KR101664291B1 - NUCKS1, Novel Coactivator regulating the transcriptional activity of HIV-1 Tat - Google Patents

Abstract

The present invention relates to a composition for treating and preventing HIV-1 infection comprising siRNA against a novel coactivator NUCKS1 that modulates the transcriptional activity of HIV-1 Tat. The present invention also relates to a composition for inhibiting reactivation of latent infectious HIV-1 virus comprising siRNA against NUCKS1. By using a composition comprising siRNA for NUCKS1 according to the present invention, it is possible to treat and prevent the HIV virus and effectively inhibit the reactivation of the HIV virus in latency period. In addition, the compositions according to the present invention can be used to screen HIV infection treatment agents and HIV re-activation inhibitors of the latent period of infection. By describing the mechanism of regulation of Tat, a major factor of HIV protein, HIV treatment target including various cell factors of host cell related to Tat protein applicable to HIV treatment and therapeutic possibility of HIV using said target Respectively.

Description

The novel coactivator NUCKS1 (NUCKS1, Novel Coactivator regulating the transcriptional activity of HIV-1 Tat), which regulates the transcriptional activity of HIV-1 TAT,

The present invention relates to a composition for the treatment and / or prevention of HIV-1 infection comprising an siRNA against a novel coactivator NUCKS1 which regulates the transcriptional activity of HIV-1 Tat or a composition for inhibiting reactivation of latent infectious HIV- ≪ / RTI > The present invention relates to a method of treating and / or preventing HIV-1 infection using the composition or inhibiting the reactivation of latent infectious HIV-1 virus. The present invention also relates to a method of screening for an HIV-1 infection therapeutic agent and a reactivation inhibitor of latent infectious HIV-1 infection.

The Tat protein of the HIV-1 virus is a transactivator protein that is encoded by HIV-1 (human immunodeficiency virus type 1), and plays an essential role in the effective transcription and disease progression of HIV- (Dayton AI, Sodroski JG, Rosenca, Goh WC, Haseltine WA. Cell, 44: 941-947, 1986). Tat cooperatively binds to transcriptional-transactivation-responsive (TAR) RNA located at the 5 'end of the HIV-1 viral RNA as a cell factor, followed by an extension of the transcription. The cellular factors of various hosts are essential for the transcriptional activity of Tat. Cyclin T1 (Cyc T1) complexes with Tat, Cyclin Dependent Kinase 9 (CDK) and positive transcription factor b (P-TEFb). The resulting complex is transferred to TAR RNA, and CDK9 bound to cyclin T1 induces phosphorylation of the C-terminal domain of RNA polymerase II, thereby effecting an effective transcriptional process of HIV-1. Proteins interacting with Tat, such as Tip 60, HT2A, CA150, TFIID, Tat-SF1, Tip30, p300, CREB-binding proteins, PC4 and Tip110, which regulate the interaction of Tat with TAR RNA and / or other host factors (Yamamoto T, Horikoshi M., The Journal of Biological Chemistry, 272: 30595-30598, 1997). In addition, cell transcription factors such as SP1, NF-KB, TBP, and nuclear factor of activated T cells (NFAT) activate HIV-1 LTR promoter activity through target components located upstream of TAR RNA in the HIV-1 LTR promoter . Tat regulates HIV-1 gene expression in a variety of host cells and various external stimuli by forming a complex regulatory network with interactions with host cell factors, transcription factors and epigenetic factors. Thus, identification of key factors for Tat-mediated HIV-1 gene expression is essential for the potential target screening of HIV replication and antiviral agents (Jeang KT, Xiao H, Rich EA, The Journal of Biological Chemistry, 274: 28837-28840, 1999).

On the other hand, NUCKS1 (Nuclear Ubiquitous Casein and Cyclin-dependent Kinase Substrate 1) is a protein that is highly phosphorylated by casein kinase II (CK II), cyclin-dependent kinase 1 (Cdk1) (Grundt K, Haga IV, Huitfeldt HS, Ostvold AC. Biochimica et biophysica acta, 1773: 1398-1406, 2007). In addition, although NUCKS1 is predicted to be an important factor involved in the regulation of various key factors in the cell, its precise role and mechanism of action have not been clearly elucidated.

Thus, the present inventors observed the Tat regulation mechanism related to major factors in the host cell using the yeast two-hybrid screening system, and found that the Tat protein accumulated in the TAR RNA region of the HIV-1 LTR promoter, 1 virus, we confirmed that NUCKS1 is a novel partner interacting with Tat, and confirmed that the composition containing NUCKS1 siRNA inhibited HIV-1 infection and / or HIV-1 recurrence in the incubation period The present invention has been completed. In addition, an effective method of treating HIV-1 infection using the composition according to the present invention and inhibiting the recurrence of HIV-1 in the latent period of infection has been proposed.

Non-Patent Document 1: Cell 1986, 44, 941-947. Non-Patent Document 2: The Journal of Biological Chemistry 1997, 272, 30595-30598 Non-Patent Document 3: The Journal of Biological Chemistry 1999, 274, 28837-28840 Non-Patent Document 4: Biochimica et biophysica acta 2007, 1773: 1398-1406 Non-Patent Document 5: Proc Natl Acad Sci USA 2002, 99, 5515-5520. Non-Patent Document 6: Science 2002, 296, 550-553. Non-Patent Document 7: Nature Biotechnology 2002, 20, 505-508. Non-Patent Document 8: Nature Biotechnology 2002, 20, 500-505. Non-Patent Document 9: RNA 2002, 8, 1454-1460.

It is an object of the present invention to provide a method for screening a therapeutic agent for HIV-1 infection and / or a reactivation inhibitor of an infection latent period.

It is still another object of the present invention to provide a method and / or composition for treating HIV-1 infection, a method for preventing recurrence by inhibiting HIV-1 reactivation of the latent period of infection, and / or a composition.

In order to accomplish the object of the present invention, the present invention provides a method for screening an HIV-1 infection treatment agent and / or an HIV-1 virus reuptake inhibitor of a latent period of infection by measuring the expression of NUCKS1 in one embodiment.

The present invention provides a pharmaceutical composition for treating and preventing an HIV-1 viral infection comprising an antisense oligonucleotide and / or an siRNA having reciprocity for the NUCKS1 gene sequence represented by SEQ ID NO: 2 in one embodiment. In this embodiment, the pharmacological activity of Tat inhibits the interaction of Tat with NUCKS1, inhibits the transcriptional activity of the HIV-1 LTR promoter mediated by Tat, and inhibits the accumulation of Tat on the HIV-1 LTR promoter Lt; / RTI >

In one embodiment of the present invention, there is provided a composition for inhibiting the HIV-1 viral reactivation of a latent period of infection comprising an siRNA against the NUCKS1 gene. In this embodiment, the present invention provides a composition characterized in that the HIV-1 virus-infected cell of the latent period of infection is an ACH2 cell or a J 1.1 cell.

"SiRNA" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 And WO 00/44914). siRNA is a short double stranded RNA strand composed of about 19 to 23 nucleotides and targets gene mRNA of the gene to be treated complementary to these nucleotide sequences and suppresses gene expression. The siRNA of the present invention can be readily prepared by those skilled in the art using methods known in the art. For example, a method of directly synthesizing siRNA (Sui G et al., Proc Natl Acad Sci USA 99: 5515-5520, 2002), a method of synthesizing siRNA using in vitro transcription (Brummelkamp TR et al , Science 296: 550-553, 2002), a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme (Paul CP et al., Nature Biotechnology 20: 505-508 , 2002), expression through an intracellular delivery of siRNA expression plasmids or viral vectors (Lee NS et al., Nature Biotechnology 20: 500-505, 2002) and polymerase chain reaction (siRNA expression cassette) (Castanotto D et al., RNA 8: 1454-1460, 2002), but are not limited thereto.

A composition comprising an siRNA for the NUCKS1 gene according to the present invention can be used to screen for a therapeutic agent for HIV-1 viral infection and / or an inhibitor against recurrence of HIV-1 latent infection, / RTI > and / or can effectively inhibit reactivation of the HIV-1 virus at latency of the infection. In addition, by clarifying the mechanism of regulation of Tat, a major factor of HIV-1 protein, treatment of HIV-1 infection including various cell factors of host cell related to Tat protein applicable to treatment of HIV-1 infection Target and the possibility of treating HIV using the target.

Figure 1 shows the interaction between NUCKS1 and Tat protein according to an embodiment of the present invention.
Figure 2 shows the results of promoting Tat-mediated transcriptional activity in HIV-1 LTR by NUCKS1 according to an embodiment of the present invention (2a and 2b), suppression of NUCKS1 expression by NUCKS1 siRNA (2c) 1 < / RTI > LTR (2d).
FIG. 3 shows the results of a decrease in Tat binding to TAR RNA by a composition comprising NUCKS1 siRNA according to an embodiment of the present invention.
Figure 4 relates to the results of a decrease in the proliferation of HIV-1 virus by a composition comprising NUCKS1 siRNA according to an embodiment of the present invention.
5 shows the inhibition of the expression of NUCSK1 (5a and 5b) in the HIV-1 latent infectious cell line by the composition comprising NUCKS1 siRNA according to an embodiment of the present invention, the reactivation of latent infectious HIV-1 virus (Fig. 5c) And the result of inhibiting the reactivation of latent infectious HIV-1 virus by the composition (FIG. 5d).

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention and are not intended to limit the scope of the invention.

Example

Vertical culture, virus plasmid production and analysis.

Example 1.1 Cell Culture

The HeLa, HEK293, TZM-bl, A3.01, ACH-2, Jurkat and J1.1 cell lines were purchased from the NIH AIDS research and Reference Reagent program and the American Type Culture Collection (ATCC). HeLa, HEK293 and TZM-bl cell lines were cultured in DMEM medium (Dulbecco's modified Eagle Medium; Gibco-BRL, Invitrogen). ACH-2 and J1.1 cell lines, HIV-1 LAV strain virus-infected cell lines, A3.01 and Jurkat, non-chronic infected cell lines, were cultured in RPMI medium (Gibco-BRL). All cell lines were cultured in 10% fetal bovine serum (GIBCO-BRL) supplemented with 1% L-glutamine and 1% penicillin-streptomycin.

Example 1.2 Plasmid

The HIV-1 molecular clone pNL4-3 (plasmid for producing CXCR4-tropic virus) was obtained from the NIH AIDS research and Reference Reagent program, and pAD8 plasmid (plasmid for producing CCR5-tropic virus) was obtained from Dr. Kim Youngbong of Konkuk University. Tat-expression plasmids were prepared by inserting wild-type Tat (full-length form derived from the HXB2 strain) cDNA into pcDNA3-Flag (Invitrogen) plasmid. The NUCKS1-expression plasmid was prepared by inserting the NUCKS1 gene into the pcDNA3-V5 (Invitrogen) plasmid. The pGL3-LTR plasmid is a plasmid prepared by inserting the HIV-LTR promoter region into plasmid pGL3 (plasmid expressing luciferase gene, purchased from Promega), and is a plasmid containing a luciferase gene expressed by HIV-LTR , And trans-activation assay mediated by Tat.

Example 1.3 NUCKS1 knockdown

NUCKS1 knockdown was performed using the following three oligonucleotide sequences corresponding to NUCKS1 (SEQ ID NO: 3: 5'-GAAGGUUGUUGAUUACUCACAGUUU-3 ', 5'-GGAUGAUUCUCACUCAGCAGAGGAU-3' and 5'-GGCGGCAUCUAAAGCAGCUUCUAAA -3 ') and non-targeting control siRNA (si-con) purchased from Invitrogen. Briefly, HeLa cells were transfected using 200 picomoles of NUCKS1 siRNA and Lipofectamine 2000 (Invitrogen). After 24 hours, the transfected HeLa cells were co-transfected with plasmids of pcDNA3-Flag-Tat, pGL3-LTR, and pCMV-LacZ using lipofectamine 2000. After 48 hours, the whole cell lysate was recovered and the luciferase activity was measured on a SpectraMax M5 microplate luminescence measurement device (Molecular devices) using a luciferase delivery system (Promega).

NUCKS1 knockdown on cells in the latent phase of HIV-1 infection (ACH-2 and J 1.1 cell lines) was performed by electroporation using Nucleofector ™ kit X (Amaxa Biosystems). Expression levels of NUCKS1 were analyzed by Western blotting using an antibody (Novus Biologicals) that binds to the NUCKS1 protein.

Example 1.4 Luciferase Delivery Analysis

HeLa cells (2.5x10 ⁵ cells / well) were cultured in 12-well plates for 1 day before transfection. 0.05 μg of pcDNA3-Flag-Tat, 0.2 μg of pGL3-LTR-Luc and another amount of pcDNA3-V5-NUCKS1 plasmid were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen). The plasmid pCMV-LacZ (0.02 μg) (Promega) was co-transfected as a control for transfection. The luciferase assay was performed as follows.

In summary, cell culture medium was replaced with fresh medium 4 hours after transfection. After 24 hours of transfection, luciferase activity was measured using a luciferase transporter assay system (Promega) in a DTX 880 MultiMode Detector (Beckman Coulter). Luciferase activity was normalized to beta-galactosidase activity and the results were expressed as mean ± standard deviation.

Example 1.5 Co-immunoprecipitation < RTI ID = 0.0 >

HEK293 cell lines were transfected with Tat-expression plasmids (pcDNA3-Flag-Tat) and NUCKS1-expression plasmids (pcDNA3-V5-NUCKS1) using lipofectamine 2000 (Invitrogen). After 48 hours, the transfected cells were used in immunoprecipitation buffer (250 mM NaCl, 0.5% NP-40, 50 mM Tric-HCl [pH 7.5], 5 mM EDTA, 1 mM Na ₃ VO ₄ , 10 mM NaF, protease inhibitor cocktail) Lt; / RTI > Cell lysates were incubated with anti-Flag monoclonal antibody (M2, Sigma-Aldrich) at 4 ° C. After overnight incubation, protein A / G agarose beads (Santa Cruz Biotechnology) was added and the mixture was incubated at 4 ° C for 1 hour and then centrifuged at 4 ° C, 2500 xg for 2 minutes. The supernatant was discarded and the beads were cleanly washed four times using Immunoprecipitation buffer. Finally, the beads were resuspended in 5X SDS sample buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol and 2% beta-mercaptoethanol) and analyzed by Western blotting.

Example 1.6 Western blotting

The whole cell lysate was dissolved in a digestion buffer containing 50 mM (pH 7.4) Tris-HCl, 1% Nonidet P-40, 150 mM sodium chloride, 2.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor (Roche) ≪ / RTI > Cell lysates were mixed with 5X sample buffer and loaded onto gel for SDS-PAGE analysis. The protein separated from the gel was transferred to a PVDF (polyvinylidenedifluoride) membrane (Millipore). Membranes were blocked using 5% defatted milk and incubated overnight at 4 ° C with the primary antibody. The primary antibodies used were the p65 units (Cell Signaling Technology), anti-IkB-alpha (Sigma-Aldrich), anti-V5 (Invitrogen) (Santa Cruz Biotechnology), anti-beta-actin (Sigma-Aldrich), anti-alpha-tubulin (Sigma-Aldrich) and anti-lamin B (Santa Cruz Biotechnology). The secondary antibodies used were HRP (horseradish peroxidase) -linked-rabbit anti-mouse antibody and goat anti-rabbit antibody (Sigma-Aldrich). The ECL western blotting detection formulation (Millipore) was used for signal detection using HRP-conjugated anti-mouse and anti-rabbit IgG. Chemiluminescence was visualized using Image Lab ^TM software (Bio-Rad). The relative intensities of the NUCKS1 proteins were measured using a Fujifilm image gauge version 4.0 program (Fujifilm, Tokyo, Japan).

Example 1.7 Chromatin Immunoprecipitation (ChIP) Assay and Quantitative Real-Time PCR

Chromatin immunoprecipitation assays were performed on HeLa and TZM-bl cell lines using a Chromatin Immuno-Precipitation Kit (Cell Signaling Technology). NUCKS1 knockdown cells were transfected with the Tat-expression vector (pcDNA3-Flag-Tat). Chromatin was obtained from cells fixed with formaldehyde and immunoprecipitation was performed on the chromatin obtained using an anti-Flag antibody or a control mouse IgG antibody. The eluted material was reverse cross-linked, the DNA was ethanol precipitated and separated by spin column purification. The purified DNA was analyzed by real-time PCR using a primer set corresponding to the TAR region of the HIV-1 LTR. The primers used were TAR-F (5'-AGCTTTCTACAAGGGACTTTCCGC-3 ') and TAR-R (5'-ATTGAGGCTTAAGCAGTGGGTTCC-3'). Real-time PCR was performed using the SYBR Green PCR kit (TAKARA) in a 7500 real-time PCR system (Applied Biosystem) using the 2.0.1 version of 7500 software. Solubility curve analysis was performed to confirm amplification of a single product. Results for real-time PCR were analyzed according to the relative Ct (concentration of target) method, which did not use the standard curve, and were standardized for IgG control and expressed as the difference in binding to control RNA. The relative amount and Comparative Ct method (RQ) = 2- ^{△△ Ct} in, and △ Ct (Ct variation) = Ct _Target -Ct _Control, △△ Ct is Ct = △ _treated -Ct _untreated.

Example 1.8 Analysis of Microarray Data on Gene Expression

Gene expression microarray analysis was performed using Illumina Human HT-12 v4 expression bead chip (Illumina, Inc.). Biotinylated cRNA was prepared from 0.55 [mu] g of total RNA using Illumina whole-made RNA amplification kit (Ambion). After grinding, 0.75 [mu] g of cRNA was mixed with the hybridization buffer and hybridized with the bead chip. After incubation in a hybridization oven, the hybridized array was washed. Microarray images were scanned using a Illumina bead array decoding confocal scanner. The export process of the array data was performed using Illumina GenoeStudio v2009.2 (gene expression module v1.5.4). All data sets were standardized using the quintile normalization method. Average values were used for duplicate data sets. Z-values were calculated by subtracting population mean from individual expression levels and dividing the difference by population standard deviation (SD).

Interaction of NUCKS1 and Tat

Hybrid screening was performed using a matchmaker GAL4 to Hybrid System 3 (Clontech). The region containing the full-length HIV-1 Tat (amino acids 1-86) was generated by PCR and cloned downstream of the GAL4 DNA binding domain of pGBKT7 (pGBKT7-Tat / bait, Clontech) to be regulated by other GAL promoters Was introduced into PBN204, a yeast species containing three transporter genes (URA3, ADE2 and LacZ). Transformants containing Frey selected from Tat bait and the human thymus DNA activation domain library were screened in a selection medium lacking leucine, tryptophan and uracil in which the bait plasmid and pre-protein interact only with each other Growth of yeast was observed. After the selection of the putative Tat binding protein NUCKS1, the amplified NUCKS1-expressing pre-plasmid was reintroduced into the yeast strain PBN204 containing the Tat bait plasmid to confirm the interaction between Tat-NUCKS1 (Fig.

A Flag-tagged Tat (Flag-Tat) expression plasmid and a V5-labeled NUCKSl (V5-NUCKS1) expression plasmid were introduced into HEK293 cell line (together with a public vector) or in combination, respectively, and coimmunoprecipitation and Western blotting to determine the interaction of Tat and intracellular NUCKS1. In the cell line co-expressing Flag-Tat and V5-NUCKS1, Tat co-immunoprecipitated with NUCKS1, whereas the cell line in which Flag-Tat and V5-NUCKS1 alone were expressed did not show the co-immunoprecipitation of Tat and NUCKS1 1b). Immunoprecipitation of endogenous NUCKS1 and Tat was also confirmed in the HEK293 cell line expressing Flag-Tat (FIG. 1C).

Regulation of Tat Transcriptional Activity by NUCKS1

Luciferase expression plasmids expressed by HIV-1 LTR and NUCKS1 expression plasmids were expressed as Tat expression vectors to confirm the transcription activation and NUCKS1 relationship in HIV-1 LTR by Tat. Tat alone significantly increased transcription of HIV-1 LTR, but the expression of Tat and NUCSK1 coexpression increased the transcriptional activity of HIV-1 LTR by 2.3-fold compared with that of Tat alone. However, transcriptional activity of HIV-1 LTR was not increased by NUCKS1 alone expression (Fig. 2a). In addition, it was confirmed that the transcriptional activity of HIV-1 LTR was increased according to the expression level of NUCKS1 even when the Tat expression level was constant (FIG. 2B). Conversely, when NUCKS1 expression was inhibited by NUCKS1 siRNA of SEQ ID NOS: 3 to 5 (Fig. 2C), LTR activity by Tat was greatly decreased, but without Tat, there was no decrease of LTR activity by NUCKS1 expression inhibition 2d).

NUCKS1 expression and chromatin immunoprecipitation assay (ChIP assay) were performed to search for targets in the TAT activity of HIV-1 LTR mediated by NUCKS1. NUCKS1 was transfected into HIV-1 LTR transduced HeLa cell line, and the chromatin was immunoprecipitated with Tat. Inhibition of NUCKS1 expression significantly reduced binding of Tat to TAR RNA (Fig. 3a). Similar results were also observed in TZM-bl cell lines in which β-galactosidase and luciferase transporter cassettes expressed by HIV LTR were combined (FIG. 3b).

Regulation of HIV-1 proliferation and reactivation by NUCKS1

Example 4.1 NUCKS1 in HIV-1 proliferation

HeLa cells (2x10 ⁵ cells / well) were transfected with NUCKS1 siRNA to perform a single round HIV-1 replication assay. After 24 hours, the cells were transfected with pNL4-3 (300 ng) or pAD8 (300 ng). After one day, the supernatant containing the virus was recovered daily for 5 days and filtered to remove the debris. The amount of virion in the supernatant was measured using an HIV-1 p24 ELISA kit (Perkin Elmer). All samples were diluted with media and 200 μl of each diluted sample was used for analysis. The culture medium of the cells not transfected was used as a negative control. The results were quantified as floating absorbance values and compared with the standard curve. All analyzes were repeated three times. Results were expressed as mean ± standard deviation.

Infectious molecular clone pNL4-3 (CXCR4- tropic) in transfected HeLa cells in which the culture medium within the viral antigen p24 ^gag amount pAD8 (OCR5- tropic) a HeLa cell culture medium virus antigen was transfected in combination with siRNA p24 ^gag of NUCKS1 Were compared. The production of CXCR4 (pNL4-3) tropic virus (FIG. 4A) and CCR5 (pAD8) -tropic virus production (FIG. 4B) were greatly reduced by the inhibition of NUCKS1 expression, Of NUCKS1.

Example 4.2 NUCKS1 in HIV-1 reactivation of the latent period of infection

HIV-1 latency is characterized by a very long time silent infection, and this feature is related to the present invention. After the tropic virus infection of Example 4.1, HIV-1 latency was formed by the incorporation of the provirus into the host chromosome, limiting the levels of various host factors and interfering with the transcription process. Thus, mRNA microarray analysis was performed on various HIV-1 incubating cell lines. In addition, the protein expression levels of NUCKS1 in normal HIV-1 infected cell lines and incubated HIV-1 infected cell lines were compared to examine the relationship between virus reactivation and HIV-1 proliferation by NUCKS1 in incubating HIV-1 infections. It was confirmed that the amount of NUCKS1 mRNA expression (Figure 5a and protein expression level 5b) in latent HIV-1 infected cell lines (ACH-2 and J 1.1) was significantly reduced compared to normal maternal cells (A3.01 and Jurkat). Positive Tat regulators such as CCNT1, protein dephosphorylase 1 (PP1) and p300 / CBP did not decrease expression in latent cells compared to normal cells (Fig. 5a) It is not related. Tat-regulatory factors such as BanF1, BRD7, and thioredoxin reductase-1 (TXNRD-1) did not have a consistent pattern of expression between normal and latent infectious cells (Fig. Of these factors, only the positive Tat-regulatory factor NUCKS1 was expressed at a reduced level in cells of the latent infectious group compared to normal cells (Fig. 5A), suggesting that the NUCKS1 low expression was associated with a latency And that it is a major factor in the maintenance of infection. In addition, when the HIV-1 provirus was reactivated by PMA treatment, the level of NUCKS1 protein in latent HIV-1 infected cells (ACH-2 and J 1.1) increased significantly over time, but normal cells A3.01 The level of NUCKS1 protein in jurkat was invariably constant (Figure 5c). In addition, the expression of NUCKS1 was suppressed by electroporation of NUCKS1 siRNA against latent HIV-1 infected cell lines (ACH-2 and J1.1) treated with PMA to activate the provirus. Inhibition of NUCKS1 expression significantly reduced p24 expression (Figure 5d, ACH-2: 60%, J1.1: 82%), indicating that NUCKS1 is a major regulator of HIV-1 latency and HIV- In the presence of HIV-1.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

<110> KOREA CENTERS FOR DISEASE CONTROL & PREVENTION <120> NUCKS1, Novel Coactivator regulating the transcriptional activity          of HIV-1 Taste <130> IPDB51859 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 243 <212> PRT <213> HUMAN <400> 1 Met Ser Arg Pro Val Arg Asn Arg Lys Val Val Asp Tyr Ser Gln Phe   1 5 10 15 Gln Glu Ser Asp Asp Ala Asp Glu Asp Tyr Gly Arg Asp Ser Gly Pro              20 25 30 Pro Thr Lys Lys Ile Arg Ser Ser Pro Arg Glu Ala Lys Asn Lys Arg          35 40 45 Arg Ser Gly Lys Asn Ser Gln Glu Asp Ser Glu Asp Ser Glu Asp Lys      50 55 60 Asp Val Lys Thr Lys Lys Asp Asp Ser His Ser Ala Glu Asp Ser Glu  65 70 75 80 Asp Glu Lys Glu Asp His Lys Asn Val Arg Gln Gln Arg Gln Ala Ala                  85 90 95 Ser Lys Ala Ala Ser Lys Gln Arg Glu Met Leu Met Glu Asp Val Gly             100 105 110 Ser Glu Glu Glu Glu Glu Glu Glu Asp Glu Ala Pro Phe Gln Glu Lys         115 120 125 Asp Ser Gly Ser Asp Glu Asp Phe Leu Met Glu Asp Asp Asp Asp Ser     130 135 140 Asp Tyr Gly Ser Ser Lys Lys Lys Asn Lys Lys Met Val Lys Lys Ser 145 150 155 160 Lys Pro Glu Arg Lys Glu Lys Lys Met Pro Lys Pro Arg Leu Lys Ala                 165 170 175 Thr Val Thr Pro Ser Pro Val Lys Gly Lys Gly Lys Val Gly Arg Pro             180 185 190 Thr Ala Ser Lys Ala Ser Lys Glu Lys Thr Pro Ser Pro Lys Glu Glu         195 200 205 Asp Glu Glu Pro Glu Ser Pro Glu Lys Lys Thr Ser Thr Ser Pro     210 215 220 Pro Pro Glu Lys Ser Gly Asp Glu Gly Ser Glu Asp Glu Ala Pro Ser 225 230 235 240 Gly Glu Asp             <210> 2 <211> 732 <212> DNA <213> HUMAN <400> 2 atgtcgcggc ctgtcagaaa taggaaggtt gttgattact cacagtttca ggaatctgat 60 gatgcagatg aagattatgg aagagattcg ggccctccca ctaagaaaat tcgatcatct 120 ccccgagaag ctaaaaataa gaggcgatct ggaaagaatt cacaggaaga tagtgaggac 180 tcagaagaca aagatgtgaa gaccaagaag gatgattctc actcagcaga ggatagtgaa 240 gatgaaaaag aagatcataa aaatgtgcgc caacaacggc aggcggcatc taaagcagct 300 tctaaacaga gagagatgct catggaagat gtgggcagtg aggaagaaca agaagaggag 360 gatgaggcac cattccagga gaaagattcc ggcagcgatg aagatttcct aatggaagat 420 gatgacgata gtgactatgg cagttcgaaa aagaaaaaca aaaagatggt taagaagtcc 480 aaacctgaaa gaaaagaaaa gaaaatgccc aaacccagac taaaggctac agtgacgcca 540 agtccagtga aaggcaaagg gaaagtgggt cgccccacag cttcaaaggc atcaaaggaa 600 aagactcctt ctcccaaaga agaagatgag gaaccggaaa gcccgccaga aaagaaaaca 660 tctacaagcc ccccacccga gaaatctggg gatgaagggt ctgaagatga agccccttct 720 ggggaggatt aa 732 <210> 3 <211> 25 <212> RNA <213> HUMAN <400> 3 gaagguuguu gauuacucac aguuu 25 <210> 4 <211> 25 <212> RNA <213> HUMAN <400> 4 ggaugauucu cacucagcag aggau 25 <210> 5 <211> 25 <212> RNA <213> HUMAN <400> 5 ggcggcaucu aaagcagcuu cuaaa 25

Claims (13)

1) contacting a sample to be analyzed with an HIV-1 virus infected cell comprising the NUCKS1 protein of SEQ ID NO: 1;
2) measuring the amount of the NUCKS1 protein;
3) screening comprising the step of determining the regulation of proliferation of the HIV-1 virus in an HIV-1 virus infected cell comprising the NUCKS1 protein when the amount of said NUCKS1 protein is determined to be down regulation Way.
1) contacting a sample to be analyzed with an HIV-1 virus infected cell comprising the NUCKS1 gene represented by SEQ ID NO: 2;
2) measuring the amount of protein expressed from the NUCKS1 gene;
3) When the amount of protein expression from the NUCKS1 gene is determined to be down-regulated, the sample is identified as a therapeutic agent for an HIV-1 virus-infected cell comprising the NUCKS1 gene.
1) contacting a sample to be analyzed with HIV-1 virus-infected cells of the incubation period containing the NUCKS1 protein represented by SEQ ID NO: 1;
2) measuring the amount of the NUCKS1 protein;
3) determining that the sample is a reactivation inhibitor for a cell infected with the HIV-1 virus of a latent period of infection comprising the NUCKS1 protein when the amount of the NUCKS1 protein is determined to be down regulation Screening method.
1) contacting a sample to be analyzed with the HIV-1 virus-infected cell of the incubation period containing the NUCKS1 gene represented by SEQ ID NO: 2;
2) measuring the amount of protein expression from the NUCKS1 gene according to SEQ ID NO: 2;
3) When the amount of protein expression from the NUCKS1 gene according to SEQ ID NO: 2 is measured to be down-regulated, the sample contains HIV-1 for HIV-1 virus-infected cells of the incubation period containing the NUCKS1 gene And determining the virus as a virus reactivation inhibitor.
A siRNA having complementarity to the NUCKS1 gene sequence represented by SEQ ID NO: 2, wherein the siRNA is selected from the group consisting of SEQ ID NOS: 3-5.
delete 6. The method of claim 5,
Wherein said composition inhibits the interaction of Tat and NUCKS1.
6. The method of claim 5,
Wherein said composition inhibits the transcriptional activity of an HIV-1 LTR promoter mediated by Tat.
6. The method of claim 5,
Wherein said composition inhibits the accumulation of Tat on the HIV-1 LTR promoter.
A siRNA having complementarity to the NUCKS1 gene sequence represented by SEQ ID NO: 2, wherein the siRNA is selected from the group consisting of SEQ ID NOS: 3-5.
delete 11. The method of claim 10,
Wherein said HIV-1 virus is in a latent state of infection.
13. The method of claim 12,
Wherein the cells infected with the HIV-1 virus at the latency of infection are ACH2 cells or J 1.1 cells.

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