WO2008105604A1

WO2008105604A1 - Recombinant mammalian cell for screening a substance preventing and treating aids and screening method using the same

Info

Publication number: WO2008105604A1
Application number: PCT/KR2008/001074
Authority: WO
Inventors: Ji Chang You
Original assignee: Avixgen Inc.
Priority date: 2007-02-27
Filing date: 2008-02-25
Publication date: 2008-09-04

Abstract

The invention relates to recombinant mammalian cells for screening a substance for preventing and treating AIDS and a method of screening the substance using the cells. More particularly, disclosed are recombinant mammalian cells, transfected with an HIV NC expression cassette, which expresses an HIV NC protein, and with a reporter gene expression cassette, which expresses an HIV Psi (Ψ) sequence and a reporter gene, as well as a screening method using the cells. The recombinant mammalian cells and the screening method provide a system capable of screening a substance, which inhibits the interaction between the HIV NC protein and the HIV Psi sequence, using eukaryotic cells. Accordingly, the disclosed invention provides novel means capable of screening a novel substance, which inhibits the interaction between the HIV NC protein involved in the packaging of HIV and the HIV Psi interaction. In addition, the present invention provides a novel method for screening a substance for preventing and treating AIDS.

Description

Invention Title

RECOMBINANT MAMMALIAN CELL EOR SCREENING A SUBSTANCE PREVENTING AND TREATING AIDS AND SCREENING METHOD USING

THE SAME

Technical Field

The present invention relates to recombinant mammalian cells for screening a substance for preventing and treating AIDS and a method of screening the substance using the cells, and more particularly to recombinant mammalian cells, transfected with an HIV NC expression cassette, which expresses an HIV NC protein, and with a reporter gene expression cassette, which expresses an HIV

Psi (Ψ) sequence and a reporter gene, as well as a screening method which uses the cells.

Background Art

AIDS (Acquired Immune Deficiency Syndrome) , identified for the first time in the early 1980s, is one of the most important diseases in the world. AIDS- treating drugs, developed to date, include protease inhibitors, such as saquinavir, indinavir and ritonavir, and reverse transcriptase inhibitors, such as AZT, ddl, ddC, d4T, 3TC and nevirapine. It is known that when such treating drugs are used alone, they have no significant effect, but when two reverse transcriptase inhibitors, such as AZT and 3TC, and one protease inhibitor, are used in combination, they show a high therapeutic effect.

However, the drug combinations do not improve disease conditions in all patients who were administered them, and moreover they have problems in that they are expensive and have serious side effects, including vomiting and high fever, and appearance of variant viruses having tolerance to these drugs appear.

Accordingly, for better therapy, there is a need to develop a novel class of therapeutic agents, which are more effective and have low toxicity.

AIDS is caused by infection with HIV (human immunodeficiency virus) , and at 3-6 weeks after infection, the person suffers from symptoms such as cold and fatigue, for about 1-2 weeks, and then recovered from the symptoms. Thereafter, the long latent period of HIV is lasting for about 10 years. During the long latent period, HIV virus destroys the immune cells of the infected person while it continues to proliferate. Thus, the immune function of the patient is gradually impaired, so that AIDS symptoms appear at the last stage of the latent period.

The HIV 1 gene has 9 ORFs, three of which are Gag, Pol and Env, and the remaining six ORFs perform regulatory functions. Particularly, Gag and Env are structural components, in which Gag expresses four kinds of proteins, including MA (matrix) , CA (capsid) , NC (nucleocapsid) and pβ, and Env expresses two kinds of proteins, including SU (surface or gpl20) and TM

(transmembrane or gp41). Also, Pol expresses three kinds of proteins, including PR (protease) , RT (reverse transcriptase) and IN (integrase) , which show enzymatic activity.

Among HIV proteins, an NC (nucleocapsid) protein is a basic protein consisting of 55 amino acids and having a molecular weight of about 6 kDa, and contains two zinc finger domains. The NC protein performs not only a structural function of forming virus individuals, but also an important function in the viral life cycle. The major functions of the HIV NC protein are as follows. First, the NC protein is involved in viral genomic encapsidation. This function is attributable to two zinc finger domains consisting of a unique Cys-X2-Cys-X4-His- X4-Cys motif (CCHC motif) , and it is known that the domains are highly conserved in all retroviruses and are essential for HIV RNA packaging and infectious virus production. Second, the NC protein is known to promote tRNA primer annealing and strand transfer during viral reverse transcription (RT) , and this suggests that the NC protein plays an important role in viral replication. Third, the NC protein has nucleic acid chaperone activity necessary for the viral life cycle, and recently, it was reported that, even when the viral DNA is inserted into the host cell chromosome, the NC protein plays a certain role . Accordingly, studies focused on impairing the functions of the NC protein to make HIV-I impotent have been conducted (see Lener, D. et al., FEBS Letters. ,361:85-88, 1995; Dexter, T. K. et al., J. Virol. , 70:6607-6616, 1996; Tanchou, V. et al., J. Virol. ,72:4442-4447, 1998; Rong, L.et al., J. Virol. ,72:9353-9358, 1998; Berthoux, L. et al., J. Virol. ,71:6973-6981, 1997; Berthoux, L. et al., J. Virol. ,73:10000-10009, 1999; Clever, J. L.et al., J. Virol. ,74:541-546, 2000; Cimarelli, A. et al., J. Virol. ,74:3046-3057, 2000). For example, Korean Patent Application No. 01-52594 discloses RNA linkable with the NC, but the RNA has a shortcoming in that, because it Ls difficult to operate, it cannot be practically used for the treatment of HIV-I.

The genomic encapsidation of HIV virus by the NC protein is involved in the interaction between the NC protein and the HIV Psi sequence. The HIV Psi sequence consists of 125 bases located at the 5' terminal end of HIV genomic RNA and binds specifically to the NC protein to perform HIV genomic packaging.

Disclosure Technical Problem

Accordingly, the present inventors have conducted studies on the physiological activity of the HIV NC protein, and as a result, have found that the interaction between the HIV NC protein and the HIV Psi sequence shows an activity of inhibiting the translation process in mammalian cells. On the basis of this finding, the present inventors have developed recombinant mammalian cells and a method for screen a substance for preventing and treating AIDS using the same, thereby completing the present invention.

Therefore, it is an object of the present invention to provide recombinant mammalian cells for screening a substance for preventing and treating AIDS and a method of screening the substance using the same.

Technical Solution

To achieve the above object, in one aspect, the present invention provides recombinant mammalian cells, transfected with a polynucleotide, which expresses the

HIV NC protein, and with a polynucleotide, which expresses the HIV Psi(Ψ) sequence and a reporter gene.

In another aspect, the present invention provides a method for screening a substance for preventing and treating AIDS, the method comprising using said cells to screen a substance affecting the interaction between the HIV NC protein and the HIV Psi (Ψ) sequence.

Hereinafter, the present invention will be described in detail. The cells of the present invention are recombinant mammalian cells transfected with: an HIV NC expression cassette, which comprises a promoter and a polynucleotide encoding a polypeptide comprising an HIV NC (nucleocapsid) protein operably linked to the promoter; and a reporter gene expression cassette, which comprises a promoter, an HIV Psi (Ψ) sequence linked to the promoter, and a reporter gene. The present inventors have found that the transcription of a gene present downstream of the HIV Psi sequence was inhibited due to the interaction between the HIV NC protein and the HIV Psi sequence in mammalian cells, and this finding was found for the first time by the present inventors. In each of Examples 1 and 2, the present inventors prepared an expression vector having a reporter gene, which was located downstream of the HIV Psi sequence and made it possible to examine the expression of the target gene in mammalian cells, and an expression vector capable of expressing the HIV NC protein. The prepared expression vectors were transfected into the same mammalian cells in Example 3. The present inventors confirmed whether the reporter gene was expressed by the interacting between the HIV NC protein and the HIV Psi sequence.

As a result, it was confirmed whether the expression of the reporter gene located downstream of the HIV Psi sequence was reduced due to the interaction between the HIV Psi sequence and the HIV NC protein. For comparison with this result, when a vector containing no HIV Psi sequence was used it was observed that the reporter gene was well expressed. This result was found because the translation of the gene located downstream of the HIV Psi sequence was inhibited due to the interaction between the HIV NC protein and the HIV Psi sequence.

The present inventors previously found that the expression of the reporter gene present downstream of the HIV Psi sequence is reduced due to the interaction between the HIV Psi sequence and the HIV NC protein in prokaryotic E. coli (Korean Patent Registration No. 360,275). However, in the present invention, this phenomenon was found for the first time in eukaryotic human T-Rex293 cells (a kind of 293 cells), and this finding is not obvious to those skilled in the art, in view of the fact that the translation process in prokaryotes differs from the translation process in eukaryotes .

The translation processes in prokaryotes and the translation process in eukaryotes differ from each other with respect to factors which are involved in the mechanisms. In the translation process in prokaryotes, IF (initiation factor) 1, IF2-GTP and IF3 bind to a ribosomal 3OS subunit, to which IF3-induced mRNA and IF2- induced fMet-tRNA bind to form a 3OS initiation complex, and then a ribosomal 5OS subunit binds thereto to form a 70S initiation complex. However, in the case of the translation process in eukaryotes, eIF3 and elFIA bind to a ribosomal 4OS subunit, to which an eIF2-GTP-Met-tRNAi complex then binds to form a 43S complex, and mRNA binds thereto by the aid of eIF4 to form a 48S complex. In the 48S complex, an initiation codon site was screened by scanning mRNA while consuming ATP, and then a 60S subunit binds to the 48S complex by the aid of eIF5 to form an initiation complex. As described above, the translation process in prokaryotes and the translation process in eukaryotes differ from each other with respect to factors involved therein, the process of formation of the initiation complex, and the method for scanning the initiation codon. Accordingly, it is not considered that the translation inhibitory mechanism applied in prokaryotes is also applied in eukaryotes in the same manner .

Furthermore, in the present invention, the interaction between the HIV NC protein and the HIV Psi sequence inhibits the process of scanning the 48S complex. Because this scanning process is a mechanism which is not present in the translation process in prokaryotes, the translation inhibitory activity in the present invention is not obvious to those skilled in the art.

As used herein, "HIV NC protein" means the nucleocapsid protein of HIV (Human Immunodeficiency Virus) causing AIDS (Acquired Immune Deficiency Syndrome. This protein strongly binds to virus genomic RNA to form a ribonucleoprotein core complex.

Preferably, the HIV NC protein may have an amino acid sequence represented by SEQ ID NO: 1 and may be the NC protein set forth in Genbank Accession Nos. P03349, P03366, P04585, P03367, P12497, P03369, P04587, P04584, P35963, P24740, P05961, P04591, Q73368, P20892, P20875, P12498, P05888, P12493, Q9QBZ5, Q9QBY3, 089940, Q9WC63, Q9WC54, Q75002, P24736, Q9QBZ1, 089290, Q9QBZ6, Q9QBY4, P12499, P05959, P18802, P04588, P04589, P05960, Q70622, P20889, P12494, P03347, Q9QSR3, Q9Q720, Q9IDV9, Q9WC62, Q9WC53, Q9Q721, Q74230, Q73367, 012157, P35962, P18800, P04592, P20873, P05887, P03348, P04593, Q89928, Q79666, Q77373, 041798, 093215, 091080, P05962, Q9QBZ2, Q9QC00, 089291, P05891, P15833, P17757, P18096, Q9QSR4, Q9IDV8, Q75001, 089939, P18095, P05890, P04594, P12495, Q76634, P20876, P18042, P0C1K7, Q79665, Q77372, 093182, 091079, P05889, P12451, P24107, Q76633, P12450, P18041, P15832, P24106, P17756, Q69383, Q74120, Q74119, P20874, P04590, P03363, Q0R5R2, Q1A268, Q1A250, Q1A267, P03353, Q0R5R3 and Q1A249. For example, in bhe case^' of P03349, the NC protein may be a sequence consisting of amino acid residues 380^th to 434^th.

In addition, Both the HIV NC protein itself, and a polypeptide containing the HIV NC protein, have transcription inhibitory activity by the interaction between the HIV NC protein and the HIV Psi sequence (see Example 3) . Accordingly, the screening method of the present invention can also preferably be carried out using the polypeptide containing the HIV NC protein. Examples of the polypeptide may include, but are not limited to, HIV GAG and HIV GAG+POL.

The HIV Psi (Ψ) sequence is a sequence consisting of 125 bases located at the 5' terminal end of the HIV genomic RNA, and it binds specifically to the HIV NC protein, so that it is involved in HIV genomic packaging. Preferably, the HIV Psi sequence may have a nucleotide sequence represented by SEQ ID NO: 2.

The reporter gene may be a conventional reporter gene, the expression of which can be measured, and examples of the reporter gene may include, but are not limited to, EGFP (enhanced green fluorescent protein) , GFP (green fluorescent protein) , luciferase, gaussia luciferase, gaussian luciferase, β-galactosidase, and alkaline phosphatase. However, any reporter gene may be used without limitation in the present invention, as long as it possesses a suitable^' antibody, because the expression of the gene can be measured using a known immunohistochemical analysis method or the like.

The expression cassette refers to a nucleic acid structure capable of expressing the target structural gene, and may comprise a suitable promoter and a structural gene operably linked thereto. The expression cassette may comprise at least one coding nucleotide and may additionally comprise other factors (e.g. polyadenylation sequences, sequences encoding a membrane- insertion signal or a secretion inducer, ribosome entry sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.), and the like), as desired. In addition, the expression cassette may also be constructed together with expression regulatory elements, including a replication origin, a selection marker, an operator, an initiation codon, a stop codon, a polyadenylation signal and an enhancer. In the present invention, the HIV NC protein expression cassette and the reporter gene expression cassette are preferably present in separate expression vectors, but may also be present as an expression cassette incorporated in one expression vector.

The HIV NC protein expression cassette is preferably pLPl, pLPl/GAG (ΔPOL) or pLPl/optiNC, and the reporter gene expression cassette is preferably pcDNA4T0- Psi (Ψ) - (-ATG) EGFP (-extra) , pcDNA4TO-R-U5-Psi (Ψ) - (- ATG)EGFP^'(-extra) , pcDNA4TO-Psi (Ψ) - (-ATG) -Flue (-extra) or pcDNA4TO-Psi (Ψ) - (-ATG) -Gaussia (-extra) . Herein, the pLPl/GAG (ΔPOL) vector or the pLPl/optiNC vector are vectors prepared based on a commercial pLPl vector (Invitrogen, USA), and the pcDNA4TO-Psi (Ψ) - (-ATG) EGFP (- extra), pcDNA4TO-R-U5-Psi (Ψ) - (-ATG) EGFP (-extra) , pcDNA4TO-Psi (Ψ) - (-ATG) -Flue (-extra) or pcDNA4TO-Psi (Ψ) - (- ATG) -Gaussia (-extra) vector is a vector prepared based on a commercial pcDNA4TO vector (Invitrogen, USA) . The methods of preparing each of the vectors are described in detail in Examples 1 and 2.

As used herein, the term "promoter" means a DNA sequence regulating the expression of an operably linked nucleic acid sequence in a certain host cell, in which the term "operably linked" means that one nucleic acid fragment is linked with another nucleic acid fragment, such that the function or expression thereof is affected by the other nucleic acid fragment. In addition, such control sequences may include an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome-binding site, and sequences controlling the termination of transcription and translation. As the promoter, a constitutive promoter that constitutively induces the expression of the target protein, or an inducible promoter that induces the expression of the target gene at a specific site and time, may be used, and examples thereof include an SV40 promoter, a CMV promoter, a CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991; Monahan et al., Gene Therapy, 7:24-30, 2000;, a CaMV 35S promoter, (Odell et al., Nature 313:810-812, 1985), an Rsyn7 promoter (US Patent Application No. 08/991,601), a rice actin promoter (McElroy et al., Plant Cell 2:163-171, 1990), an uiquitin promoter (Christensen et al., Plant MoI. Biol. 12:619-632, 1989), an ALS promoter (US Patent Application No. 08/409,297), etc. Also usable promoters are disclosed in US Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, etc.

Meanwhile, standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989) ; by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley- lnterscience (1987) . In another aspect, the present invention provides a method for screening a substance for preventing and treating AIDS using the transfected recombinant mammalian cells of the present invention..

The screening method of the present invention is a method of screening a substance influencing the interaction between the HIV NC protein and the HIV Psi (Ψ) sequence. Accordingly, since the interaction between the HIV NC protein and the HIV Psi sequence is a phenomenon necessary for packaging in the process of proliferation of HIV, when the recombinant mammalian cells of the present invention are used in order to confirm whether or not their interaction is inhibited, a substance influencing the interaction between the HIV NC protein and the HIV Psi sequence can be screened, thus screening a substance for preventing and screening AIDS.

Specifically, the screening method of the present invention comprises the steps of:

(a) treating the transfected recombinant mammalian cells of the present invention with a candidate substance and culturing the treated cells; and

(b) measuring the expression level of a reporter gene in the cultured cells.

As used herein, the term "expression level of the reporter gene" refers to the expression amount of the reporter gene. It can be measured directly or indirectly depending on the kind of reporter gene using various methods known in the art. For example, when EGFP

(enhanced green fluorescent protein) or GFP (green fluorescent protein) is used as the reporter gene, the expression level of the reporter gene can be measured by measuring the amount of emitted fluorescence. When luciferase or β-galactosidase is used as the reporter gene, the expression level of the reporter gene can be measured according to a known method of measuring the luciferase or β-galactosidase activity. In addition, the expression level of the reporter gene can also be measured by co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA) , immunohistochemical assay, Western blotting, and fluorescence activated cell sorting (FACS).

Also, the inventive screening method targeting the interaction between the HIV NC protein and the HIV Psi sequence can be performed using high throughput screening (HTS) . The HTS is a method for screening the biological activities of a number of candidate substances simultaneously or almost simultaneously by testing the candidate substances simultaneously. In a certain embodiment, cell lines are cultured in a 96-well microtiter plate or a 192-well microtiter plate and treated with a number of candidate substances, and then measured for the expression of the HIV NC protein- containing polypeptide in the cells using an immunohistochemical method. In this format, 96 independent tests may be simultaneously performed in a single 8 cm x 12 cm plastic plate containing 96 reaction wells. The wells require an assay volume of 50-500 μJL typically. In addition to the plate, a number of gauges, instruments, pipetters, robots, plate washers and plate readers are commercially available in order to make the 96-well format suitable for a wide range of homogeneous and heterogeneous assays.

In one Example of the present invention, the following vectors were prepared: a pcDNA4TO-Psi (Ψ) - (- ATG) EGFP (-extra) vector, a pcDNA4TO-R-U5-Psi (Ψ) - (- ATG) EGFP (-extra) vector, a pcDNA4TO-Psi (Ψ) - (-ATG) -Flue (- extra) vector and a pcDNA4TO-Psi (Ψ) - (-ATG) -Gaussia (- extra) vector, which contain the HIV Psi sequence; and a pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector, a pcDNA4TO-R-U5- Psi(Ψ) -EGFP (-extra) vector, a pcDNA4T0-Psi (Ψ) -Flue (- extra) vector and a pcDNA4TO-Psi (Ψ) -Gaussia (-extra) vector, which contain no HIV Psi sequence.

In another Example of the present invention, a pLPl/GAG(POL) vector and a pLPl/optiNC vector, which express a polypeptide containing the HIV NC protein, were constructed.

In still another Example of the present invention, each of combinations of the vectors was used to examine whether the reporter gene was expressed in the interaction between the HIV NC protein and the HIV Psi sequence. As a result, it was confirmed that, in the case where there was an interaction between the HIV NC protein and the HIV Psi sequence, the reporter gene was not expressed, and in the case where there was no interaction between the HIV NC protein and the HIV Psi sequence, the reporter gene was expressed. This suggests that the interaction between the HIV NC protein and the HIV Psi sequence has translation inhibitory activity (see Example 3 and FIGS. 1 to 3 and FIG. 7) . In still another Example of the present invention, the substance inhibiting the interaction between the HIV NC protein and the HIV Psi sequence was used to examine the effect of the interaction on translation inhibitory activity. As a result, it was confirmed that the expression of the reporter gene reoccurred, leading to a decrease in translation inhibitory activity. This suggests that whether the reporter gene is re-expressed can be used to screen a substance for inhibiting and preventing AIDS (see Example 4 and FIG. 4) .

Advantageous Effects Accordingly, the recombinant mammalian cells and screening method of the present invention provide a system capable of screening a substance, which inhibits the interaction between the HIV NC protein and the HIV Psi sequence, using eukaryotic cells. Thus, the present invention provides novel means for screening a novel substance inhibiting the interaction between the HIV NC protein and the HIV Psi sequence, which is involved in the packaging of HIV. In addition, the present invention provides a novel method capable of screening a substance having the effect of preventing and treating AIDS.

Description of Drawings

FIG. 1 shows the translation inhibitory effect of the interaction between the HIV NC protein and the HIV

Psi sequence, measured using the inventive pcDNA4TO-

Psi (Ψ)- (-ATG)EGFP (-extra) vector (A) and pcDNA4TO- ΔPsi (Ψ) -EGFP (-extra) vector (B).

FIG. 2 shows the translation inhibitory effect of the interaction between the HIV NC protein and the HIV

Psi sequence, measured using the inventive pcDNA4TO-R-U5- Psi (Ψ) - (-ATG)EGFP (-extra) vector (A) and pcDNA4TO-R-U5-

Psi (Ψ) -EGFP (-extra) vector (B).

FIG. 3 shows the translation inhibitory effect of the interaction between the HIV NC protein and the HIV

Psi sequence, measured using the inventive pcDNA4T0- Psi (Ψ) - (-ATG) -Flue (-extra) vector (A) and pcDNA4TO-

Psi (Ψ) -Flue (-extra) vector (B).

FIG. 4 shows that, when the interaction between the HIV NC protein and the HIV Psi sequence in a screening system comprising the inventive transfected cells is inhibited, translation occurs.

FIG. 5 schematically shows a process of preparing the inventive pcDNA4TO-Psi (Ψ) - (-ATG) -Gaussia (-extra) vector.

FIG. 6 schematically shows a process of preparing the inventive pcDNA4TO-Psi (Ψ) -Gaussia (-extra) vector.

FIG. 7 shows the translation inhibitory effect of the interaction between the HIV NC protein and the HIV Psi sequence, measured using the inventive pcDNA4TO-Psi (Ψ) - (- ATG) -Gaussia (-extra) vector (A) and pcDNA4TO-Psi (Ψ) - Gaussia (-extra) vector (B). Best: Mode

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative only, and the scope of the present invention is not limited thereto.

Example 1: Preparation of reporter gene expression vector

<1-1> Preparation of pcDNA4T0-Psi (Ψ) - (-ATG) EGFP (- extra)

The pcDNA4T0-Psi(Ψ)- (-ATG)EGFP (-extra) vector of the present invention, based on the pcDNA4TO vector (Invitroren, USA) and comprised of the Psi (Ψ) sequence and EGFP (enhanced green fluorescent protein) without ATG, was prepared in the following manner.

In order to remove a site, which interferes with the formation of the secondary structure of the HIV Psi sequence, from the pcDNA4TO vector, a fragment between sites digested with restriction enzymes Ndel(483) and HindIII(978) was amplified by PCR using ^' the pcDNA4TO vector (Invitrogen, USA) as a template with a forward primer of SEQ ID NO: 3 (483-NdeI: 5- GTATCATATGCCAAGTACGCCCCCTATT-S) and a reverse primer of SEQ ID NO: 4 (859-ffindIII : 5- GCGGATCCAAGCTTTCTCTATCACTGATAGGGAG-S), and the amplified product (376 bp) was inserted into the Ndel and HindIII restriction enzyme sites. The resulting vector was named "intermediate vector 1". Herein, the insertion of the PCR product into the restriction enzyme sites was performed by treating fragments to be ligated, with restriction enzymes, and then ligating complementary cohesive ends with each other. In the following context, the insertion of PCR products will have the same meaning as described above, unless otherwise mentioned.

The Psi (Ψ) sequence was amplified by PCR using HIV ARV-2/SF2 (SEQ ID NO: 5; CTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACAGCAAGAGGCGAGGGGCGGCG ACTGGTGAGTACGCCAATTTTTGACTAGCGGAGGCTAGAAGGAGAGAGAGATGGGTG CGAGAGCGTCGGTATTA) as a template with a forward primer of SEQ ID NO: 6 (ffindlll: 5-CCAAGCTTACGCAGGACTCGGCTTGCTG-S) and a reverse primer of SEQ ID NO: 7 (BamRI: 5- CCGGATCCTAATACCGACGCTCTCGCAC-3) , and then the amplified PCR product was inserted into the HindIII and BamHI restriction enzyme sites of the intermediate vector 1. The resulting vector was named "intermediate vector 2".

The EGFP (enhanced green fluorescence protein) sequence was amplified by PCR using the pEGFP-Cl vector

(BD Science, USA) as a template with a forward primer of

SEQ ID NO: 8 (BamRI: 5-CCGGATCCGTGAGCAAGGGCGAGGA-S) and a reverse primer of SEQ ID NO: 9 (Xhol: 5-

TACTTCTCGAGCTCTGTACATGTCCGCGG-3) , and then the amplified PCR product was inserted into the BamHI and Xhol restriction enzyme sites of the intermediate vector 2, thus obtaining a pcDNA4TO-Psi (Ψ) - (-ATG) EGFP (-extra) vector. The obtained vector was named "intermediate vector 3".

Herein, in order to remove the initiation sequence ATG from EGFP, the primers used in the PCR amplification were designed such that a region which is removed ATG from EGFP would be amplified.

<l-2> Preparation of pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector

The pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector used as a control group, that is, the vector, in which the Psi (Ψ) sequence was not inserted in the pcDNA4TO-Psi (Ψ) - (-

ATG) EGFP (-extra) and from which the initiation sequence of EGF was not removed, was prepared in the same manner as Example <1-1>, except that the step of inserting the Psi (Ψ) sequence was excluded from the method of preparing the pcDNA4TO-Psi(Ψ)- (-ATG)EGFP (-extra) vector and that the amplification was performed using a forward primer of

SEQ ID NO: 10 (5-CCGGATCCATGGTGAGCAAGGGCGAGGA-S) and a reverse primer of SEQ ID NO: 9, such that the EGFP fragment in place of the (-ATG)EGFP fragment was amplified by PCR.

<l-3> Preparation of pcDNA4TO-R-U5-Psi (Ψ) - (- ATG)EGFP (-extra) vector

A vector, in which the Psi (Ψ) sequence in the pcDNA4TO-Psi (Ψ) - (-ATG)EGFP (-extra) of Example <1-1> was replaced with a R-U5-Psi (Ψ) sequence, was prepared in the following manner. The R-U5-Psi (Ψ) sequence was obtained by performing PCR amplification using the pNL4-3 vector as a template with a forward primer of SEQ ID NO: 11 (Hindlll: 5- CCAAGCTTGGGTCTCTCTGGTTAGACCA-3) and a reverse primer of SEQ ID NO: 12 (BamHI: 5-CCGGATCCTAATACCGACGGTCTCGCAC-3) . Herein, the R-U5-Psi (Ψ) sequence is a region corresponding to the 454 to 813 bp region of the pNL4-3 vector, and the pNL4-3 vector is a vector capable of producing recombinant HIV virus, because it contains all HIV-I genes, except that half of the Nef gene was replaced with the EGFP reporter gene. The pNL4-3 vector is described in detail in the published literature (Lee et al., 1997, Biochem. Biophy. Res. Comm. , 233: 288-292).

Each of the PCR amplified fragment and the pcDNA4TO-Psi (Ψ) - (-ATG) EGFP (-extra) vector was digested with Hindlll and BamHI, and the amplified R-U5-Psi (Ψ) sequence was inserted into the pcDNA4TO-Psi (Ψ) - (- ATG) EGFP (-extra) vector instead of Psi (Ψ) , thus preparing a pcDNA4TO-R-U5-Psi(Ψ)- (-ATG)EGFP (-extra) vector. <l-4> Preparation of pcDNA4TO-R-U5-Psi (Ψ) -EGFP (- extra) vector

In order to insert an R-U5 fragment into the pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector of Example <l-2>, the R-U5 fragment was amplified by PCR using a forward primer of SEQ ID NO: 13 (R-U5-F; 5'- CCAAGCTTGGGTCTCTCTGGTTAGACCA-3 ' ) and a reverse primer of SEQ ID NO: 14 (R-U5-R; 5'-CCGGATCCCGAGAGATCTCCTCTGGCTT- 3'), and the PCR amplified fragment was digested with HindIII and BamHI and inserted into the pcDNA4TO-ΔPsi (Ψ) - EGFP (-extra) vector, thus preparing a pcDNA4TO-R-U5- Psi (Ψ) -EGFP (-extra) vector. <l-5> Preparation of pcDNA4TO-Psi (Ψ) - (-ATG) -Flue (- extra)

In order to prepare a pcDNA4T0-Psi (Ψ) -( -ATG) -Flue (- extra) vector comprising luciferase instead of EGFP as the reporter gene, luciferase in place of EGFP as the reporter gene was inserted into the vector of Example <1-

1> in the following manner.

Luciferase was amplified by PCR using pGL3 (Promega, USA) as a template with an (ATG) luc-F primer of SEQ ID NO: 15 (5'-CCGGATCCGAAGACGCCAAAAACATAAA-S') and an luc-R primer of SEQ ID NO: 16 (5'-TACTCGAGTTACACGGCGATCTTTCCGC- 3'). The amplified PCR product was inserted into the BamHI and Xhol restriction enzyme sites of the vector of Example <1-1>.

<l-6> Preparation of pcDNA4TO-Psi (Ψ) -Flue (-extra) vector

In order to prepare a pcDNA4TO-Psi (Ψ) -Flue (-extra) vector comprising luciferase instead of EGFP as the reporter gene in the vector of Example <l-2>, the luciferase fragment obtained in Example <l-5> was inserted into the vector of Example <l-2> according to the method of Example <l-2>, thus preparing the pcDNA4TO- Psi (Ψ) -Flue (-extra) vector. <l-7> Preparation of pcDNA4TO-Psi (Ψ) -Gaussia (- extra) vector

A pcDNA4TO-Psi (Ψ) - (-ATG) -Gaussia (-extra) (shown as pcDNA4/T0/atg. Psi/-Gau (-ex) in the figure), comprising Gaussia luciferase as a reporter gene, was prepared in the following by inserting the Psi (Ψ) seguence into a pcDNA4T0-Psi (Ψ) -Flue (-extra) vector (shown as pcDNA4/TO/del.psi/F-luc (-Extra) in the figure) and replacing the F-luc gene with a Gaussia luciferase fragment.

For this purpose, pcDNA4TO-Psi (Ψ) -Flue (-extra) was first digested with HindIII and Xhol restriction enzymes to prepare a fragment (fragment I) from which the F-luc gene has been removed. The Psi (Ψ) sequence was amplified by PCR using the pcDNA4/TO vector (Invitrogen, USA) as a template with a pair of primers of SEQ ID NO: 18 (Psi HindIII-F, 5¹- CCAAGCTTACGCAGGACTCGGCTTGCTG-3' ) and SEQ ID NO: 19 (Psi BamHI-R_r 5-CCGGATCCTAATACCGACGGTCTCGCAC-3 ' ) . The PCR ^'amplified fragment was digested with HindIII and BamHI restriction enzymes, and then ligated with the fragment I. The resulting fragment was named "fragment II". Then, the Gaussia luciferase fragment was obtained by performing PCR amplification using a pBasicGau vector (Targeting system Inc.) as a template with a pair of primers of SEQ ID NO: 20 (Gau BamHI-F, 5- CCGGATCCGGAGTCAAAGTTCTGTTTGC-3) and SEQ ID NO: 21 (Gau Xbal-R, 5 ' -AATCTAGATGCATGCTCGAGCGGCCGCT-S ')• The PCR amplified fragment was digested with BamHI and Xhol restriction enzymes, and then ligated with the fragment II, thus preparing a pcDNA4T0-Psi (Ψ) - (-ATG) -Gaussia (- extra) vector of the present invention.

<l-8> Preparation of pcDNA4T0-Psi (Ψ) -Gaussia (- extra) vector

In order to prepare a pcDNA4TO-Psi (Ψ) -Gaussia (- extra) vector (shown as pcDNA4/T0/del .psi/gaussia (-extra) in the figure) , which comprises Gaussia luciferase as a reporter gene and does not comprise Psi (Ψ) sequence, a Gaussia luciferase fragment was inserted into a region, which was located between the BamHI and Xbal restriction enzyme sites of the pcDNA4TO~Psi (Ψ) -Flue (-extra) vector prepared in Example <l-β> and from which the luciferase fragment has been removed, thus preparing the pcDNA4TO- Psi (Ψ) -Gaussia (-extra) vector. Herein, the inserted Gaussia luciferase fragment was obtained by performing PCR according to the method of Example <l-7> and digesting the PCR amplified fragment with BamHI and Xbal restriction enzymes.

All the above-described vectors were verified through nucleotide sequencing after the preparation thereof in order to confirm whether these vectors were orderly prepared (data not shown) . Example 2: Preparation of HIV NC protein expression vector

<2-l> Preparation of pLPl/GAG (POL) vector The pLPl vector (Invitrogen, USA) was digested with restriction enzymes BcII (2994) and BspEI(5672) to remove the POL sequence, and then treated with the Klenow fragment, followed by self-ligation. The resulting vector was named "pLPl/GAG (POL) " .

<2-2> Preparation of pLPl/optiNC vector A pLPl/optiNC vector was prepared in the following manner. A codon-optimized HIV NC gene (OptiNC DNA) of SEQ ID NO: 17 was digested with restriction enzymes EcoRI and HindIII, and the digested fragment was cloned into a pUC57 vector (Genescript, USA) , which was digested with the restriction enzymes EcoRI and HindiII in advence. The resulting vector was named "pUC57 /OptiNC" . The pUC57/OptiNC and pcDNA4/TO (Invitrogen, USA) were digested with HindiII and EcoRI, and then ligated to each other, thus constructing pcDNA4/TO/OpicNC. The pcDNA4/TO/OptiNC vector was digested with Hindi11 and Notl, and then the digested, HIV NC gene-containing DNA fragment was treated with the Klenow fragment, thus obtaining a blunt-end fragment. Also, the pCMV(-HA) vector (Clontech Laboratories, Inc., USA) was digested with EcoRI and Notl, and then the digested DNA fragment was treated with the Klenow fragment, thus obtaining a blunt-end fragment. The two fragments, obtained by treatment with the Klenow fragment, were ligated to each other, and the resulting vector was named "pCMV(- HA)/OptiNC". The pLPl vector (Invitrogen, USA) was digested with Pmll/Avrll/BspEI to remove the GAG-POL gene, and the OptiNC polynucleotide was obtained from the pCMV(-HA) /OptiNC vector by treating Xmal/EcoRI. The pLPl vector and the OptiNC polynucleotide were all treated with the Klenow fragment to obtain blunt-end fragments, which were then subjected to blunt end ligation. The resulting vector was named "pLPl/optiNC" .

Example 3: Examination of interaction between HIV NC protein and HIV Psi sequence

<3-l> Examination of interaction between HIV NC protein and HIV Psi sequence

The pcDNA4TO-Psi(Ψ)- (-ATG)EGFP (-extra) vector, prepared in Example <1-1> or the pcDNA4TO-ΔPsi (Ψ) -EGFP (- extra) vector, prepared in Example <l-2> was co- transfected into cells with each of pLPl, pLPl/GAG (POL) and^' pLPl/optiNC, prepared in Example 2, ^'and whether EGFP was expressed in the cells was analyzed to examine the interaction between the HIV NC protein and the HIV Psi sequence .

For transfection, T-Rex293 cells (Invitrogen, USA) were cultured in DMEM (Dulbecco's Modified Eagle Medium), containing 10% (v/v) fetal bovine serum (FBS) and 1% streptomycin/penicillin, in conditions of 37 ^°C and 5% CO₂. After the cells were cultured to a confluency of about 60-70%, the cells were co-transfected with each of the vector using jetPEI transfection reagent (Polyplus, France) according to the manufacturer's instruction. Herein, the co-transfected vectors were as follows: a combination of the pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector (1 μg) and each of a control vector pLP/con (prepared by digesting a pLPl vector with restriction enzymes PmII (1321) and BspEI(5672) to remove the GAG-POL sequence, treating the vector fragments so as to have blunt ends, and then subjecting the vector fragments to blunt end ligation), the pLPl vector, the pLPl/GAG (POL) vector and the pLPl/optiNC vector, used in varying amounts (0.5 μg, 1 μg and 2 μg) ; and the pcDNA4TO-Psi (Ψ) - (-ATG) EGFP (- extra) vector (1 μg) and each of a control vector pLP/con, the pLPl vector, the pLPl/GAG (POL) vector and the pLPl/optiNC vector, used in varying amounts (0.5 μg, 1 μg and 2 μg) . After the co-transfected cells were cultured for 16-18 hours, the cells were treated with 1 μg/ml of tetracycline and' cultured overnight.

EGFP in the cultured cells was observed using a fluorescent microscope (Axiovert 200M, Zeiss, Germany) .

As a result, as shown in FIG. 1, in the T-REx293 cells (A) transfected with the pcDNA4TO-ΔPsi (Ψ) -EGFP (- extra) vector without Psi sequence, the expression of EGFP in the cells was not greatly affected, and thus high fluorescence was detected. However, in the T-REx293 cells (B) transfected with the pcDNA4TO-Psi (Ψ) - (- ATG) EGFP (-extra) containing the Psi sequence, the NC protein or the GAG+POL or GAG protein containing the NC protein interacted with the Psi sequence, and thus the expression of EGFP in the cells was inhibited in a concentration-dependent manner.

<3-2> Measurement of interaction between HIV NC protein and HIV Psi sequence

The interaction between the HIV NC protein and the HIV Psi sequence was measured in the same manner as in Example <3-l>, except that the pcDNA4TO-R-U5-Psi (Ψ) - (- ATG) EGFP (-extra) vector of Example <l-3> was used instead of the pcDNA4TO-Psi(Ψ) - (-ATG)EGFP (-extra) vector, the pcDNA4TO-R-U5-Psi (Ψ) -EGFP (-extra) vector of Example <l-4> was used instead of the pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector, and the HIV NC expression vector was used in varying amounts of 0.5 μg, 0.75 μg and 1.0 μg.

As a result, as shown in FIG. 2, in the T-REx293 cells (A) transfected with the pcDNA4TO-R-U5-Psi (Ψ) - EGFP (-extra) vector without Psi sequence, the expression of EGFP was not greatly affected, and thus high fluorescence was detected. However, in the T-REx293 cells (B) transfected with the pcDNA4TO-R-U5-Psi (Ψ) - (- ATG) EGFP (-extra) vector containing the Psi sequence, the NC protein or the GAG+POL or GAG protein containing the NC protein interacted with the Psi sequence, and thus the expression of EGFP in the cells was inhibited in a concentration-dependent manner. Also, the HIV Psi sequence additionally containing R-U5 showed results similar to those in Example <3-2>.

<3-3> Measurement of interaction between HIV NC protein and HIV Psi sequence using luciferase

The interaction between the HIV NC protein and the HIV Psi sequence was measured in the same manner as in Example <3-l>, except that the pcDNA4TO-Psi (Ψ) - (-ATG) - Flue (-extra) vector of Example <l-5> was used instead of the pcDNA4TO-Psi (Ψ) - (-ATG)EGFP (-extra) , and the pcDNA4T0- Psi (Ψ) -Flue (-extra) vector of Example <l-6> was used instead of the pcDNA4TO-ΔPsi (Ψ) -EGFP (-extra) vector.

Herein, the each amount of the vectors used in transfection was as follows: pcDNA4TO-Psi (Ψ) - (-ATG) - Flue (-extra) vector: 500 ng; pcDNA4TO-Psi (Ψ) -Flue (-extra) vector: 200 ng; control vector: 2 μg; pLPl : 2 μg; pLPl/GAG(POL) : 2 μg; and pLPl/optiNC: 2 μg.

The activity of luciferase detected in the transfected cells was measured in the following manner according to the manufacturer's instruction (Promega,

USA) . The tranfected T-Rex293 cells were washed with PBS

(phosphate buffered saline) , and then the PBS was completely removed. The medium containing the cells was shaken along with a PLB (passive lysis buffer) reagent at room temperature for 15 minutes. The medium was centrifuged at 4 "C at 12000 rpm for 30 seconds, and the supernatant was placed in a test tube to perform a dual- luciferase assay. 20 μl of the cell supernatant was mixed with LAR II (luciferase assay reagent II) in the test tube. The activity of firefly luciferase was measured using a luminometer (TD-20/20, Turner designs, USA) . Then, 100 μl of the Stop & GIo reagent was added to the test tube in order to increase the transfection efficiency, and the renilla luciferase activity in the cells was measured. Accordingly, the luciferase activity was determined "by the ratio of firefly luciferase activity to renilla luciferase activity.

As a result, as shown in FIG. 3, in the case where no HIV Psi sequence existed, the ratio of luciferase activity was not significantly reduced compared to the control group, even when the HIV NC protein or the GAG or GAG+POL protein containing the HIV NC protein was expressed. However, in the case where the HIV Psi sequence existed, the HIV NC protein or the GAG or GAG+POL protein containing the HIV NC protein interacted with the HIV Psi sequence, and thus the ratio of luciferase activity ^'was greatly reduced compared to the^' control group. Meanwhile, it could be seen through this experiment that the interaction between the HIV NC protein and the HIV Psi sequence could also be measured using luciferase activity instead of EGFP. <3-4> Measurement of interaction between HIV NC protein and HIV Psi sequence using Gaussia luciferase The interaction between the HIV NC protein and the HIV Psi sequence was measured in the same manner as in Example <3-l>, except that the pcDNA4TO-Psi (Ψ) - (-ATG) - Gaussia (-extra) vector of Example <l-7> was used instead of the pcDNA4T0-Psi (Ψ) - (-ATG) EGFP (-extra) vector, and the pcDNA4T0-Psi (Ψ) -Gaussia (-extra) vector of Example <l-8> was used instead of the pcDNA4T0-ΔPsi (Ψ) -EGFP (-extra) vector.

Herein, each of the pcDNA4T0-Psi (Ψ) - (-ATG) - Gaussia (-extra) vector and the pcDNA4TO-Psi (Ψ) -Gaussia (- extra) vector was used in an amount of 1 μg, and the control vector (pLP/Con) and the pLP/OptiNC vector were used in varying amounts of 500 ng, 1 μg, 1.5 μg and 2 μg. For transfection, T-Rex293 cells (Invitrogen, USA) were cultured in DMEM (Dulbecco's Modified Eagle Medium), containing 10% (v/v) fetal bovine serum (FBS) and 1% streptomycin/penicillin, in conditions of 37 ^°C and 5% CO₂. The cells were cultured to a confluency of about 60-70%, and then transfected with each of the vector combinations using jetPEI reagent (Polyplus, France) according to the manufacturer's instruction. After the transfected cells were cultured for 16-18 hours, the cells were treated with 2 μg/ml of tetracycline and cultured overnight. Then, the Gaussia luciferase activity in the cultured cells was measured. As a result, as shown in FIG. 7, in the case where no HIV Psi sequence existed (FIG. 7A), the Gaussia luciferase activity was not significantly reduced compared to the control group, even when the HIV NC protein was expressed. However, in the case where the HIV Psi sequence existed (FIG. 7B), the HIV NC protein and the HIV Psi sequence interacted with each other, and thus the Gaussia luciferase activity was greatly reduced to the control group. Meanwhile, it could be seen through this experiment that the interaction between the HIV NC protein and the HIV Psi sequence could also be measured using Gaussia luciferase activity.

Example 4: Screening of substance inhibiting interaction between HIV NC protein and HIV Psi sequence

In order to confirm whether cells co-transfected with the inventive expression vector, which expresses the HIV NC protein, and the HIV Psi sequence-containing expression vector, can be used to screen a substance inhibiting the interaction between the HIV NC protein and the HIV Psi sequence, the expression of the reporter gene in the cells, which occurred when the cells were treated with an HIV NC protein inhibitor that is an zinc chelator represented by Chemistry Figure 1 (William G. Rice et al., Science, 270: 1194, 1995), was measured in the following manner: Chemistry Figure 1

As described in Example 3, T-Rex293 cells were transfected with 250 ng of the pcDNA4T0-Psi (Ψ) - (- ATG) EGFP (-extra) vector and 1 μg of the control vector described in Example <3-l>, and then were cultured. Then, the expression of EGFP in the cells was observed with a fluorescent microscope. In addition, as described in Example 3, T-Rex293 cells were transfected with 250 ng of the pcDNA4TO- Psi (Ψ) - (-ATG)EGFP (-extra) vector and 1 μg of the pLPl/optiNC vector, and then cultured. Then, the expression of EGFP in the cells was observed with a fluorescent microscope.

In order to examine the effect of the zinc chelator, the T-Rex293 cells, transfected with 250 ng of the pcDNA4TO-Psi(Ψ) -(-ATG) EGFP (-extra) vector and 1 μg of the pLPl/optiNC vector, were cultured together with varying amounts (0.1 μM and 1 μM) of the zinc chelator in the same manner as in Example <3-l>. Herein, the zinc chelator was added to the cells when the cells were treated with tetracycline.

As a result, as shown in FIG. 4, in the control group not expressing the HIV NC protein, EGFP fluorescence was clearly observed (FIG. 4A) , but in the case where the HIV NC protein was expressed, EGFP was not expressed due to the interaction between the HIV NC protein and the HIV Psi sequence (FIG. 4B) . Also, in the case where the zinc chelator, that is, an HIV NC protein inhibitor, was added to the cells, fluorescence caused by the expression of EGFP was observed depending on the concentration of the zinc chelator (FIG. 4C) . Accordingly, the use of the system according to the present invention makes it possible to screen a substance inhibiting the interaction between the HIV NC protein and the HIV Psi sequence.

Industrial Applicability As described above, the inventive recombinant mammalian cells and the screening method using the cells provide a system capable of screening a substance, which inhibits the interaction between the HIV NC protein and the HIV Psi sequence, using eukaryotic cells. Accordingly, the present invention provides novel means capable of screening a novel substance, which inhibits the interaction between the HIV NC protein involved in the packaging of HIV and the HIV Psi interaction. In addition, the present invention provides a novel method for screening a substance for preventing and treating AIDS.

Claims

CLAIMS Claim 1

Recombinant mammalian ceils transfected with:

(a) an HIV NC expression cassette, which comprises a promoter and a polynucleotide encoding a polypeptide comprising an HIV NC (nucleocapsid) protein operably linked to the promoter; and

(b) a reporter gene expression cassette, which comprises a promoter, an HIV psi (Ψ) sequence operably linked to the promoter, and a reporter gene.

Claim 2

The cells of Claim 1, wherein the transfected cells are T-Rex293 cells.

Claim 3

The cells of Claim 1, wherein the HIV NC protein has an amino acid sequence represented by SEQ ID NO: 1.

Claim 4

The cells of Claim 1, wherein the HIV NC protein expression cassette is selected from the group consisting of pLPl, pLPl/GAG(POL) and pLPl/optiNC.

Claim 5

The cells of Claim 1, wherein the HIV psi (Ψ) sequence has a base sequence represented by SEQ ID NO: 2. Claim 6

The cells of Claim 1, wherein the reporter gene is selected from the group consisting of EGFP (enhanced green fluorescent protein) , GFP (green fluorescent protein), luciferase, gaussia luciferase, gaussian luciferase, β-galactosidase and alkaline phosphatase.

Claim 7 The cells of Claim 1, wherein the reporter gene expression cassette is selected from the group consisting of pcDNA4TO-Psi (Ψ) - (-ATG)EGFP (-extra) , pcDNA4TO-R-U5- Psi(Ψ)- (-ATG)EGFP (-extra) , pcDNA4TO-Psi (Ψ) - (-ATG) -Fluc(- extra) and pcDNA4TO-Psi (Ψ) - (-ATG) -Gaussia (-extra) .

Claim 8

A method for screening a substance for preventing and treating AIDS, the method comprising the steps of:

(a) treating the cells of Claim 1 with a candidate substance and culturing the treated cells; and

(b) measuring the expression level of the reporter gene in the cultured cells.

Claim 9 The method of Claim 1, wherein the expression level of the reporter gene is measured using a method selected from the group consisting of fluorescence detection, luciferase activity measurement, β- galactosidase activity measurement, alkaline phosphatase activity measurement, co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA) , immunohistochemical assay, Western blotting, and fluorescence activated cell sorting (FACS) .