Patent Application: US-47568103-A

Abstract:
the present invention relates to repressors suppressing production of acquired immune deficiency syndrom viral genome and methods repressing transcription thereof . the invention , more specifically , relates to fusion proteins repressing aids viral genomes of a protein selected from group consisting of proteins strongly repressing activities of proteins such as sp1 , nf - kb , proteins repressing transcriptional activities by strongly condensing chromatin , and proteins which are able to bind around aids viral promoter ; and protein recognizing short rna strand and methods repressing transcription using same . the said fusion proteins dramatically show effect repressing production of hiv - 1 genome by delivering repressing proteins like sp1 , nf - k b to hiv - 1 ltr using protein recognizing short rna strand as a carrier . the invention shows transcription inhibitory effect of hiv by targeting transcription - repressing fusion proteins to hiv ltr .

Description:
the present invention will be explained in terms of exemplary embodiments described in detail with reference to the accompanying drawings , which are given only by way of illustration and thus are not limitative of the present invention . regulation of synthesizing hiv rna is complicated , and it requires interaction with cis - acting elements in various viral ltrs , viral transactivators and cellular proteins . this interaction controls the basic level of rna transcription , and induces expression of high amount of viral genes . transcription in hiv - ltr promoters is mainly regulated by cellular protein sp1 transcription factors for recognizing adjacent promoters in the absence of viral protein tat . various signals inducing activation of nf - κb activates transcription by interacting with cis - acting elements existing in upstream of sp1 - associated gc - box . other proteins ( cofactors ) promote transcription and replication in cells having latent hiv provirus by regulating activity of proteins such as sp1 or nf - κb . tat proteins which hiv codes activate transcription by increasing the initiation or elongation of transcription , and they are important for replication . tat requires tar , which is a cis - acting rna element existing in the 5 ′ end of viral transcripts . tat also highly promotes production of viral rna having tar stem - loop rna structure in the 5 ′ end of all hiv transcripts . as a result of the recent study , sp1 is repressed by interaction with hdac and poz - domain through zinc - finger dna binding domain , and also by an interaction with mecp2 . the study has demonstrated that a poz - domain transcription factor referred to as fbi - 1 inhibits hiv transcription by interacting with sp1 and tat and binding to an ist region ( short strand inductive region ) of hiv - ltr . these proteins interact with corepressor proteins such as msin3a , smrt / n - cor and had for inhibiting transcription by condensing chromatin . accordingly , if these proteins are targeted in hiv - 1 ltr , the activity of transcription by sp1 and tat may be regulated by blocking activity of sp1 , condensing chromatin around hiv promoters and targeting polypeptides able to bind hiv promoter region . expression plasmid fused transcription inhibitory protein with tatwt is prepared in order to target transcription inhibitory proteins to core promoter regions . inventors in the present invention make tatwt fusion proteins not inhibit transcription in hiv ltr ( promoters ) in cell and tatwt fusion proteins function as transcription activation factor of viral rna by tatwt part of fusion proteins . function of transcription stimulatory factor of transcription inhibitory proteins fused with tatwt is regarded that tatwt potently functions in transcription elongation step enough to compensate with function of inhibitors inhibiting transcription initiation . however , although fusion proteins consist of two different kinds of proteins , they may be targeted to tar by tatwt part . as a result , tat proteins may be used as a targeting means to a core ltr promoter region . accordingly , the present invention uses tat protein mutant ( tatdmt : tatk28a & amp ; k50a ) strongly binding to tar but lacking an interaction with tak ( or referred to as ‘ ptef ’) which is an important cellular factor in transcription activation by tat . the longest tat of hiv consists of 101 amino acids . because there are many diverse mutants , it is difficult to distinguish which kind of tats a wild type . the present invention uses tat proteins wherein lys - 28 and lys - 50 are substituted with alanine , and uses a tat polypeptide consisting of 72 or 73 amino acids . however , fragments of the tat polypeptide or other tat mutants besides the above - mentioned tat have the similar function as described above . tatdmt fusion proteins dramatically inhibit transcription of hiv genomes in simian cv - 1 cells even when excessive tat ( 300 ng of expression plasmid ) is expressed . this experimental result is an epoch - making discovery of a protein having a dominant - negative form . most tat fusion proteins function regardless of their directions binding to tatdmt . particularly hdac - tatdmt , fbi - 1 - tatdmt fusion proteins strongly inhibit the transcription ( see fig3 b ). the fusion proteins strongly act as inhibitors in hela cells wherein hiv - 1 ltr - chloramphenicol acetyl transferase gene is inserted into human genomes . although tat protein itself activates transcription by 46 times , mecp2 , hdac , poz -, or fbi - 1 - tatdmt fusion proteins strongly inhibit the transcription activation by tatwt even under 300 ng of tatwt over expression plasmids condition . hdac - tatdmt and fbi - 1 - tatdmt among the fusion proteins completely inhibit the transcription activation by tat in hela cells too . this result shows that the fusion proteins may repress the transcription of hiv inserted into human genomes as provirus state , in consequence effectively inhibit the proliferation of hiv . because the fusion proteins of the present invention repress transcription in hiv ltr promoter and rna necessary to produce all components for viral replication is not produced , it necessarily follows that the fusion proteins repress expression of viral proteins and replication of hiv . hiv - 1 ltr cat fusion plasmid ( puc3r - iii cat ) was prepared by cloning about 720 bp of hiv - ltr to pcat - basic plasmid ( promega co .). hiv - 1 ltr luciferase fusion plasmid ( puc3r - iii - luc ) was prepared by cloning 720 bp segments of puc3r - iii cat digested with xho i hidiii restriction enzymes to pgl3 - basic plasmid ( xho i hidiii , promega co .). the fusion proteins of the present invention fused with tat ( consisting of 86 amino acids ), tatdmt ( consisting of 73 amino acids ), hdac1 , mecp2 , fbi - 1 , poz - domain and tatwt or mutant tatdmt ( tatk28ak50a ) were prepared by amplifying the corresponding gene by a pcr method , and then cloning the genes to a mammalian expression vector pcdna3 . 0 ( inivtorgen ). in order to prepare pcdna3 . 0 tatwt ( 86 amino acids ), the genes were amplified by a pcr method using pet - 15b - tatwt ( 86aa , provided by phd . park jinseo in hallym univ ., korea , hivhbx2r type ) as a template and the reaction conditions were as follows : template denaturation at 94 ° c . for 3 minutes , 30 cycles of amplification reaction ( 94 ° c . 30 sec . ; 60 ° c . 1 min . ; 72 ° c . 30 sec . ), and then post - amplification reaction at 72 ° c . for 3 minutes . after pcr products and expression vector pcdna3 . 0 were digested with two restriction enzyme ecor1 and xba1 , the digested products were ligated using t4 dna ligase , and then introduced into e . coli dh5a by a transformation method and pcdna3 . 0 tatwt ( 86 amino acids ) plasmid was prepared by an alkali lysis method . next , in order to prepare pcdna3 . 0 tatdmt ( 73aa ), methods similar to the above mentioned methods were used . however , pcdna3 . 0tatdmt was prepared by a pcr amplification using hiv tat gene ( kiernan et al ., embo j . 18 : 6106 - 6118 , 1999 ) as a template and 5 ′ primer : gat cga att cat gga gcc agt aaa tcc tag cct ag , 3 ′ primer : gatctctagatcagctttgatagagaaacttgatg ( containing stop codon ). in order to prepare hdac , mecp2 , poz -, or fbi - 1 fusion proteins of x - tatdmt , non - tat portions were prepared by amplifying the corresponding human cdna using pcr method and then by cloning the amplified genes to pcdna3 . 0 tatdmt . the reaction conditions were as follows : template denaturation at 95 ° c . for 3 minutes , 30 cycles of amplification ( 95 ° c . 30 sec . ; 62 ° c . 1 min . ; 72 ° c . 7 min .) and post - amplification reaction at 72 ° c . for 3 minutes , and pcr reaction was carried out using the following pcr primers : mecp2 5 ′ primer : gatcggatccaccatggtagctgggatgttagggctcag 3 ′ primer : gatcgaattcgctaactctctcggtcacgggcgtccg hcac1 5 ′ primer : gatcaagcttaccatggcgcagacgcagggcacccggagg 3 ′ primer : gatcgaattcggccaacttgacctcctccttgacccctttg poz - domain 5 ′ primer : gatcaagcttaccatggccggcggcgtggacggccccatc 3 ′ primer : gatcgaattcctgccggtccaggaggtcggcgcacacg fbi - 1 5 ′ primer : gatcaagcttaccatggccggcggcgtggacggccccatc 3 ′ primer : gatcagattcggcgagtccggctgtgaagtt . the amplified products were digested with bamh1 - ecor1 ( mecp2 ) or hindiii - ecor1 ( hdac1 , poz , fbi - 1 ), and then cloned to pcdna3 . 0 tatdmt / bamh1 - ecor1 or pcdna3 . 0 tatdmt / hindiii - ecor1 . in order to prepare hdac , mecp2 , poz -, fbi - 1 fusion proteins of pcdna3 . 0 tatdmt - x , methods similar to the above mentioned methods were used . however , pcdna3 . 0 tatdmt ( 73aa ) was prepared by pcr amplification using hiv tat gene ( kiernan et al ., embo j . 18 : 6106 - 6118 , 1999 ) as a template and non - tat portions of pcdna3 . 0 tatdmt - x family were prepared by amplifying the corresponding human cdna in the same condition the above mentioned and by digesting the amplified products with ecor1 - xba1 and by cloning the digested products to pcdna3 . 0 tatdmt / ecor1 - xba1 . primer sets used in amplification reaction were as follows : mecp2 5 ′ primer : gatcgaattcatggtagctgggatgttagggctca 3 ′ primer : gatctctagatcagctaactctctcggtcacgggc hdac1 5 ′ primer : gatcgaattcatggcgcagacgcagggcacccgga 3 ′ primer : gatctctagatcaggccaacttgacctcctccttg poz - domain 5 ′ primer : gatcgaattcatggccggcggcggcgtggacggcc 3 ′ primer : gatctctagatcactgccggtccaggaggtcggcg fbi - 1 5 ′ primer : gatcgaattcatggccggcggcggcgtggacggcc 3 ′ primer : gatctctagatcaggcgagtccggctgtgaagtt . cv - 1 cells were cultured in a dmem culture medium with 10 % fbs . when the cells grew enough to occupy 50 - 60 % of bottom area in culture vessel , the mixtures consisting of 0 . 6 μg of phiv - ltr - luciferase plasmid , pcmv - β galactosidase plasmid , tatwt , and a mammalian expression pcdna3 . 0 plasmid selected from the group consisting of hdac1 - tatdmt , mecp2 - tatdmt , fbi - 1 - tatdmt , poz - domian - tatdmt , tatdmt - hdac1 , tatdmt - mecp2 , tatdmt - fbi - 1 , and tatdmt - poz - domain were introduced into cell using a lipopectamin plus reagent ( gibco - brl ). in order to perform an experiment for repressing genome expressions in hela cells which hiv - ltr - cat was inserted into genome , the mixtures consisting of 300 ng of tat expression plasmid and 300 ng of various fusion proteins expression plasmid of the present invention were introduced into cells were cultured in dmem culture medium with 10 % fbs using the lipopectamin plus reagent ( gibco - brl ). the cells were cultured for 24 hours , and the expression of reporter gene was analyzed . the efficiency for introducing plasmid into cell and the deviation for recovering cell extracts were standardized using activity of simultaneously introduced and expressed β - galactosidase . the results of the present invention are shown in fig2 ˜ 4 . in fig2 the fusion proteins fused with tatwt may not inhibit genome expression in hiv - ltr at transcriptional level , but the fusion proteins may be targeted into hiv ltr promoter region by tatwt part , thereby enhancing expression by promoting transcription elongation . in fig3 the fusion proteins fused with tatdmt ( mutants consisting of 72 or 73 amino acids wherein 2 amino acid sequences of tat proteins are mutated ) potently inhibit genome transcription in hiv - ltr in the presence of 300 ng of tatwt expression plasmid , thereby reduce the expression by the low level in the absence of tat . the result reveals that transcription in hiv - ltr like provirus state in hela cell strongly inhibit by targeting fusion protein to tar region by tat . in fig4 fbi - 1 itself does not show transcription inhibitory function . tatdmt shows only competitive inhibition that competitively binds to the same site ( tar ). when the same amount of fbi - 1 and tatdmt is provided , about 50 % of inhibitory function is shown ( b8 ). but fbi - 1 - tatdmt shows inhibitory function at 1 ng , and 50 % inhibitory function at 3 ng . fbi - 1 - tatdmt shows the same inhibitory function at 81 ng as that of the absence of tatwt , thereby completely inhibiting transcription by tatwt . however , fbi - 1 - tatdmt shows a lower level of expression suppression at 243 ng than in the absence of tatwt . within cell nucleus , host cellular proteins such as sp1 , nf - κb act upon hiv transcription regulatory regions . as a result , viral rna of short rna strand ( short transcript ) is produced and it stays around the regions ( representing rna strands binding to polymerase ii ). if viral protein such as tat binds to tar region , rna synthesis is highly promoted . as a result , a large amount of viral genome ( rna ) is produced , and protein components necessary for viral proliferation are produced by using the resulting viral genome ( rna ). accordingly , the most effective method to inhibit viral proliferation is to regulate function of sp1 , nf - k b , tat . the present invention provides the method to potently inhibit production of viral rna ( transcription process ) in viral ltr , when protein for recognizing hiv short transcript regions ( e . g . tar ) is targeted to hiv transcription regulatory promoter region , by fusing the protein with the protein selected from the group consisting of proteins repressing transcription factor like sp1 , nf - κb ; corepressor or proteins interacting with corepressor ; hdac , or proteins interacting with hdac repressing transcriptional activity by strongly condensing chromatin ; and proteins such as zinc finger having binding activity to viral promoter region . the present invention is the first study showing the method of potently inhibiting transcription activity in hiv ltr by targeting transcription inhibitory protein groups to hiv - ltr . the present invention also basically inhibits production of components ( genomes , proteins ) necessary for viral proliferation by repressing expression of viral rna genome from dna type viral ltr ( promoter ) existing as proviral state in host cellular genome . accordingly , the present invention may basically inhibit the proliferation of virus and production of resistant virus . particularly , hdac - tatdmt and fbi - 1 - tatdmt fusion proteins completely block the process of producing viral genome ( rna ). therefore , the present invention to overcome the problems of the conventional drugs as aids therapeutic agents is the effective protein or gene therapeutic agent to treat aids . met glu pro val asn pro ser leu glu pro trp lys his pro gly ser his cys gln val cys phe ile thr lys ala leu gly ile ser tyr gly met glu pro val asn pro ser leu glu pro trp lys his pro gly ser his cys gln val cys phe ile thr lys ala leu gly ile ser tyr gly met val ala gly met leu gly leu arg glu glu lys ser glu asp gln ser gly arg ser ala gly lys tyr asp val tyr leu ile asn pro gln gly lys ala phe arg ser lys val glu leu ile ala tyr phe glu lys glu pro trp lys his 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