analysis showed an unclear serum reaction (1+ reactivity only with gp160) (Sanofi Fujirebio Diagnostics, Japan) (strip 1 in FIG. 1). The results of the cellodia anti-HIV gelatin particle aggregation (PA) test (Fujirebio, Japan) and enzyme immunoassay (VIDAS Ab-EIA, Bio Merieux, France) showed a weak seropositive with a titer of 1:16. This obscure test result for this patient persisted for 18 months (strip 1-10 and A and B in FIG. 1). This patient's husband (GM46) was found to be HIV-1 positive in April 1996, the Western blot result was clearly positive (Figure 1B), and the antibody titer from the PA test was 1:> 10 It was ⁴ . GM46 is probably sexually infected with HIV-1 in Thailand before 1995, but the exact route of infection is not clear. Newborns who gave birth to GM43 by cesarean section on May 17, 1996 were HIV-negative. GM43 did not receive antiretroviral therapy. The patient had no risk factors other than sexual contact with her husband (GM46), but the exact time of infection was unknown. Both GM43 and GM46 remained healthy with no clinical symptoms. There are no signs of HIV-2 infection.

(2) Preparation of PBMC and detection of RNA content in plasma As previously reported, peripheral blood mononuclear cells (PBMC) were collected from the blood of patient GM43 collected in citrate phosphate dextrose by ficoll-hypaque gradient centrifugation. Separated (January 1996-April 2002). PBMCs were dissolved in lysis buffer containing proteinase K (10 mg / ml) (1x physiological sodium citrate (SSC), 1 mM EDTA, 10% sodium dodecyl sulfate) for 3 hours at 60 ° C and then phenol / chloroform DNA was extracted using ethanol. DNA was concentrated to 1 μg / 5 μl in TE buffer (pH) and stored at −20 ° C. until analysis. Plasma HIV-1 RNA levels were measured by an ultrasensitive method using Amplicor HIV-1 monitor kit (version 1.5) (Roche Diagnostics, NJ). According to this, HIV-1 RNA such as CRF01 (AE can be detected with high sensitivity not only from HIV-1 subtype B strain but also from non-subtype B strain (Triques, K., et al., 1999. Efficiencies of four versions of the AMPLICOR HIV-1 MONITOR test for quantification of different subtypes of human immunodeficiency virus type 1 J Clin Microbiol. 37: 110-6).

(3) Amplification and nucleotide sequencing of HIV-1 genome
The nef-LTR region of the HIV-1 proviral genome is expressed as Env43F14 (sense; 5'-GAGTTAGGCAGGGATACTCAC (SEQ ID NO: 2); nucleotide positions 7922-7912) and 3'LTR43R16 (antisense; 5'-TAAGCACTCAAGGCAAGC-3 '(sequence 3); position 9202-9185) outer primer and Env43F15 (sense; 5'-AGCCTG-TGCCTCTTCAGCTACCA-3 '(SEQ ID NO: 4); positions 52-8083) and MSR5 (reverse; 5'-GCACTCAAGGCAAGCTTTATTGAGGCT-3' ( Amplified by nested PCR using SEQ ID NO: 5), inner primer at positions 9199-9173). The nucleotide numbering of each primer is HIV-1 CRF01 (AE reference strain TH.CM240 (Carr, JK, et al. 1996. Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand J Virol. 70: 5935-43) The nucleotide sequence was determined using the Prism 310 DNA Sequencer (Applied Biosystems) by the Big Dye terminator cycle sequencing method.

(4) Data analysis Nucleotide sequences were determined using CLUSTAL W version 1.4 (Thompson, JD, et al., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res. 22: 4673-80) was used to align with the reference strain and corrected so that the reading frame was not altered by the gap. 100 bootstraps by Kimura's two-parameter distance matrix (Saitou, N., et al., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol. 4: 406-25) A phylogenetic tree was constructed with a copy (Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39: 783-791). Analysis was performed using PHYLIP version 3.573 (Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle).

  The nucleotide sequence determined in the present invention is described in SEQ ID NO: 1 in the sequence listing.

(B) Results (1) Identification of HIV-1-infected individuals with abnormally weak serum responses
According to the serologic test (at the first visit) for a female patient GM43 at the 18th week of pregnancy in January 1996, the HIV-1 serum response was determined in the cellodia anti-HIV gelatin particle agglutination (PA) test (Figure 1A) and the VIDUS ELISA. It was positive but close to the limit (1:16 titer). In Western blots, HIV-1 serum response was unclear (1+ reactivity only with gp160) (strip 1 in FIG. 1B). This unusually low seropositive reaction in GM43 was confirmed in additional samples taken after 5 days and 1 month (strips 2 and 3 in FIG. 1B). This obscure test result persisted for 18 months (strips 1-10 and A and B in FIG. 1B).

Empirically, in most cases of HIV-1 infection, antibody titers from PA testing exceed 1:10 ³ within the first 2 weeks and reach 1:10 ⁴ one month after seroconversion. However, in the case of patient GM43, a low PA titer (less than 1:10 ³ ) persists for 1.5 years, and it takes 5.5 years or more to reach an antibody titer of 1:10 ⁴ (FIG. 1A). In parallel with this abnormally low seroconversion process in the patient, HIV-1 plasma RNA levels were measured for 4.5 years (until May 24, 2000) by ultrasensitive testing using Amplicore version 1.5. Sustained values below the detectable level of <50 copies / ml (FIG. 1A). In July 1997 (1.5 years after the first blood collection), only the env (C2 / V3) region was amplified by nested PCR from HIV-1 proviral DNA from the patient's PBMC DNA. About 2 years after the first blood collection), both the gag (p17) and env (C2 / V3) regions were amplified by nested PCR and HIV-1 proviral DNA was amplified from the patient's PBMC DNA. It was finally confirmed that 1 was infected (FIG. 1A). The CD4 count in patient GM43 decreased gradually from 1,074 to 600 cells in 1 μl of blood during the first 3 years as the plasma virus titer gradually increased. The level of 400-600 was stably maintained without showing symptoms (FIG. 1A).

(2) HIV-1 CRF01 (progressive deletion of nef-LTR region of AE genome) To investigate the abnormally slow mechanism of antibody titer increase in patient GM43, it is known to be associated with the slow progression of the disease. The structural features of the nef-LTR region have been investigated (Deacon, NJ, et al., 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients [see comments] Science. 270 : 988-91; Kirchhoff, F., TC Greenough, DB Brettler, JL Sullivan, and RC Desrosiers 1995. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection [see comments] N Engl J Med. 332: 228-32; and Salvi, R., et al., 1998. Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor J Virol. 72: 3646-57). The nef-LTR region of the proviral genome was subjected to nested PCR from DNA extracted from PBMC of patients collected at the following five time points: The five time points were 1997 (18 months after the first visit) (I); December 1998 (35 months later) (II); April 2000 (51 months later) ( III); January 2001 (57 months later) (IV); February 2002 (74 months later) (V) (Figures 1 and 2) nef-LTR predicted from the complete genome of HIV-1. The size of the amplicon in the region was 1,140 bp, but the size of the amplified fragment ranged from 500 to 1,000 bp (Figure 2A), such an amplicon size range was approximately 100--100 in the nef-LTR region. This shows the presence of a 500 bp deletion (FIG. 2A).

  With the aim of identifying genetic alterations in the nef-LTR region at the sequence level, obtained from samples at designated time points (time points I to V in FIGS. 2 and 3) between July 1997 and February 2000 The PCR product was molecularly cloned. Nucleotide sequences were determined for 16-39 clones at each time point, and CRF01_AE reference strain CM240 (McCutchan, FE, et al., 1992. Genetic variants of HIV-1 in Thailand AIDS Res Hum Retroviruses. 8: 1887-95) Aligned with the nucleotide sequence (Figure 3). Nucleotide sequence alignment revealed the presence of a time-lapse deletion in the nef-LTR region of GM43 (FIG. 2B). An 84-bp deletion (designated Δ84) was detected at time point I (18 months after the first visit), at which time the HIV-1 proviral genome was amplified for the first time (FIGS. 2B and 3). The size of the deletion in the nef-LTR region was continuously increased. That is, the deletion of 201-342-bp (mostly Δ215 and Δ323) at time II (35 months later), and the deletion of 369-623-bp (most major species are Δ391 and Δ402 at time III (51 months later)). ), Deletion at 369-391-bp at time IV (after 57 months) (major species are Δ369 and Δ391), and deletion at 391-503-bp at time V (after 74 months) (Δ369 for major species) , Δ391 and Δ403) were observed, respectively (see FIGS. 2B and 3).

  Replication competent HIV-1 strain (GM43-23) was isolated for the first time at time point V (74 months after the first blood collection) (FIGS. 1 and 2). GM43-23 was found to have a 391-bp deletion (Δ391) in the nef-LTR region (FIG. 3F). Thus, HIV-1 quasispecies lacking 391-bp are considered to be a major functional (replication competent) HIV-1 provirus group in GM43 (FIG. 3F).

(3) Phylogenetic relationship between GM43 and putative contagious person (GM46) The structural features of the HIV-1 genome in a female patient husband (GM46) were examined. Most of the GM46 HIV-1 proviruses did not retain a detectable defect in the nef-LTR region (Figures 2A and 3G), but a small portion (less than 5%) of the GM46 nef-LTR sequence. A unique set of deletions was detected (Figure 3G). However, none of these deletions matched any of the defective genomes identified with GM43 (FIG. 3G).

  Based on the results of phylogenetic tree analysis based on the nucleotide sequence of env (C2 / V3) and nef-LTR region, the HIV-1 sequences obtained from GM43 and GM46 have a high boot trap rate (100% and 98%, respectively). (It was found that a single strain cluster was formed in the AE (Figs. 4A and 4B). The GM43 / GM46 cluster was collected in the same area where GM43 and GM46 live (near Tokyo). Other CRF01 (different from the AE region control sequence (sequence enclosed by the squares in Figure 4A)), indicating that patient GM43 was actually infected from his husband (GM46) and that the defective genome in GM43 was It was suggested that it evolved from a minor virus quasispecies in GM46.

(4) Deletion gene structure in nef-LTR region
The gene structure of nef / LTR deletion in GM43 and GM46 is summarized in FIG. Two major large deletions were detected. The first major deletion was located on the amino terminal side of the nef gene that did not overlap with the LTR. The second major deletion was located in the nef / U3 overlap region. Following these major deletions were one or two other small deletions. Most of the first major deletions removed the highly conserved acidic (EEEE) domain and (Pxx) ₄ motifs essential for Nef function, while downstream deletions nef Even when localized in the / U3 region, the polypurine region (3'-PPT) essential for HIV-1 replication, the NFκB and SP-1 binding sites, and the TATAA box remain (Figs. 3 and 5). This was reported in previous studies on a defective subtype B strain in the US-European population (Deacon, NJ, et al., 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients [see comments] Science. 270: 988-91; Kirchhoff, F., TC Greenough, DB Brettler, JL Sullivan, and RC Desrosiers 1995. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection [see comments] N Engl J Med. 332: 228-32; and Salvi, R., et al., 1998. Grossly defective nef gene sequences in a human immunodeficiency vir us type 1-seropositive long-term nonprogressor J Virol. 72: 3646-57).

(5) Characteristic of nucleotide sequence in nef-LTR deletion
The overall structural arrangement of the nef-LTR deletion in GM43 (belonging to CRF01_AE) is surprisingly similar to that of the attenuated HIV-1 variant C18 reported in the Sydney blood bank cohort (belonging to HIV-1 subtype B) (Fig. 3H) (Deacon, NJ, et al., 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients [see comments] Science. 270: 988-91) (Fig. 3 And 5). The nucleotide sequence containing the first 42 amino acids of the nef open reading frame and the 3′-polypurine region (3′-PPT) is retained in both GM43 and C18 (FIG. 5). Similar to C18, the majority of GM43 quasispecies retain highly conserved sequence elements required for efficient replication of HIV-1, such as TCF-1α, NFκB and SP1 binding sites ( Figures 3 and 5). In GM43, GABP motif (5'-ACTTCCG-3 '), single NFkB, abnormal TATA box (5'-TAAAA-3') and 2 nucleotide bulge in TAR stem (3 nucleotide bulge 5'-UCU-3 ' CRF01_AE (Carr, JK, et al., 1996. Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand J Virol. 70: 5935-43; Gao , F., et al., 1996. The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A / E) recombinant of African origin J Virol. 70: 7013-29; and Kusagawa, S., et al., 2002 Isolation and characterization of replication-competent molecular DNA clones of HIV type 1 CRF01_AE with different coreceptor usages AIDS Res Hum Retroviruses. 18: 115-22) (Figure 5). Nef-LTR region (Salvi, R., et al., 1998. Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor J Virol. 72: 3646-57) The direct repeats that were detected were not detected in the quasispecies in GM43.

(C) Discussion In this example, CRF01 (a special case of AE infection (GM43) in which the increase in HIV-1 antibody titer was abnormally slow and virus titer could not be detected over a long period of time was described. , Was found to have an attenuated virus mutant with a large deletion in the nef-LTR region, which was very similar to that seen in some previously reported LTNPs, which was nef / LTR region This is the first case of HIV-1 non-subtype B infection that has slowed disease progression due to major genetic alterations.

As shown in Fig. 3, with regard to the composition of genetic modification in the nef-LTR region, a remarkable similarity was observed between subtype B and CRF01 (AE, ie, essential for Nef function regardless of strain differences Most of the sequence elements of, such as the highly conserved acidic region (EEEE) required for MHC class I regulation, and interactions between signaling molecules such as Nef and PAK-1 / 2, cell activation (Saksela , K., et al., 1995. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef + viruses but not for down-regulation of CD4 Embo J 14: 484-91), MHC class I regulation, apoptosis inhibition (Wolf, D., et al., 2001. HIV-1 Nef associated PAK and PI3-kinases stimulated Akt-independent Bad-phosphorylation to induce anti-apoptotic signals Nat Med 7: 1217-24) intervening (PxxP) ₃ motifs and downstream sequences are due to genetic modification in the nef region. (Figure 3) There were a number of deletions in the region of nef that overlapped U3 in the LTR, both of which were most important for viral replication. There was no effect on cis-acting elements known to be, such as the 3′-polypurine region (3′-PPT), the U3 terminal sequence, the TATAA box, and the binding site of NFkB and SpI. This suggests that the defective HIV-1 genome has strong selection pressure to maintain minimal replication ability.

  Phylogenetic analysis showed that GM43 acquired HIV-1 from his husband (GM46) (FIG. 4). Although most of the HIV-1 genome in GM46 appeared to be intact, GM46 was found to carry an HIV-1 quasispecies carrying a unique combination of nef / LTR deletions (Figure 3G). . GM43 is thought to have acquired a virus variant with a defective nef allele from GM46. However, it is rather surprising that GM43 did not select the nef allele during transmission from the husband (GM46) and / or during infection colonization. This is because, in rhesus monkeys experimentally infected with nef-deficient SIV, the functional form of the nef allele is selected instantaneously and efficiently (Kestler, HW d., Et al., 1991. Importance of the nef gene for maintenance of high virus loads and for development of AIDS Cell. 65: 651-62). In certain patients, the attenuated virus mutant may have a selective advantage over the HIV-1 strain with the intact nef allele. An immune response appears to be effective in selecting nef-defective viruses that can escape CTL recognition that efficiently controls viral replication (Mariani, R., et al., 1996. High frequency of defective nef alleles in a long -term survivor with nonprogressive human immunodeficiency virus type 1 infection J Virol. 70: 7752-64).

  The above results demonstrate that HIV-1 non-subtype B may also convergently evolve into an attenuated mutant carrying a large nef deletion. This unique case of CRF01_AE infection systematically studies long-term, non-progressive infections, and the inheritance of viruses and hosts in epicenters where HIV-1 development has not progressed in developing countries, such as in Asia Research to elucidate the confounding between factors and immune factors will be facilitated.

FIG. 1 shows changes in parameters over time in serum tests and virus tests on patient GM43. (A) Change in profile of serum test and virus test parameters. (Upper) Detection of proviral HIV-1 DNA by nested PCR of gag (p24) and env (C2 / V3) regions: negative (-); positive (+). The antibody titer (PA titer) of the cellodia gelatin particle agglutination test is shown. (Middle) CD4 + number of cells in 1 ml blood (open circles). (Lower) HIV-1 blood RNA level (copy number / μl (log scale)) (black squares). The proviral HIV-1 genome was analyzed for samples collected at designated time points (A-E). In February 2002 (sample E), HIV-1 was isolated from GM43 for the first time. (B): Western blot analysis (Genelabs Diagnostics HIV 2.2) on serum continuously collected from GM43. Strips 1-9: Serum collected continuously from January 1996 to October 1996. Strips A and B: serum collected in July 1997 (sample collection time A) and serum collected in December 1998 (sample collection time B) FIG. 2 shows the progressive deletion of the nef-LTR region in GM43. (A): Analysis of PCR amplified fragments of HIV-1 nef-LTR region from PBMC of patients collected at designated time points (A to E). DNA products amplified by nested PCR were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. CRF01 (amplified fragment size expected from the intact genome of AE (GM46 used as control) is 1140 bp. (B): Deletion size (y-axis) relative to sampling time point (x-axis) The bar graph shows the percentage of deletions of different sizes in the nef-LTR region at the specified time points. FIG. 3 shows the evolution of the genomic organization of the nef-LTR region in patient GM43. The genomic structure of HIV-1 CRF01 (AE TH.CM240 for the corresponding region is shown at the top. The position of the nested PCR primer is indicated by an arrow. The black box represents the normal sequence, and the blank represents the deletion. Nucleotide positions are shown relative to M240, numbers in the blank area represent the size of the deletion, HIV-1 subtype B as described in patient GM43 and her husband GM46 and Sydney Blood Bank Cohort The genomic organization of each HIV-1 isolate from the attenuated mutant HIV-1C18 is shown for comparison. FIG. 4 shows a phylogenetic analysis based on the nucleotide sequences of the env (C2 / V3) (A) and nef-LTR (B) regions. The phylogenetic tree was constructed by the adjacent joint method based on Kimura's two parameter distance matrix. SIVCPZGAB is used as an outgroup. The bootstrap value (> 80) is shown in the corresponding node. The boot trap value for the GM43 / 36 cluster is circled. On the right side of each phylogenetic tree is the name of the subtype and CRF. FIG. 5 shows the nucleotide sequence of the nef-LTR region in GM43-20, a major quasispecies (Δ391) of patient GM43, and HIV-1 subtype B attenuated mutant described in the Sydney Blood Bank Cohort. A comparison with the nucleotide sequence of 1C18 is shown. (A) Sequence landmarks in the nef-LTR region are shown. C18 carries a 23-bp replica consisting of a pair of NFkB and SpI sequences. (B) Sequence landmark and site of deletion in the nef-LTR region of GM43-20.

SEQUENCE LISTING
<110> National Institute of Infectious Diseases
<120> Attenuated HIV-1 nucleotide sequence
<130> A51025A
<160> 5
<210> 1
<211> 8586
<212> DNA
<213> Human immunodeficiency virus
<400> 1
ggacttgaaa gcgaaagtta atagggactc gaaagcgaaa gttccagaga agttctctcg 60
acgcaggact cggcttgctg aggtgcacac agcaagaggc gagagcggcg actggtgagt 120
acgccaaatt ttgactagca ggggctagaa ggagagagat gggtgcgaga gcgtcagtat 180
taagtggggg aaaattagat gcatgggaaa aaattcggtt acggccaggg ggaaagaaaa 240
aatataggct aaaacattta gtatgggcaa gcagagagtt agaaagattc gcacttaacc 300
ctggcctttt agaaacagca gaaggatgtc aacaaataat agagcagtta cagtcaactc 360
tcaagacagg atcagaagaa cttaaatcat tatttaatac agtagcaacc ctctggtgcg 420
tacaccaaca gataagggta aaagacacca aggaagcttt agataaacta gaggaagtac 480
aaagtaagag ccagcaaaag acacagcagg cagcagctgg cacaggaagc aacagcaaag 540
tcagccaaaa ttaccctata gtgcaaaatg cacaagggca aatgacacat cagcctatat 600
cacctagaac tttgaatgcc tgggtgaaag tagtagaaga aaagggtttt aacccagaag 660
taatacccat gttctcagca ttatcagagg gaggcacccc acaagattta aatatgatgc 720
taaatataat agggggacac caggcagcaa tgcaaatgtt aaaagagacc atcaatgagg 780
aagctgcaga atgggatagg gtacacccag tacatgcagg gcctattcca ccaggccaga 840
tgagggaacc aaggggaagt gacatagcag gaactactag tacccttcaa gaacaaatag 900
gatggatgac aaacaatcca cctatcccag tgggagacat ctataaaagg tggataatcc 960
tgggattaaa taaaatagta agaatgtata gccccattag cattttagac ataagacaag 1020
ggccaaagga acccttcaga gactatgtag ataggttcta taaaactctc agagcggaac 1080
aagctacaca ggaggtaaaa aactggatga cagaaacctt gctagtccaa aatgcgaatc 1140
cagactgtaa gtccatttta aaagcattag gaacaggagc tacattagaa gaaatgatga 1200
cagcatgcca gggagtggga ggacctagcc ataaagcaag ggttttggct gaggcaatga 1260
gccaagcaca acatgcaaat ataatgatgc agagaggcaa ttttaagggc cagaaaagca 1320
ttaagtgctt caactgtggc agagaaggac acctagccag aaattgcagg gcccctagaa 1380
aaaagggttg ttggaaatgt gggaaggaag gacatcaaat gaaagactgc actgagagac 1440
aggctaattt tttagggaaa ttttggcctt cccacaaggg aaggccaggg aattttcctc 1500
agagcagacc agagccaaca gccccaccag cagaaaactg ggggatgggg gaagagacac 1560
cctcctcact gaggcaggag cagaaggaca agcaacagcc tcctccttta atttccctca 1620
aatcactctt tggcaacgac cccttgtcac agtaaaaata ggaggacagc tgaaagaagc 1680
tctattagat acaggagcag atgatacagt attagaggaa ataaatttgc caggaaaatg 1740
gaaaccaaaa atgatagggg gaattggagg ttttatcaag gtaaggcaat atgatcaaat 1800
acttatagaa atttgtggaa aaagagctat aggtacagta ttagtaggac ctacacctgt 1860
caacataatt ggacgaaata tgttgactca gctgggttgt actttaaatt tcccaattag 1920
tcctattgac actgtaccag tagcattaaa gccaggaatg gatggaccaa gggttaaaca 1980
gtggccattg acagaagaaa aaataaaagc attaacagaa atttgtaaag agatggaaga 2040
ggagggaaaa atctcaaaaa ttgggcctga aaatccatac aatactccag tatttgctat 2100
aaagaaaaag gacggcacca aatggaggaa attagtagac ttcagagagc ttaataaaag 2160
aactcaggac ttttgggaag ttcaattagg aataccgcat ccagcaggtt taaaaaagaa 2220
aaaatcagta acagtactag atgtggggga tgcatatttt tcagttcctt tacatgaaag 2280
ctttagaaag tatactgcat tcaccatacc tagtataaac aatgagacac caggaatcag 2340
atatcagtac aatgtgctgc cacagggatg gaaaggatca ccagcaatat tccagaatag 2400
catgacaaaa attttagagc cctttagaat aaaaaatcca gaaatggtta tctatcaata 2460
catggatgac ttgtatgtag gatctgattt agaaataggg cagcacagag caaaaataga 2520
ggagctgaga gctcatctaa tgagctgggg atttactaca ccagacaaaa agcatcaraa 2580
ggarcctccg ttcctttgga tgggrtatga actccatcct gacagatgga cagtccagcc 2640
tatagaactg ccagaaaaag ayagctggac tgtcaatgat atacagaaat tagtgggaaa 2700
actaaattgg gcaagccaaa tttatccagg gattaaagta aagcaactat gtaaactcct 2760
caggggagct aaggcactga cagacatagt gccactgact gaagaagcag aattagagtt 2820
ggcagagaac agggagattc taaaaacccc tgtgcatgga gtatattatg acccatcaaa 2880
agacttaata gcagaagtgc agaaacaagg gcaggatcaa tggacatatc aaatatttca 2940
agagccattt aaaaatctga aaacaggaaa atatgccaga aaaaggtctg ctcacactaa 3000
tgatgtaaga caattagcag aagtggtgca aaaaatagcc acagaaggca taataatatg 3060
gggaaagacc cctaaattta gactacccat acacagagaa acatgggaaa catggtggat 3120
ggagtattgg caggctacct ggattcctga atgggagttt gttaataccc ctcctctagt 3180
aaaattatgg taccaattag aaaaagaccc catagtagga gcagagactt tctatgtaga 3240
tggggcagct agtagggaga ctaagctagg aaaagcagga tatgtcactg acagaggaag 3300
acaaaaggta gtttccctaa ctgagacaac aaatcaaaag actgaattac atgcaatcca 3360
tttagccttg caggattcag gatcagaagt aaatatagta acagactcac aatatgcatt 3420
aggaatcatt caggcacaac cagacaggag tgaatcagaa gtagtcaacc aaataataga 3480
ggagctaata aaaaaggaaa aggtctacct gtcatgggta ccagcacaca aggggattgg 3540
aggaaatgaa caagtagata aattagtcag ttcaggaatc agaaaggtgc tatttttaga 3600
tgggatagat aaggctcaag aagagcatga aagatatcac agcaattgga gaacaatggc 3660
tagtgatttt aatttgccac ctatagtagc aaaggaaata gtagccaact gtgataaatg 3720
tcaactaaaa ggggaagcta tgcatggaca rgtggactgt agtccaggga tatggcaatt 3780
agattgcaca catctagaag gaaaagtcat cctggtagca gtccacgtgg ccagtggata 3840
tatagaagca gaagttatcc cagcagaaac aggacaggag acagcatact ttctgctaaa 3900
attagcaggg agatggccag taaaagtaat acacacagac aacggtagca atttcaccag 3960
cgctgcagtc aaagcagcct gttggtgggc caatgtccga caggaatttg ggatccccta 4020
caatccccaa agtcaaggag tagtagaatc tataaataag gaattaaaga aaatcatagg 4080
gcagrtaaga gagcaagctg aacaccttaa gacagcagta caaatggcag tattcattca 4140
caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa taatagacat 4200
aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa tycaaaattt 4260
tcgggtttat tacagggaca gcagagaccc aatttggaaa ggaccagcaa agctactctg 4320
gaaaggtgaa ggggcagtag taatacaaga caatagtgat ataaaaatag taccaagaag 4380
aaaagcaaag atcattaggg attatggaaa acagatggca ggtgatgatt gtgtggcagg 4440
tagacaggat gaggattaga acatggaaca gtttagtaaa acatcatatg tacgtctcaa 4500
agaaagctaa aaagtggttt tatagacatc attatgaaag ccagaatcca aaggtaagtt 4560
cagaagtata tattccacta ggagaggcta gattagtaat aagaacatat tggggtctgc 4620
agacaggaga aaagractgg caattgggcc atggagtctc catagaatgg aggcagagaa 4680
catatagcac acaaatagat cctgacctcg cagaccaact gattcatcta cactattttg 4740
actgtttttc agactctgcc ataaggaaag ccatattagg acaagtagtt agacgtaggt 4800
gcgaataccc atcaggacat aacaaggtag gatccctaca atatttggca ctgaaagcat 4860
taacaacacc aaaaaggata aggccacctc tgcctagtgt taagaaatta acagaagata 4920
gatggaacaa gccccagaag atcaggggcc acagagagaa cccttcaatg aatggrcakt 4980
agaactgtta gaggagctta aaaatgaagc tgttaggcat tttcctaggc cctggctcca 5040
tggcttagga cggtacatct ataacactta tggagatact tgggaagggg ttgaagctat 5100
artaagaatt ttgcaacaac tactgtttgt tcatttcaga attgggtgtc aacatagcag 5160
aataggcatt ttgccaggga gaggaggcag gaatggagcc agtagatcct aacctagagc 5220
cctggaatca tccgggaagt cagcctacaa ctgcttgtag caagtgttac tgtaaaatat 5280
gttgctggca ttgtcaacta tgctttctga aaaaaggctt aggaatctcc tatggcagga 5340
agaagcggaa gcaccracga gaaactcctc agaacagtaa ggatcatcaa aatcatatac 5400
caaagcagta agtaataagt atatgtaatg acacctttgg aaattagtgc aatagtagga 5460
ctgatagtag cgctaatctt agcagtagta gtgtggacta tagtagctat agaaatagtg 5520
agaatactag agcaaagraa aatagacagg ttagttaaaa gaataagaga aagagcagaa 5580
gacagtggaa atgagagtga aggagacaca gakgaattgg ccaaacttgt ggaaatgggg 5640
gactttggtc cttgggttgg tgataatttg tagtgcctca gacaacttgt gggttacagt 5700
ttattatggr gttcctgtgt ggagagatgc agagaccacc ctattttgcg catcagatgc 5760
caaagcacat gagacagaag tgcacaatgt atgggccaca catgcctgtg tacccacaga 5820
ccccaaccca caagaaatac ccctggaaaa tgtaacagaa aattttaaca tgtggaaaaa 5880
taacatggta gagcagatgc aggaggatgt aatcagtcta tgggatcaaa gtctaaagcc 5940
atgtgtaaag ttaactcctc tctgcgttac tttaaattgt accaatgcta ctttgaccaa 6000
tgttactttg accagagrca ctaacatawc aggtgactat aacataggaa atgtaacaga 6060
tgaagtaaga aactgttctt ttaacatgac cacagaaata agagataaga agcagaatkt 6120
ctatgcactt ttttacaagc ttgatatagt acaaatggat aakagtratg astrtaggtt 6180
aataaattgt aatacttcag tcattaagca ggcttgtcca aagatgtcct ttgatccaat 6240
tcctatacat tattgtactc cagctggttt tgcgctttta aagtgtaata rtaagaattt 6300
caatgggaca gggccatgta amaatgtcag ctcagtacaa tgcacacatg gaattaagcc 6360
agtggtatca actcaattac tgttaaatgg cagtctagca gaagacgaga taataatcag 6420
atctgaaaak ctcacagaca atgccaaaac cataatagtg caccttaata aatctgtaga 6480
aatcaattgt accagaccct ccaacaatac aagaacaagt atacctatag gaccaggaca 6540
agtattctat agaacaggag acataacagg agatataaga aaagcatatt gtaagattaa 6600
tggaacaaaa tggarggaag ttttagcaca ggtagctgga aaattaaaag agcratttaa 6660
taagacaata gtcttccaac caccctcagg aggagatcta gaaattacaa cgcatcattt 6720
tacttgtaga ggggaatttt tctattgcaa tacaacacaa ctgtttaata gtacttgcaa 6780
trgtacttgc acaggagata atggcactat cacacttcca tgcagaataa agcaaattat 6840
aaacatgtgg cagggagcag gacaagcaat atatgctcct cccgtcagtg gaanaattaa 6900
ttgtgtatca aacattacag gaatactatt gacaagagat ggtggtgtta ataatagtgc 6960
aactaacgag accttcagac ctggaggagg aaatataaag gacaattgga gaagtgaatt 7020
atataaatat aaagtagtac aaattgaacc actaggaata gcacccacca gggcaaagag 7080
aagagtggtg gaaagagaaa aaagagcagt gggaatagga gctatgatct ttgggttctt 7140
aggagcagcc ggaagcacta tgggcgcggc gtcaataacg ctgacggtac aggccagaca 7200
attattgtct ggtatagtgc aacagcaaag caatttgctg agggctatag aggcgcagca 7260
gcatctgttg caactcacag tctggggcat taaacagctc caggccagag tcctggctgt 7320
ggaaagatac ctaaaggatc aaaagttcct aggactttgg ggctgctctg gaaaaatcat 7380
ctgcaccact gctgtgccct ggaactccac ttggagtaat aaatcttttg aagagatttg 7440
gaacaatatg acatggatag aatgggagag agaaattagc aattacacaa cacaaatata 7500
tgagatactt acaaaagcgc aggaccagca ggacagaaat gaaaaggatc tgttagaatt 7560
ggataattgg gcaagtctgt ggaattggtt tgacataaca aattggctgt ggtatataaa 7620
aatatttata atgatagtag gaggtttaat aggtttaaga ataatttttg ctgtgctttc 7680
tatagtaaat agagttaggc agggatactc acctttgcct ttccagaccc ctacccatca 7740
gcagagggaa cccgacagac ccgaaagaat cgaagaagga ggtggcgagc aaggcagaga 7800
cagatccgtg cgattagtga ccggattctt agcacttgcc tgggacgatc tacggagcct 7860
gtgcctcttc agctaccacc gcttgagaga cttcatcttg gttgcagcga ggattgtgga 7920
acttctggga cgcagcagtc tcaagggact gagacggggg tgggaaggcc tcaaatatct 7980
ggggaacctt ctgttatatt ggggacagga actaaaagct agtgctattt ctttgtttga 8040
tgctacagca atagcaatag cgggrtggac agatagggtt atagaagtag cacaaagagc 8100
ttggagagcc attctccaca tacctagaag aatcagacag ggcttagaaa gggctttgct 8160
ataacatggg aagcaagtgg tcaaaaagta acatagtggg atggcctcct tttaaaagaa 8220
aaggggggac tggatgggct agtttagtag aggagaacaa caaagaagaa gacaactgcc 8280
tgtyacaccc cataagccag catggaatag atgatgaaga aagagaagtg ctgatgtgga 8340
agtttgacag tgccagaact gcacccagag ttctataaag actgctgaca aagaagtttc 8400
taactaggac ttccgctggg gactttccag gggaggtgtg gccggggcgg agttggggag 8460
tggctaaccc tcagatgctg cataaaagca gccgctttcc gcttgtactg ggtctctctt 8520
gttagaccag gtcgagcccg ggagctctct ggctagcaag ggaacccact gcttaagcct 8580
caataa 8586
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 2
gagttaggca gggatactca c 21
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 3
taagcactca aggcaagc 18
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 4
agcctgtgcc tcttcagcta cca 23
<210> 5
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 5
gcactcaagg caagctttat tgaggct 27

Claims (5)

DNA having any of the following base sequences:
(1) the base sequence set forth in SEQ ID NO: 1; or (2) in the base sequence set forth in SEQ ID NO: 1, wherein one to a plurality of bases are deleted, substituted and / or added, Base sequence encoding an attenuated HIV-1 virus variant: A recombinant vector comprising the DNA according to claim 1. A transformant comprising the DNA according to claim 1 or the recombinant vector according to claim 2. A virus particle prepared by culturing a transformant obtained by transforming the DNA of claim 1 or the recombinant vector of claim 2 into a host. An HIV vaccine comprising the viral particle of claim 4.