KR101049858B1 - HIV-1 latent cell lines, methods for their preparation and diagnostic methods for latent HIV infection - Google Patents

Abstract

The present invention relates to a latent HIV-infected cell line, a method for preparing the same, and a method for diagnosing latent HIV infection. ) Latent HIV-infected cell line NCHA-3 characterized in that the gene is fused. For this reason, the latent HIV-infected cell lines of the present invention contain HIV proviruses and exhibit molecular molecular characteristics different from those of conventional latent cell lines, which are useful for studies on the latent nature and reactivation of HIV proviruses. Furthermore, by effectively identifying the latent pathology of HIV, it can ultimately contribute to the treatment and prevention of AIDS in HIV / AIDS patients.

Cell line, HIV-1, latent infection, decreased receptor expression, histone H3 modification

Description

HIV-1 latent cell line, a method for preparing the same, and a method for diagnosing latent HIV infection {HIV-1 latent cell line, its manufacturing process, and diagnostic method of HIV-1 latency}

The present invention relates to a latent HIV infected cell line and a method for preparing the same.

Since the HIV virus, an AIDS causative agent, was isolated in the early 1980s, many researchers around the world have tried to overcome AIDS, but no effective preventive and therapeutic vaccines have been developed. In clinical terms, highly active antiretroviral therapy (HAART) treatment for HIV and AIDS patients significantly reduced AIDS incidence and AIDS-related mortality, but the resting CD4 + T cells of HIV-infected patients, namely latent HIV-infected hospitals ( Persistent presence of HIV reservoirs has been a major barrier to the eradication of HIV virus from the host (Ho et . Al . , 1995; Chun et . Al ., 2000). These latent infected cells serve as a critical cause of HIV virus reactivation after HAART discontinuation (Chun et . Al ., 1999). The biggest challenge to be solved in the field of HIV research around the world is 'How can HIV stay latent in the host and how can we effectively eliminate the HIV-infected pathogen in the host?' It is a buzzword. Thus, a detailed understanding of the molecular mechanisms regulating the latency and reactivation of HIV proviruses will provide clues to the cure of AIDS disease through the removal of intracellular HIV pathogens.

According to a recent research report, HIV Pro latent cells with the virus may be removed by treatment with various chemical drugs, such as Kin -2 (interleukin-2) and PMA (phorbol myristyl acetate) to inter (Fraser et. Al. , 2000; Alexaki et . Al ., 2007). However, these chemicals are very toxic and are not effective in removing HIV-1 pathogens from HIV infected people. In addition, it has been reported that intracellular proteins including proteasome, histone deacetylase (HDAC) and TRAIL are associated with the latent nature of HIV (Lum et . Al ., 2001; Krishnan and Zeichner, 2004; Williams et. . al;. 2006). It is reported that latent infected cells such as ACH-2, J1.1 and U1 cells, which are currently used all over the world, show significant differences in gene expression patterns from normal cells (Krishnan and Zeichner (2004)). HDAC inhibitors such as valporic acid and trichostatin A have been evaluated to reactivate HIV pathogens to stop chronic HIV infection (Lehrman et . Al ., 2005; Vandergeeten et. al . , 2007). In particular, the HIV chronic infectious mechanism by HDAC has been suggested in HIV latent induction models by CBF-1 / HDAC interaction and c-Myc / Sp1 / HDAC complex (Tyagi M and Karn J, 2007; Jiang et . al . , 2007). Recent epigenetic studies have shown that intracellular histone protein inhibitors such as HAT (histone acetyltransferase), HDAC (histone deacetylase) and HMT (histone methyltransferase) inhibitors can be used as therapeutic agents for pathogenic infections (Mai, 2007). . However, most of the determinant cell factors and detailed mechanisms involved in HIV latent infection have yet to be identified, and there are limitations on the use of HIV chronically infected cell lines.

Accordingly, in the present invention, three different HIV types that can represent various infection states, including cell lines capable of maintaining a more stable level of chronic infection and maintaining high levels of virus reactivation compared to existing infected cell lines. Chronic infected cell lines were prepared, the molecular biological characteristics of the prepared chronically infected cell lines were investigated, and the mechanism of HIV infection by chromatin-modified proteins was studied.

An object of the present invention is a novel latent HIV-infected cell line NCHA-3 (New chronic HIV-infected A3. 01-derived cell-3), to provide a cell line useful in the field of research on the latent and reactivation of HIV provirus.

The present invention provides a latent HIV-infected cell line NCHA-3, wherein a human immunodeficiency virus (HIV) gene is fused to p14.2 of chromosome 7 of a human T-lymphocyte A3.01 cell line.

In addition, a) infecting human T-lymphocyte A3.01 cell line with HIV, fusing the gene of HIV to p14.2 of chromosome 7;

b) when the viability of HIV infected A3.01 cells is less than 5%, screening for a potential latent HIV infected cell line via marginal dilution cloning. To provide.

The present invention also provides a method for diagnosing latent HIV infection, characterized by identifying an HIV gene fused to p14.2 of chromosome 7 of a T lymphocyte cell line taken from a human.

Preferably, a method of diagnosing latent HIV infection is characterized in that HIV is a pNL4-3 virus.

Preferably, the method for diagnosing latent HIV infection is characterized in that the T lymphocyte cell line is an A3.01 cell line.

Preferably, the method provides a method for diagnosing latent HIV infection, wherein the cell line is treated with PMA to reactivate the HIV-provirus and then the amount of HIV-1 p24 antigen produced is measured.

Preferably, the cell line is treated with TNF-a or TSA to provide a method for diagnosing latent HIV infection, characterized by measuring acetylation of histone H3 protein.

The latent HIV infected cell line for HIV / AIDS treatment according to the present invention includes HIV provirus, and can maintain a high level of virus reactivation while maintaining a more stable state of chronic infection as compared to conventional infected cell lines, and It is an HIV chronically infected cell line that can represent an infection state. As a result, it is useful for studies on the latent nature and reactivation of HIV proviruses, and is effective in identifying the latent pathology of AIDS, and ultimately contribute to the treatment and prevention of AIDS.

Hereinafter, the present invention will be described in detail. Those skilled in the art will appreciate that various modifications and changes can be made in the present invention without departing from the spirit and scope of the invention as set forth in the claims below.

Example

Example  One: HIV  Generation of latent cells

<1-1> Obtaining Cell Line

Control cell lines (A3.01, ACH-2) were purchased through the National Institutes of Health AIDS Research and Reference Reagent Program. ACH-2 is a chronic HIV-1 infected cell line, including HIV-1 LAV standard strain, and A3.01 cells are parental cell lines uninfected with homologous standard strain. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS (fetal bovine serum), 5% penicillin-streptomycin and 5% CO ₂ , 2 mM glutamine at 37 ° C. under a 95% air-humidity controlled incubator. Cell concentration of the culture flask was maintained at 1 x 10 ⁶ cells / ml. Human T-lymphocyte A3.01 cells infected with HIV-1 pNL4-3 standard strain (Genebank Acc. No. AF324493) with multiplicity of infection (MOI) 0.3 were cultured at 37 ° C. After 1 hour of exposure to virus, fresh medium was added to the infected cells and 1 × 10 ⁶ cells / ml in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 5% penicillin-streptomycin and 2 mM glutamine. The cells were cultured at the concentration of. Live and dead cell numbers were measured over time after virus infection, and supernatants containing virus were collected.

<1-2> HIV -One DNA Screening of Cell Lines Containing

HIV-1 pNL4-3 570 potential A3.01-derived HIV latent infected cell lines were prepared by limiting dilution cloning when the viability of HIV infected A3.01 cells was less than 5% after 50 days of virus infection.

Total genomic DNA was isolated by DNeasy Midi Kit and Blood & Cell Culture DNA Kit (Qiagen, Valencia, California), respectively, according to the manufacturer's protocol. The concentration and purity of the total DNA were measured by spectrophotometer, and the separated genomic DNA was stored in 100 µl of RNase-free distilled water and 100 µl of Tris-EDTA buffer (pH 8.0) until further use. . Screening of potential HIV infected cells was carried out via HIV DNA PCR and HIV p24 antigen ELISA using SK38 / SK39 primers.

New chronic HIV-infected A3.01-induced cells (NCHA) from 570 potential HIV latent infected cell lines by HIV-1 gag PCR and real-time PCR using GeneAmplimer® HIV-1 Control Reagents (Applied Biosystems, Foster City, California) : New chronic HIV-infected A3.01-derived cells) were obtained. This included fusion proviruses (FIG. 1A) and microexpressed HIV-1 p24 antigen (FIG. 1B). In Figure 1 A3.01 shows HIV-1 uninfected cells (negative control cell line); ACH2 is an HIV-infected cell line (positive control cell line); NCHA (A3.01 cell-derived HIV-1 chronically infected cell line) is a generated cell line and results in amplification of DNA size as expected. Data is presented as the mean (± standard error) of three measurements.

Example  2: HIV -1 fusion ( integration Inverse to the contiguous sequence of the Inverse ) PCR

Analysis of the HIV-1 fusion site was performed by modifying Dr. Siliciano's reverse-PCR method. Restriction Enzyme Pst I (New England Biolabs, Beverly, Massachusetts) was used to cut 50 ng of genomic DNA from the cell line of the invention. Pst I digested DNA was incubated overnight at 37 ° C. and then Pst I restriction enzyme was inactivated and DNA was purified by ethanol-precipitation method followed by ligation with DNA T ₄ ligase (Roche Diagnostics Corporation, Indianapolis, Indiana). Conjugated circularized genomic DNA was purified by ethanol-precipitation method and the HIV-1 contiguous sequence was amplified with primers developed by Han et al. In Dr. Siliciano's laboratory.

The sequence of the first primer pair is the forward primer (Outer LTR) 5'-TAA CCA GAG AGA CCC AGT ACA GGC-3 'and the reverse primer 5'-GGT CAG CCA AAA TTA CCC TAT AGT G -3' ; The second primer sequence is the forward primer (Inner LTR) 5'-TGG TAC TAA CTT GAA GCA CCA TCC A -3 'and the reverse primer (Inner gag) 5'- TGT TAA AAG AGA CCA TCA AGT AGG AAG-3'. 50 µl of the PCR mixture was 5.0 µl of cyclic genomic DNA; 5.0 μl 10 × -EF-Taq buffer; 0.4 μl (2.5 U) of EF-Taq polymerase; 1.5 μl of each 10 μM primer for HIV-1 DNA; 1.5 μl each 10 mM dNTP and 35.1 μl distilled water. PCR was repeated 30 times for 3 minutes at 94 ° C, 30 seconds at 94 ° C, 1 minute at 65 ° C, and 2 minutes at 68 ° C, and final extension was performed for 4 minutes at 68 ° C. Second reverse PCR was performed using 5.0 μl of the nested primer and the first reverse PCR product developed by Dr. Han et al. Under the same conditions as the first reverse PCR. Nested PCR bands were isolated from gels and sequenced by Inner LTR primers using an ABI PRISM 3730XL DNA analyzer and BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, California).

In order to reconfirm the HIV-1 fusion site obtained from the modified reverse PCR, a specific primer was prepared using the site where the nucleotide sequence was determined, and the result was confirmed by performing direct genomic DNA PCR. The HIV-1 fusion site was determined using the UCSC Bioinformatics Human Genome Database. In Table 1, a means the junction site sequence between the 5 'end of the HIV-1 LTR (TTCCA) and the host cell DNA, b means the integration site, and UN (unknown) is unknown. it means.

As shown in Table 1, HIV-1 proviruses in three NCHA cell lines were inserted at different chromosomal positions and specific region genes, and the number of inserted HIV-1 proviruses was 1-2. NCHA-1 cell line inserts two HIV proviruses into the host chromosome; One of them fused to the GDAP2 gene on chromosome 1 and the other to the gene on chromosome X. Each NCHA-2 and NCHA-3 cell line was fused with one HIV provirus to the host chromosome. In NCHA-2 cell line, it was fused to AK096155 on chromosome 11 and in NCHA-3 cell line to ANL gene on chromosome 7.

Example  3: HIV -One DNA For cell lines containing PMA  Inductive analysis

Chronic HIV infected cell lines (ACH-2 cells) and uninfected parental cell lines (A3.01 cells) were used as positive and negative controls, respectively. All cell lines were washed twice with PBS (phosphate-buffered saline) to remove the p24 antigen present in the supernatant prior to PMA treatment. PMA 20 ng / ml was used to stimulate HIV infection and uninfected cells, and the number of cells used for this test was 1 x 10 ⁵ cells / ml. HIV chronically infected cells and maternal cells were divided into groups treated with PMA and groups not treated with PMA. Cell supernatants from each cell line were collected for p24 antigen concentration measurements at 0, 24, 48 and 72 hours after PMA treatment. The p24 antigen concentration present in the cell supernatant was measured according to the manufacturer's protocol using the HIV-1 p24 antigen capture assay kit (Vironostika® Biomeriex, Boxtel, the Netherlands). Four independent PMA induction experiments were performed for biological replicates.

The amount of HIV-1 p24 antigen produced at various time points (0, 24, 48 and 72 hours) after PMA treatment was measured and shown in FIG. 2. White and gray bars in FIG. 2 indicate the amount of HIV-1 p24 antigen produced in the supernatant before and after PMA treatment at each time point. All test results are expressed as the mean (± standard error) of the three measurements. ACH2 cells and NCHA cell lines significantly increased HIV-1 p24 antigen production after PMA treatment (FIG. 2; gray bars). The rate of HIV-1 replication was very different in ACH2 cells and NCHA cell lines, and in particular, the production of HIV-1 p24 antigen after PMA treatment in NCHA-1 and NCHA-2 cell lines was significantly increased compared to that before PMA treatment. Without PMA treatment, HIV-1 p24 antigen production in ACH2 cells gradually increased over time, whereas HIV-1 p24 antigen production in NCHA-1 and NCHA-2 cell lines was significantly higher than that of ACH2 and NCHA-3 cell lines. Low (FIG. 2; white bar).

Example  4: HIV -1 infectivity of progeny virus

An HIV infectivity test was performed on target T cells of the HIV-1 descendant virus produced by the cell line of the present invention using an A3.01 cell line. The target cell number and HIV-1 p24 antigen production used for the HIV infection test were 2 × 10 ⁵ and 10 ng, respectively. The cell supernatant volume required for 10 ng of HIV-1 p24 antigen production from each cell line was calculated and the calculated amount added to the target cells (A3.01 cells). Supernatants were collected after 0, 24, 48 and 72 hours to determine HIV-1 p24 antigen production over time after the addition of 10 ng of HIV-1 p24 antigen production. HIV-1 p24 antigen concentration was measured according to the manufacturer's protocol using the HIV-1 p24 antigen capture assay kit (Vironostika® Biomeriex, Boxtel, the Netherlands) and three independent experiments were performed for biological repeats. The test results are expressed as the mean (± standard error) of the three measurements. Both HIV-1 progeny virus generated in ACH2 cells and NCHA cell lines showed infectivity to A3.01 cells, which are target cells (FIG. 3). The time when HIV-1 progeny virus was highly infectious was when HIV-1 progeny generated in ACH2 and NCHA-3 cell lines was 24 hours after infection, whereas in NCHA-1 and NCHA-2 cell lines. The resulting HIV-1 progeny virus was 48 hours after infection (FIG. 3).

Example  5: TNF -α and NF by κB HIV  Reactivation of Provirus

The present invention relates to reactivation of fused HIV-1 provirus in NCHA chronic HIV infected cell lines. Western blots including flow cytometry, HIV-1 p24 antigen production and electrophoretic mobility shift assay (EMSA) were performed to determine the role of tumor necrosis factor alpha (TNF-α) and NFκB p65. Individual cells were collected and washed in PBS to lyse the cells and extract the nuclei using Panomix (AY 2002) nuclear extraction kit. Protein concentration was measured with a micro-BCA protein analysis kit (# 23235, PIERCE). Nuclear extracts (protein 5 ug) were incubated with biotin-labeled double stranded oligonucleotide probes. Probes were specified with EMSA kit and activator protein NFκB (5'-AGTTGAGGGGACTTTCCCAGC-3 ') obtained from Panomix (AY 1030). DNA-protein complexes were digested with 6% DNA retardation gel (EC63652; Invitrogen). Gels were transferred to a Biodyne B nylon membrane (P / N 60201, PALL) and detected using streptavidin-HRP and a chemiluminescent substrate. Blots were imaged by exposure to x-ray film; NFκB p65 (CST-3034), Phospho-NFκB p65 (Ser536) (CST-3031), Phospho-NFκB p65 (Ser276) (CST-3037), β-actin (sc-1616), Acetyl-Histone H3 (Lys9) (CST-9671), Di-Methyl-Histone H3 (Lys9) (CST-9753), Di-Methyl-Histone H3 (Lys27) (CST-9755), and Acetyl-Histone H3 (Lys27) (Upstate, 07-360 ). All test results are expressed as the mean (± standard error) of the three measurements.

As shown in FIG. 4A, the amount of HIV-1 p24 antigen produced in the cells after 48 hours of treatment with TNF-α increased by three-fold in ACH2 cells, but did not show a significant increase in NCHA cell lines. On the other hand, after 48 hours of treatment with TNF-α shown in FIG. 4B, the amount of HIV-1 p24 antigen present in the supernatant was significantly increased in all HIV-1 chronically infected cells compared with TNF-α treatment. In particular, the increase rate of HIV-1 p24 antigen production by TNF-α in NCHA-1 and NCHA-2 cell lines was significantly higher than that of ACH2 cells and NCHA-3 cell lines.

In addition, as shown in FIG. 4C, phosphorylated NFκB p65 protein expression of NFκB p65 and serine 276 residues in the NCHA-1 cell line was significantly reduced compared to other cells. In the electrophoretic mobility shift assay (EMSAA) assay shown in FIG. 4D, the NCHA-1 and NCHA-2 cell lines reduced the serine 276 residue phosphorylated NFκB p65 protein and NFκB complex compared to A3.01 cells, whereas ACH2 cells and NCHA- There was no significant difference in the three cell lines.

Example  6: NCHA  In cell lines HIV Surface Density Measurement of --1 Receptors and Coreceptors

The HIV-1 receptor (CD4 receptor) and co-receptor (CXCR4 / CCR5 co-receptor) are essential for the influx of HIV virus into target cells and their expression patterns were measured in NCHA cell lines. Cell surface and intracellular staining for CD3 receptor, CD4 receptor and CXCR4 co-receptor, CCR5 co-receptor were stained using monoclonal antibodies (mAbs) for the following molecules: CD3 (UCHT1 [phycoerythrin-Cyanine5]; Beckman Coulter), CD4 (13B8.2 [fluorescein isothiocyanate]; Beckman Coulter), CXCR4 (12G5 [R-phycoerythrin]; Beckman Coulter), CCR5 (2D7 / CCR5 [phycoerythrin-Cyanine5]; Beckman Coulter). Each cell was collected and washed with staining buffer (1XPBS containing 2% FBS). Cell pellets were resuspended and either antibody bound to 10 fluorochromes (used in all anti-mouse mAbs) or mouse IgG1 isoform controls were added. Subsequent incubation for 20 minutes followed by two washes and analysis of samples with FC500 flow cytometer (Beckman Coulter, Flullerton, CA). As shown in FIG. 5A, A3.01 cells not infected with HIV-1 showed high CD3-CD4 + T subsets (60.7%) and low CD3 + CD4- T subset (8.1%). CD3-CD4 + T subsets were rarely detected in all latent A3.01-induced cell lines, ACH2 cells and NCHA cell lines. CD3 + CD4- T subsets are four times higher in NCHA-2 and NCHA-3 cell lines compared to HIV-1 uninfected A3.01 cells (NCHA-2 cell line: 34.4%, NCHA-3 cell line: 35.7) %), Similar levels in ACH2 cells (ACH2 cells: 9.7%). However, in the NCHA-1 cell line, neither CD4 nor CD3 receptor was detected on the cell surface. As described above, CD4 surface molecules, which are HIV-1 receptors, significantly reduced expression in latent HIV infected cell lines (receptor down-regulation). Next, by examining the expression of HIV-1 co-receptors involved in HIV tropism, NCHA-1 cell lines express CCR5 and / or CXCR4 co-receptors, whereas in other cell lines, only CXCR4 co-receptors are present at the cell surface. Expressed. About a third (33.5%) subset of NCHA-1 cell lines expressed both CCR5 and CXCR4 co-receptors (FIG. 5B).

Example  7: HIV -One Latent  And Histone  Protein modifications ( Histone H3 modification Association with)

Histones play an important role in gene activity and silencing. Histone H3 acetylation was quantified using PathScan acetylated histone sandwich ELISA (Cell Signaling). The test results are expressed as the mean (± standard error) of the three measurements. Histone H3 acetylation was induced by treatment of tumor necrosis factor alpha (TNF-α) and trichostatin A (TSA) in NCHA cell lines. Histone H3 acetylation in latent cell lines was measured quantitatively after 24 hours treatment with TNF-a (A) and TSA (B). As shown in FIGS. 6B and 6D, all HIV chronically infected cell lines rapidly increased histone H3 acetylation after TSA treatment, whereas only minor changes were observed after TNF-α treatment (FIGS. 6A and 6B). This suggests that the degree of activation of HIV-1 latent infected cells by increasing histone H3 acetylation is different depending on TNF-α and TSA (trichostatin A). Further subdivided, the acetylation and methylation changes of 9 and 27 lysine residues of histone H3 protein were measured by Western blot. Only HDAC inhibitor TSA significantly induced the acetylation of 9 and 27 lysine residues of histone H3 protein. On the other hand, both TNF-α and TSA (trichostatin A) reduced methylation of lysine residue 27 (FIGS. 6C and 6D). Consistent with this, TSA induced significantly more reactivation of HIV provirus from ACH2 cells and NCHA cell lines compared to TNFα treatment (FIGS. 6E and 6F).

1 shows the results of limiting dilution screening results (A) and HIV-1 p24 antigen expression (B) of latent provirus (B) for obtaining cell line NCHA-3 according to the present invention.

2 shows the induction of HIV-1 proviral reactivation before (-) and after (+) PMA induction over time.

Figure 3 is a result confirming the infectivity of the target cells of HIV-1 progeny virus generated from the cell line according to the present invention.

4 is according to the invention by TNF-α and NFκB The results show the reactivation of HIV provirus in the cell line.

5 is a result of measuring the surface density of the HIV-1 receptor (A) and co-receptor (B) in the cell line according to the present invention.

Figure 6 is a result of finding the association between HIV-1 latent and histone H3 protein modification in the cell line according to the present invention.

Claims (9)

A latent HIV-infected cell line NCHA-3 (Accession No .: KCLRF-BP-00185) characterized by fusion of a human immunodeficiency virus (HIV) gene to p14.2 of chromosome 7 of a human T-lymphocyte A3.01 cell line. The latent HIV-infected cell line NCHA-3 according to claim 1, wherein the HIV gene is a pNL4-3 virus gene. a) infecting human T-lymphocyte A3.01 cell line to fuse the gene of HIV to p14.2 of chromosome 7; b) screening for potential latent HIV-infected cell lines through marginal dilution cloning when viability of HIV-infected A3.01 cells is less than 5%. Manufacturing method. The method of claim 3, wherein the HIV gene is a pNL4-3 virus gene. A method for diagnosing latent HIV infection, characterized by identifying an HIV gene fused to p14.2 of chromosome 7 of a T lymphocyte cell line taken from a human. The method for diagnosing latent HIV infection according to claim 5, wherein the T lymphocyte cell line is an A3.01 cell line. The method of claim 5, wherein the HIV is a pNL4-3 virus. The method according to any one of claims 5 to 7, wherein the cell line is treated with phorbol myristyl acetate to reactivate the HIV-provirus, and then the amount of HIV-1 p24 antigen is measured. How to diagnose latent HIV infection. 8. The cell line is treated with tumor necrosis factor alpha or trichostatin A to measure the acetylation of the histone H3 protein. How to diagnose latent HIV infection.

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