Patent Application: US-51680195-A

Abstract:
upon entry into a host cell , retroviruses direct the reverse transcription of the viral rna genome and the establishment of an integrated proviral dna . the retroviral integrase protein is responsible for the insertion of the viral dna into host chromosomal targets . the in catalyzes two specific biochemical reactions : cleavage of the 3 &# 39 ; termini of the viral dna to produce 3 &# 39 ;- oh ends , and joining of the two newly generated 3 &# 39 ;- termini to the 5 &# 39 ;- phosphates on each strand of the target sequence in a concerted strand - transfer reaction . the yeast two - hybrid system was used to identify a novel human gene product , herein designated integrase interactor 1 or ini - 1 , that binds tightly to the human immunodeficiency virus type 1 integrase in vitro . approximately 10 6 complementary dnas of the hl60 macrophage - monocytic cell line were expressed as gal4ac fusions and tested for coactivation of a reporter gene together with a gal4db in fusion . overlapping cdna clones were identified and their nucleotide sequences ascertained . nucleotide sequence analysis revealed that ini - 1 displays limited amino acid homology to the yeast snf5 protein , a transcriptional activator required for high - level expression of many disparate cellular genes . the ini - 1 gene product will prove useful for the generation of biochemical reagents and the development novel hiv - 1 antiviral agents .

Description:
this invention provides an isolated nucleic acid encoding an integrase interactor 1 gene ( ini - 1 ). in one embodiment of this invention , the isolated nucleic acid is dna encoding the integrase interactor 1 gene that is free of one or more introns present in genomic dna . in other embodiments of this invention , the isolated nucleic acid sequence described herein are cdna or genomic dna . the dna may be labelled with a detectable moiety selected from a group consisting of a fluorescent label , a radioactive atom , and a chemiluminescent label . in one embodiment of the invention replicable vectors which comprise the nucleic acid described herein are also provided . the replicable vectors include those where the nucleic acid is free of introns . suitable vectors comprise , but are not limited to , a plasmid or a virus . the dna sequence described and claimed herein is useful for the information which it can provide concerning the amino acid sequence of the polypeptide . the sequence is useful for generation new cloning and expression vectors , transforming and transfecting prokaryotic , eucaryotic and bacterial host cells , and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products . the invention further provides a purified polypeptide comprising naturally - occurring ini - 1 , the polypeptide may be the product of prokaryotic or eukaryotic expression of an exogenous dna sequence . the exogenous dna sequence is a cdna or a genomic dna sequence . the exogenous dna sequence may be carried on an autonomously replicating dna plasmid or viral vectors . in one embodiment the purified polypeptide of ini - 1 may be human ini - 1 . the invention also provides for the purified polypeptide possesses part or all the amino acid sequence of human ini - 1 as shown in fig4 or any naturally occurring allelic variant thereof . the purified polypeptide may have in vivo or in vitro biological activity of naturally occurring ini - 1 . the purified polypeptide may be covalently associated with a detectable label substance . the invention also provides a method of determining whether a compound is capable of interfering with the formation of a complex between a retrovirus integrase protein and an ini - 1 protein , which comprise the following steps : a ) incubating the compound with an appropriate ini - 1 affinity fusion protein and the retrovirus integrase protein ; b ) contacting the incubate of step ( a ) with an appropriate affinity medium under conditions so as to bind the ini - 1 affinity protein complex , if such a complex forms ; and c ) measuring the amount of the ini - 1 affinity protein complex formed in step ( b ) so as to determine whether the compound is capable of interfering with the formation of the complex between the retrovirus integrase protein and the ini - 1 protein . in one preferred embodiment , the retrovirus integrase protein may be hiv - 1 in , the affinity fusion protein may be gst - ini - 1 . the affinity medium may be glutathione - agarose beads . the amount of the affinity protein complex formed may be determined using monoclonal or polyclonal antibodies . the above method may also be performed using a retroviral integrase protein fusion . in one preferred embodiment the ini - 1 affinity protein complex or the retrovirus integrase affinity protein complex is bound to the affinity medium . the ini - 1 affinity protein complex or the retrovirus integrase affinity protein complex is purified and removed from the affinity medium and the amount of integrase protein or ini - 1 protein is determined . the amount of the integrase protein or ini - 1 protein may be determined using monoclonal or polyclonal antibodies . the above assays may be performed in vivo or in vitro . the invention also provides for a method of disrupting a retrovirus life cycle in a cell which comprises contacting the cell with a compound which is capable of disrupting a retrovirus integrase protein - ini - 1 protein interaction so as to thereby disrupt the retrovirus life cycle . the compound contacting the cell may be a soluble ini - 1 fragment , a hiv - 1 in fragment or a chemical molecule . the soluble ini - 1 fragment may be a small peptide of 4 to 20 amino acids in length , in one preferred embodiment there may be 6 to 12 amino acids . other fragments may include non - peptide mimics of ini - 1 fragments . a method of disrupting a retrovirus life cycle in a mammal which comprises administering to the mammal a compound which is capable of disrupting a retrovirus integrase protein - ini - 1 protein interaction so as to thereby disrupt the retrovirus life cycle . the compound administered to the mammal may be a soluble ini - 1 fragment , a hiv - 1 in fragment or a chemical molecule . the invention provides an isolated cdna encoding an integrase interactor 1 gene ( ini - 1 ) having a coding sequence substantially the same as the coding sequence as shown in fig4 . for the above - identified compounds and methods the retrovirus may be selected from the following groups , avian leukosisarcoma , mammalian c - type , b - type viruses , d - type viruses , htlv - blv group , lentiviruses and &# 34 ; foamy viruses . the retroviruses may also be selected from the following examples , rous sarcoma virus ( rsv ), avian myeloblastosis virus ( amv ), avian erythroblastosis virus ( aev ), rous - associated virus ( rav )- 1 to 50 , rav - 0 , moloney murine leukemia virus ( mo - mlv ), harvey murine sarcoma virus ( ha - msv ), abelson murine leukemia virus ( a - mulv ), akr - mulv , feline leukemia virus ( felv ), simian sarcoma virus , endogenous and exogenous viruses in mammals , reticuloendotheliosis virus ( rev ), spleen necrosis virus ( snv ), mouse mammary tumor virus ( mmtv ), mason - pfizer monkey virus ( mpmv ), &# 34 ; saids &# 34 ; viruses , human t - cell leukemia ( or lymphotropic ) virus ( htlv ), bovine leukemia virus ( blv ), human immunodeficiency virus ( hiv - 1 and - 2 ), simian immunodeficiency virus ( siv ), feline immunodeficiency virus ( fiv ), visna / maedi virus , equine infectious anemia virus ( eiav ), caprine arthritis - encephalitis virus ( caev ), progressive pneumonia virus , many human and primate isolates e . g ., simian foamy virus ( sfv ). this invention is also directed to pharmaceutical compositions comprising therapeutically effective amounts of compounds of the invention together with suitable diluents , preservatives , solubilizers , emulsifiers and adjuvants . administering a therapeutically effective amount refers to that amount which provides therapeutic effect for a given condition and administration regime . such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content ( e . g ., tris - hcl , acetate , phosphate ), ph and ionic strength , additives such as albumin or gelatin to prevent adsorption to surfaces , detergents ( e . g ., tween 20 , tween 80 , pluronic f68 , bile acid salts ), solubilizing agents ( e . g ., glycerol , polyethylene glycol ), anti - oxidants ( e . g ., ascorbic acid , sodium metabisulfite ), preservatives ( e . g ., thimerosal , benzyl alcohol , parabens ), bulking substances or tonicity modifiers ( e . g ., lactose , mannitol ), complexation with metal ions , or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid , polyglycolic acid , hydrogels , etc . or into liposomes , microemulsions , micelles , unilamellar or multilamellar vesicles , erythrocyte ghosts , or spheroplasts . such compositions will influence the physical state , solubility , stability , rate of in vivo release . controlled or substained release compositions include formulation in lipophilic deposits ( e . g ., fatty acids , waxes , oils ). also included in this invention are particulate compositions coated with polymers ( e . g ., poloxamers or poloxamines ). other embodiments of the compositions of the invention incorporate particulate forms protective coatings and permeation enhancers for various routes of administration , including parenteral , pulmonary , nasal and oral . the following examples are offered to more fully illustrate the invention , but are not to be construed to limit the scope thereof . to identify human proteins that bind to the hiv - 1 integrase , the yeast two hybrid system was used to screen a large library of human cdnas . in this system , the expression of two constructs in yeast -- one encoding the gal4 dna binding domain ( gal4db ) fused to one protein , and the other encoding the gal4 activator domain ( gal4ac ) fused to another protein -- results in the reconstitution of gal4 function if the two proteins bind with sufficient affinity ( fields and song , 1989 ). the appearance of gal4 function can be detected by monitoring the expression of an integrated reporter gene , such as lacz , downstream of a gal4 - dependent promotor . previously the system was used to detect several interactions between viral and host proteins ( luban et al ., 1992 ; luban et al ., 1993 ), and in particular to detect in - in multimerization ( kalpana and goff , 1993 ). to generate a library of gal4 activator domain - cdna fusions , the inserted sequences of a human cdna library derived from the hl60 macrophage / monocytic cell line were excised from the original phage vector and transferred in bulk to a plasmid vector . six different pools of plasmids were prepared , each containing 100 , 000 to 500 , 000 individual clones ( table 1 ). table 1______________________________________ number oflibrary original e . coli . in - interactingpools clones clones recovered______________________________________pool i 0 . 23 × 105 -- pool ii 0 . 5 × 105 onepool iii 5 . 00 × 105 -- pool iv 3 . 00 × 105 -- pool v 1 . 5 × 105 -- pool vi 1 . 00 × 105 two______________________________________ table 1 is a summary of recombinant clones in various pools of hl60 cdna library and positive in - interacting clones obtained from each pool in the two hybrid screen . yeast strain ggy1 :: 171 , which contains an integrated reporter gene , was transformed with a mixture of a given dna pool and an equal amount of pgal4db - in dna , encoding a fusion protein consisting of the gal4 dna binding domain and the entire hiv - 1 in ( kalpana and goff , 1993 ). cotransformants were recovered after selection for markers on both plasmid vectors , and colonies were replicated to filters and stained with x - gal . 10 blue colonies were obtained from a total of 600 , 000 transformants screened ( table 1 ). the plasmids were rescued from these colonies and retested by transformation along with the plasmid encoding gal4db - in into ggy1 :: 171 . three of these candidate clones consistently tested positive upon cotransformation : one from pool ii and two from pool vi . subsequent analysis of these clones ( see below ) showed that all three contained identical cdna inserts . thus , an single cdna was identified in this large - scale screen as encoding a protein able to interact with the hiv - 1 in . the novel gene was termed ini - 1 for integrase interactor 1 . many cdnas initially isolated as candidates in the two - hybrid system do not in fact depend on interaction with the partner hybrid protein , but instead activate expression of the indicator gene through other means ( luban et al ., 1993 ). to demonstrate a requirement of the partner for interaction , the gal4ac - ini - 1 fusions were tested for activation in several settings ( table 2 ). table 2______________________________________ β - galfusion proteins promoter operator activity______________________________________gal4db - in + gal4ac - ini gal1 uas . sub . g ++++ gal4db - in + gal4ac - ini gal1 uas . sub . g ++++ gal4db - mg + gal4ac - ini gal1 uas . sub . g - gal4db + gal4ac - ini gal1 uas . sub . g - gal4ac - ini gal1 uas . sub . g - lexadb - in + gal4ac - in gal1 uas . sub . g ++++ lexadb - in + gal4ac - ini gal1 uas . sub . g ++++ lexadb - lamin + gal4ac - ini gal1 uas . sub . g - lexadb + gal4ac - ini gal1 uas . sub . g - gal4ac - ini gal1 uas . sub . g - ______________________________________ table 2 shows the specificity of inini = 1 interaction in yeast under various conditions of promoters and dna binding domains . two plasmids , on encoding gal4db fusion and the other encoding gal4ac fusions were cotransformed into either ggy1 :: 171 ( for testing the gal4db fusions ) or cty105d ( for testing lexadb fusions ). the transformants were scored for bgal activity . fusion protein gal4dbin was encoded by plasmid pmai , gal4acin by pgadi , gal4ac ini by pd2 . 1 , lexadb by psh21 , lexadbin by pshin , and lexadblamin by plexalamin . transformation of ggy1 :: 171 by the gal4ac - ini - 1 plasmids alone , without pgal4db - in , did not activate lacz expression . cotransformation with a plasmid encoding gal4db alone also did not activate , suggesting that the ini - 1 protein did not interact directly with the gal4 dna binding domain . to confirm that the activation was not restricted to the gal4 system , the dnas were introduced into strain cty10 - 5d , containing an integrated gal1 - lacz fusion downstream of the lexa operator , along with a plasmid encoding a lexa - in fusion , or as control , lexa alone . lacz expression was detected only when the gal4ac - ini - 1 protein was present with lexa - in fusions and not with the lexa protein alone . these results indicate that activation by ini - 1 fusions was not dependent on the particular operator and binding domain used to tether the in protein to dna . to determine whether the ini - 1 protein could interact with unrelated fusion proteins , the three ini - 1 plasmids were introduced into the appropriate indicator strain along with control plasmids encoding a gl4db - moloney gag fusion or a lexa - lamin fusion protein . no lacz expression was detected in either of these settings , indicating that activation by the cdna fusions was specific for the hiv - 1 in protein ( table 1 ). thus , in contrast to other screens for interacting partners with other proteins , where many rna - binding proteins were initially detected , there were no false positive clones recovered with in . the results suggest that the original gal4 - in construct was not prone to interaction with false positives , but bound uniquely to a human protein encoded by a single cdna . the central domain of in is required for interaction with ini - 1 . the two hybrid system has been previously used to define the minimal region of in required for in - in interactions , finding that the central core region of the protein was necessary for multimerization ( kalpana and goff , 1993 ). to determine the region of in required for binding to ini - 1 , a panel of mutants of pgal4db - in were tested containing deletions and point mutations for activation in the presence of gal4ac - ini - 1 . mutants lacking either the n - terminal domain of in , containing a putative zinc finger region , or the c - terminal domain , retained their ability to bind to ini - 1 ( fig1 ). two larger c - terminal deletions , removing part of the central core and eliminating in - in interactions , did not affect in - ini - 1 interaction . in addition , a variant containing a point mutation in the c - terminal region of in that blocked in - in interaction ( kalpana and goff , unpublished date ) was still positive for in - ini - 1 interaction . thus , the in - ini - 1 interaction requires less of the in central and c - terminal domains than the in - in interaction . two mutants of in with point mutations in the n - terminal zinc finger region were also tested . while these mutants still carry out in - in interactions , they were both defective for the in - ini - 1 interaction . thus , binding to ini - 1 seems to require the n - terminal zinc finger region of in . while the two interaction domains -- that for in - in dimerization and that for in - ini - 1 interaction -- may overlap , the in - ini - 1 domain seems to be more n - terminal . the cdna inserts recovered in the gal4ac plasmids were derived from mrnas of the hl60 human monocytic - myelocytic cell line , suggesting that the gene must be expressed in at least moderate levels in this tumor line . the sequences present in the cdna insert might include only a portion of the complete mrna . to determine how widely the ini - 1 mrna was expressed , and to determine the size of the full - length transcript , rnas were isolated from hela cells , a human b - cell tumor line ( cb33 ), and a human t - cell line ( hut78 ), and analyzed by northern blot hybridization using an ini - 1 probe ( fig2 ). rnas from all three lines contained a single major species detected with the probe , migrating at approximately 2 . 0 kb . in addition , the hela and cb33 lines contained a minor species migrating at approximately 4 . 0 kb . to determine whether the ini - 1 gene was expressed in normal tissues , rnas isolated from peripheral blood lymphocytes , colon , small intestine , ovary , testis , prostate , thymus and spleen were separated by electrophoresis , blotted and probed as before ( fig2 ). all 8 tissues expressed substantial levels of the 2 . 0 kb mrna . the level of expression of the mrna was similar in all the tissues tested . in addition to the major mrna species , long exposures of the autoradiographs revealed low levels of a species migrating at 1 . 25 kb present in the spleen , and similarly low levels of a species migrating at about 4 kb in the thymus , prostate and testes . these results suggest that the ini - 1 gene is very widely , and possibly ubiquitously , expressed , and that the major transcript in all tissues is approximately 2 . 0 kb in length . additional transcripts with alternative structures , or transcripts from closely related genes , may be present in some tissues . isolation of cdnas spanning the complete ini - 1 coding region and predicted sequence of the ini - 1 protein the cdna inserts in the three gal4ac plasmids recovered were examined by restriction mapping and partial sequence analysis , and all were found to consist of the identical 1 . 0 kb fragment , presumably from sibling clones in the original phage library . to isolate longer cdnas , this fragment was excised from the plasmid and used as a probe to screen two phage cdna libraries of hela cell mrna , one made in the λzapii vector and one in λgt11 . 20 clones were recovered from approximately 600 , 000 clones of the λzapii library , and the six largest inserts were excised from the vector . four of these had overlapping restriction maps ( fig3 ) consistent with that of the probe dna and were subjected to sequence analysis . 12 clones were recovered from the λgt11 library but no inserts were larger that the earlier clones ; one of these cdnas was also sequenced . the dna sequences obtained could be readily aligned , and spanned 1 . 85 kb , nearly the size of the full - length mrna detected by northern blots ( fig3 ). the dna sequence from the clones contained several unusual features ( fig4 ). first , the sequence was extraordinarily gc - rich and included several long stretches of pure gc runs . these features made determination of the sequence by dideoxynucleotide methods difficult , and several regions could only be read from smaller subclones that presumably removed secondary structures from the dna templates . the sequence revealed a single long open reading frame of 385 codons , curiously beginning with a tandem array of four consecutive atg codons . the first atg of the array lies in a good match to the consensus sequence for translational initiation ( kozak , 1991 ). these codons are likely to represent the rue start sites for translation , since termination codons are found upstream of these atgs . the significance of the presence of these tandem methionine codons remains unclear . the one clone from the λagt11 library ( pini . gt ) contained a stretch of poly ( a ) residues at the 3 &# 39 ; junction adjacent to the vector , and three of the clones from the λzapii library had 3 &# 39 ; junctions at or upstream of this position , such that they could have been derived from a similar mrna . examination of the sequence upstream of the poly ( a ) stretch revealed the presence of a perfect consensus polyadenylation signal , aataaa , at - 25 bp relative to the poly ( a ). these results suggest that most of the ini - 1 mrnas are processed by cleavage and polyadenylation at this position . one cdna clone ( pini . 21 ), however , extended beyond this region without poly ( a ) sequences . this clone suggests that some mrnas are of extended length and arise through use of alternative poly ( a ) addition sites further downstream . these rnas could possibly account for the longer mrnas observed in northern blots of mrnas from various tissues . one clone , ( pini . 9 ), lacked a short stretch of 27 bp ( nt 206 - 232 ) near the 5 &# 39 ; end of the coding region . this clone might have arisen from an alternatively spliced mrna lacking an internal exon . the long open reading frame predicts the formation of a protein of 44 , 131 daltons containing 385 amino acids . the sequence revealed the presence of a heptad repeat of three leucine residues near the amino - terminus of the encoded protein ; these residues could potentially form a leucine zipper structure . while these sequences might be important for multimimerization , interactions with other proteins , or for the normal function of the ini - 1 , these structures can be eliminated as important for interaction with the in protein since they are not present in the original yeast plasmid clone that demonstrated binding to in . the predicted sequence includes no amino - terminal secretion signals , no transmembrane segment , and no strikingly acidic or basic regions . there are three potential sites for addition of n - linked sugars ( fig4 ). the predicted pi of the protein is 6 . 15 . comparison of the predicted sequence of ini - 1 with the known sequences in the genembl data base revealed a single significant match , the snf5 protein of s . cerevisiae , encoding a transcriptional activator protein ( abrams et al ., 1986 ; laurent et al ., 1990 ; neigeborn and carlson , 1984 ). snf5 is a nuclear protein thought to act in a complex with several other proteins including snf2 / swi2 , snf6 , swi1 , and swi3 , to activate target gene expression ( laurent et al ., 1991 ; peterson and herskowitz , 1992 ). the alignment of ini - 1 with the snf5 sequence displayed three regions of close similarity , with 33 - 55 % sequence identity and 41 - 71 % of conserved residues ( fig5 ). all three regions lay in the central portion of the snf5 sequence rich in changed amino acids and the flanking n - and c - terminal portions of the yeast gene were not conserved in the human gene . in particular , the proline - and glutamine - rich segments of the yeast protein were not retained . based on the striking similarity between the yeast and human genes in the core coding region , the ini - 1 may be a human homologue of the yeast snf5 gene . to demonstrate that ini - 1 interacts directly with in in solution , binding studies between recombinant proteins were carried out in vitro . the ini - 1 cdna from plasmid pd2 . 1 was inserted into plasmid pgex and expressed as a glutathione s - transferase fusion protein in e . coli . lysates of the bacteria were prepared , and the gst - ini - 1 protein was affinity purified on glutathione agarose beads ( g - beads ). the beads were washed extensively to remove nonspecific proteins . to ensure that the gst - ini - 1 proteins were successfully expressed and bound to the beads , the proteins on the beads were recovered by boiling in sds , and examined by sds - page ( fig6 a ). a novel protein of the expected size ( 60 kd ) was recovered from lysates containing the gst - ini - 1 protein , and represented 70 - 80 % of the total protein . the immobilized ini - 1 was used as an affinity matrix for binding of in . the hiv - 1 in protein was expressed in e . coli from the t7 promoter after induction of the t7 polymerase , and soluble in protein was extracted from inclusion bodies with buffer containing high salt . these lysates were then incubated with g - beads alone , g - beads with gst alone , or g - beads with gst - ini - 1 , the beads were washed extensively , and the bound proteins were recovered with sds . the eluted proteins were separated by sds - page , blotted to nitrocellulose , and visualized with polyclonal antibodies specific for hiv - 1 in ( fig6 b ). the results showed that the recombinant in bound efficiently to the ini - 1 beads and not to the control gst beads or the beads alone . to further characterize the in - ini - 1 interaction , binding experiments were repeated under various conditions ( fig6 b and 6c ). binding was observed over a wide range of salt concentrations , and was detected even in the presence of 1m nacl . the in was retained by the ini - 1 beads when washed with buffers containing 0 . 5 % np40 or 0 . 1 % triton x - 100 . the interaction was disrupted , however , by the addition of 0 . 1 % sds , suggesting that denatured in and ini - 1 proteins could not bind . ini - 1 acts as a transcriptional activator in yeast when expressed as a dna binding domain fusion protein the yeast snf5 protein is a transcriptional activator , required for the high - level of expression of many genes in yeast . though the protein has not been shown to bind to dna directly , it is capable of activating a reporter gene when artificially tethered to dna by fusion to the lexa dna binding domain ( laurent et al ., 1990 ). to determine whether ini - 1 could also act as a transcriptional activator in this setting , a construct encoding a fusion of gal4 dna binding domain - ini - 1 was generated and expressed in an indicator strain containing a gal1 - lacz reporter . the transformants expressed high levels of β - galactosidase as judged by staining with χ - gal , while control transformants expressing only the gal4db or gal4ac - ini - 1 protein did not . thus , like snf5 , the human ini - 1 protein can activate transcription in yeast . the two - hybrid system has been used to seek human proteins that might be involved in retroviral replication . the novel gene identified in this screen , ini - 1 , encodes a protein which is capable of binding the hiv - 1 in both in vivo and in vitro . the fact that all three clones recovered in the screen were identical , and that no other clones were identified in a large number tested , suggests that ini - 1 is the major human protein capable of binding to in . it is noteworthy that there were no false positive clones at all detected in this screen , suggesting that the gal4db - in fusion used here did not allow interactions to the gal5 region or other proteins that often produce false positives . the binding seemed to be very specific , and could be observed in the setting of several fusion constructs including either the gal4 or lexa binding domains . the interaction measured in vitro was tight and was resistant to high salt , suggesting that it may involve hydrophobic contacts on the two partners . the binding in solution was also specific , with no significant binding of in to gst or gst - cyclophilin proteins ( luban et al ., 1993 ) tested as controls . the region of in required for binding to ini - 1 was a portion of the central domain ; the very n - and c - terminal regions were dispensable . the essential region for interaction to ini - 1 was distinct from that for multimerization of in , apparently lying more toward the n - terminus of the protein . mutants of in that showed differential effects on the two interactions were readily obtained . it is possible that the ini - 1 protein can bind to a multimer of in and stabilize multimer formation , or it could block or compete for in multimerization . ini - 1 could stimulate concerted joining of both termini into target dnas , accelerating functional integration reactions ; or , alternatively , it could inhibit concerted joining of two termini of the viral dna to the target sequence , acting to restrain normal retroviral integration . the function of ini - 1 can be explored through analysis of its effects on various in vitro integration activities . the presence of a protein like ini - 1 in an infected cell able to bind the hiv - 1 in could be responsible for targeting proviral insertion to selected sites in the genome . the phenomenon of non - random integration of retroviral and retrotransposon dnas is well - established , but the mechanisms by which targeting occurs remain uncertain . insertions seem to preferentially occur into transcriptionally active regions , and perhaps into open chromatin ( rohdewohld et al ., 1987 ; vijaya et al ., 1986 ). in the case of the yeast transposon ty3 , site selection is profoundly specific , with insertions almost always occurring at a position 16 or 17 bp from the site of initiation of poliii transcripts ( chalker and sandmeyer , 1990 ; chalker and sandmeyer , 1992 ). analysis of mutant promoter sequences and of hybrid target sites strongly suggest that nuclear protein complexes including tfiii1a , tfiiib , and tfiid are responsible for site selection , and for precise positioning of the insertion into the promoter ( sandmeyer et al ., 1990 ). in the case of the transposon ty1 , site selection is more relaxed , but analysis of a large number of insertions into yeast chromosme iii suggests that insertions tend to occur within regions clustered within 400 bp of poliii genes ( ji et al ., 1993 ). such preferences might be mediated by the accessibility of stretches of dna , or by interactions of the transposon - in complex with chromatin of other dna - bound proteins . the existence of a mammalian protein with high affinity for the hiv - 1 in is consistent with its playing a similar role in site selection for retroviral insertion . snf5 is a transcriptional activator in yeast , and is required for transcription of many unrelated genes such as suc2 , ho , ino1 , pho5 , and gal1 , 7 and 10 . in addition , it is required for the function of many gene - specific activators , including gal4 , bicoid , and the glucocorticoid receptor ( laurent and carlson , 1992 ; yoshinaga et al ., 1992 ). genetic experiments suggest that the yeast snf5 protein acts in a enormous complex with the products of the swi1 , snf2swi2 , swi3 , snf6 , and possibly other genes ( laurent et al ., 1991 ; peterson and herskowitz , 1992 ), and co - immunoprecipitation studies using antibodies to yeast snf5 confirm its presence in a large complex . the snf2 subunit of the complex has domains similar in sequence to dna helicases ( davis et al ., 1992 ; laurent et al ., 1992 ), and has been shown to exhibit dna - dependent atpase activity ( laurent et al ., 1993 ). mammalian homologues of the yeast snf2 / swi2 products have recently been identified ( khavari et al ., 1993 ; muchardt and yaniv , 1993 ; okabe et al ., 1992 ) the snf and swi transcription factors may act by helping to reorganize chromatin structure ( for review , see winston and carlson , 1992 ). deletions of one copy of the genes encoding histones h2a and h2b can suppress the defects in ty and suc2 transcription caused by snf2 , and 5 mutations ( clark - adams et al ., 1988 ; happel et al ., 1991 ), and these suppressors probably act by inducing changes in the chromatin structure as assayed by microccal nuclease digestion experiments ( hirschhorn et al ., 1992 ). other suppressors of snf and swi mutations have been identified as alleles of a gene encoding histone h3 ( cited in peterson and herskowitz , 1992 ), and of a gene encoding a nonhistone dna binding protein similar to hmg1 ( kruger and herskowitz , 1991 ). these observations suggest that the normal role of the snf and swi genes may be to alter the arrangement of nucleosomes on target genes to facilitate their transcription . the unexpected sequence similarity of the ini - 1 protein to snf5 is intriguing : the similarity implies that ini - 1 may be a novel transcriptional activator in human cells , and may act in a complex to decondense chromatin . the ability of the human sequence to activate a reporter gene in yeast when tethered to dna lends further support to this notion . such a role is also consistent with its affinity for hiv - 1 in , and would suggest that ini - 1 might indeed account for the propensity of retroviral dna to insert into active genes . finally , the identification of a host protein as interacting with the hiv - 1 in raises the possibility that it may be used as a novel route to inhibit viral replication . if the protein serves to stimulate integration , then drugs which could block the interaction might retard viral spread . in addition , it might be possible to generate dominant negative alleles of ini - 1 , perhaps encoding small fragments of the protein , that bind inappropriately to in and block its activity . the retroviral integrase protein ( in ) is responsible for the insertion of the viral dna into host chromosomal targets . the two hybrid system has been used to screen a human cdna library expressed as gal4 fusion proteins in yeast for gene products that interact with the human immunodeficiency virus type 1 in . the screen led to the recovery of three independent isolates of the same gene from approximately 10 6 colonies . the protein encoded by this gene bound tightly to the hiv - 1 integrase in vitro . the sequence of the gene suggests that the novel protein is a human homologue of yeast snf5 , a transcriptional activator required for high level expression of many genes . the new gene is termed ini - 1 for integrase interactor 1 , encodes a nuclear receptor for incoming viral integration complexes , and may be a component of the long - sought mechanism for biased target site selection during provirus integration . bacterial and yeast strains : yeast strain ggy1 :: 171 ( mat αleu2 - 3 , 112 his3 - 200 met - tyrl ura3 - 52 ade2 gal4δ gal80δ ura3 :: gal1 - lacz ) ( fields and song , 1989 ) contains an integrated gal1 - lacz reporter gene ; cty 10 - 5d ( mata ade2 trpl - 901 leu2 - 3 , 112 his3 - 200 gal80 - ura3 :: lexa - lacz ) contains an integrated gal1 - lacz gene with lexa operator . β - galactosidase assays , both in liquid cultures and on nitrocellulose lifts , were carried out as published with minor modifications ( chien et al ., 1991 ). e . coli strains dh5α ( brl ), xl1blue and sure ( stratagene ) were used for subcloning plasmids ; strain bl21 ( de3 ) was used for the expression of recombinant proteins from t7 promoters . construction of plasmids pmai ( encoding gal4db - in fusion ), and pgadi ( encoding gal4ac - in ), pshin ( lexadb - in fusion ) and pma - mg ( encoding the gal4db fused to the moloney mulv gag protein ) have been previously described ( kalpana and goff , 1993 ; luban et al ., 1992 ). plasmids pgvk10 ( expressing the gst - ini - 1 fusion protein ) and pmai ( expressing gal4db - ini - 1 ) were constructed by transfer of the ecori cdna fragment of the interacting clone pd2 . 1 to the unique ecori sites of pgex - 1λt and pma424 , respectively . construction of in mutants pmahh , pmacc , pmaδn3 , pmaδc2 and pmaδc3 were described earlier ( kalpana and goff , 1993 ). the remaining in deletion mutants , pmaδ18 to pmaδ273 , were originally isolated as gal4ac fusion mutants that retained the ability to interact with gal4db - in . the bamhi - sali fragments from these mutants were excised from the gal4ac plasmid and transferred into pma424 . isolation of pmam5 , encoding a mutant in defective for in - in interaction , will be described elsewhere . construction of hl60 cdna library fused to the activation domain of gal4 the hl60 cell cdnas were excised from a λzap hl60 cdna library ( stratagene catalogue # 936214 ). the original λzap library encompassed about a million recombinant phage clones . to ensure that complexity of the original library was retained , a plate lysate of the phage library was prepared by plating 10 7 phage ; phage particles were isolated by peg precipitation and two consecutive steps of cscl gradient centrifugation . dna was isolated from the total phage by standard methods . about 100 μg of dna was digested with noti and xhoi , separated on agarose gels and inserts 0 . 2 - 3 . 0 kb in size were isolated by electroelution . the cdna inserts were ligated to the pgadnot vector ( luban et al , 1993 ) digested with noti plus sali and phosphatase - treated . dh5α cells were transformed - with the ligation products and the transformants from six individual batches of 100 , 000 to 500 , 000 colonies each were pooled separately in lb / amp ( kgli , pool i to pool vi ). this unamplified library in pgadnot vector was aliquoted into small vials and stored frozen until further use . the ration of non - recombinants to recombinants in the library was determined by comparing the number of transformants obtained with self ligated vector to that obtained with vector ligated to insert ; and by examining plasmids from several individual colonies to determine the presence of insert . both these tests indicated that there were & gt ; 95 % recombinants in the library . the plasmid library dna was isolated from 1 l cultures of each pool by quiagen columns . this dna was used for transformation into yeast strain ggy1 :: 171 . overnight cultures of ggy1 :: 171 were diluted 1 : 50 or 1 : 100 in ypad ( yepd supplemented with 30 μg / ml of adenine ) and incubated at 30 ° c . until the od 600 reached 0 . 25 - 0 . 4 . the cells were pelleted , washed once with 1 / 10th volume of 100 mm liac / 10 mm te , and resuspended in 1 / 200th volume of the same buffer . the cells were further incubated with shaking for 1 hour at 30 ° c . the competent cells were incubated with 1 - 10 μg of plasmid dnas encoding gal4db and gal4ac fusions , 20 μg of sonicated salmon sperm carrier dna ( sigma , catalogue # d - 9156 ) and 40 % peg in liac / te with agitation at 30 ° c . for 30 minutes . after the peg treatment , the cells were pelleted and resuspended in 1 ml of ypad and incubated further for 1 hour at 30 °. the post - incubated incubation step increased the efficiency of co - transformation by about 10 fold . cells were pelleted , resuspended in te and plated on selective medium . bacterial extracts containing gst - ini - 1 fusion protein were prepared as follows . overnight bacterial cultures containing the required plasmid was diluted 1 : 10 into lb / amp and incubate at 37 ° c . until the o . d .. sub .. 600 was ˜ 0 . 5 . iptg ( isopropyl - β - d - thiogalactopyranoside ) was added to a final concentration of 1 mm and incubation was continued for 3 - 5 hours . the cells were collected and resuspended in buffer y ( 50 mm tris / cl ph 7 . 5 , 50 mm nacl , 1 mm edta , 0 . 5 % np - 40 and 1 mm pmsf ). lysozyme was added to a final concentration of 1 mg / ml and incubation was continued on ice for half an hour . this lysate was subjected to sonication ( 3 × 30 sec bursts ). the lysate was clarified in a microfuge for 15 minutes , and the supernatant was transferred to a fresh microfuge tube . pre - swollen g - beads were added to the above lysate and incubated at 4 ° c . for 30 minutes with gentle rocking . the beads were spun at 1600 rpm in the microfuge for 20 sec and the resulting pellet was washed three times with excess of buffer y and resuspended in buffer y to yield a 50 % ( v / v ) slurry . bacterial extract containing hiv - 1 in was prepared as follows . overnight bacterial cultures of bl21 ( de3 ) containing either one of the plasmids , pt7f11 - in ( encodes in under the control of t7 promoter ), and pt7 - δin ( control plasmid from which in is deleted ) were diluted 1 : 10 in lb / amp and incubated at 37 ° c . for 1 hour . iptg was added to a concentration of 1 mm and incubation was continued for 3 - 5 hours . the cultures were pelleted and the pellets were resuspended in buffer y . lysozyme was added to a final concentration of 1 mg / ml and the cells were incubated on ice for 30 minutes . the lysed bacteria were sonicated and passed through a syringe with a 23 gauge needle several times . the insoluble material was collected by centrifugation and resuspended in buffer containing 1m nacl and 1 mm dtt , and the mixture was subjected to gentle rocking at 4 ° c . for 30 minutes . the resulting solution was spun in the microfuge for 30 minutes and the supernatant , referred to as 1m nacl extract of in , was used for the binding assay with gst - ini - 1 . the binding of in to gst - ini - 1 was tested by adding the washed g - beads with bound gst - ini - 1 to the 1m nacl extract of in and incubating for 30 minutes at 4 ° c . in buffer containing 1m hepes , ph 7 . 3 , 200 mm nacl , 5 mm dtt , 0 . 1 % f np - 40 , 1 mm pmsf and 10 mg / ml bsa . to test the effect of salt , the concentration of nacl was varied in the binding buffer from 200 mm to 1m . the mixture was incubated at 4 ° c . for 30 minutes and washed three times with excess of either buffer y or buffer y containing various concentrations of np - 40 and sds . the resulting beads were boiled in laemmli buffer and subjected to sds - page in duplicate . the presence or absence of in in these binding experiments was determined by western analysis using monoclonal antisera to in . screening the phage library to isolate full length recombinant clones of ini - 1 . two hela cdna libraries , one constructed in λzap ii vector ( stratagene , cat . # 936201 ) and one constructed in λgt11 , were screened using standard methods . the cdna insert from one positive interacting clone obtained in the yeast screen was purified , labelled by random priming with 32 p - dctp , and used as a probe to screen about 0 . 5 × 10 6 phage of the λzapii library . dna isolated from twenty positive cones obtained after three rounds of plague purification was subjected to restriction analysis . six positive clones that had the largest inserts were chosen for further analysis . the recombinant pbluescript phagemids from these six positive λzapii clones were subjected to in vivo excision using the m13 helper phage ( exassist / solr system , stratagene , cat # 200253 ). unfractionated mrna prepared from hela , cb33 and hut78 cell lines were subjected to northern analysis using standard methods . a northern blot of human mrnas from multiple tissues ( clontech , palo alto calif . ; catalog # 7759 - 1 ) was hybridized to a labelled ini - 1 probe using standard methods ( maniatis et al ., 1982 ). abrams , e ., neigeborn , l ., and carlson , m . 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( 1985 ). insertion mutagenesis of embryonal carcinoma cells by retrovirus infection . science 228 , 554 - 558 . kozak , m . ( 1991 ). structural features in eukaryotic mrnas that modulate the initiation of translation . j . biol . chem . 266 , 19867 - 19870 . kruger , w ., and herskowitz , i . ( 1991 ). a negative regulator of ho transcription , sin1 ( spt2 ), is a nonspecific dna - binding protein related to hmg1 . mol . cell . biol . 11 , 4135 - 4146 . laurent , b ., and carlson , m . ( 1992 ). yeast snf2 / swi2 , snf5 , and snf6 proteins function coordinately with the gene - specific transcriptional activators gal4 and bicoid . genes dev . 6 , 1707 - 1715 . laurent , b . c ., treich , i ., and carlson , m . ( 1993 ). the yeast snf2 / swi2 protein has dna - stimulated atpase activity required for transcriptional activation . genes dev . 7 , 583 - 591 . laurent , b . c ., treitel , m . a ., and carlson , m . ( 1990 ). the snf5 protein of saccharomyces cerevisiae is a glutamine - and proline - rich transcriptional activator that affects expression of a broad spectrum of genes . mol . cell . biol . 10 , 5616 - 5625 . laurent , b . c ., treitel , m . a ., and carlson , m . ( 1991 ). functional interdependence of the yeast snf2 , snf5 , and snf6 proteins in transcriptional activation . proc . natl . acad . sci . u . s . a . 88 , 2687 - 2691 . laurent , b . c ., yang , x ., and carlson , m . ( 1992 ). an essential saccharomyces cerevisiae gene homologous to snf2 encodes a helicase - related protein in a new family . mol . cell . biol . 12 , 1893 - 1902 . luban , j ., alin , k . b ., bossolt , k . l ., humaran , t ., and goff , s . p . ( 1992 ). genetic assay for multimerization of retroviral gag polyproteins . j . virol ., 66 , 5157 - 5160 . luban , j ., bossolt , k . l ., franke , e . k ., kalpana , g . v ., and goff , s . p . 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( 1992 ). roles of swi1 , swi2 , and swi3 proteins for transcriptional enhancement by steroid receptors . science 258 , 1598 - 1604 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 4 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 1867 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( iii ) hypothetical : n ( iv ) anti - sense : n ( v ) fragment type : n - terminal ( ix ) feature :( a ) name / key : cds ( b ) location : 70 .. 1225 ( d ) other information :( xi ) sequence description : seq id no : 1 : 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metmetmetmetalaleuserlysthrpheglyglnlysprovallys151015pheglnleugluaspaspglygluphetyrmetileglysergluval202530glyasntyrleuargmetpheargglyserleutyrlysargtyrpro354045serleutrpargargleualathrvalglugluarglyslysileval505560alaserserhisglylyslysthrlysproasnthrlysasphisgly65707580tyrthrthrleualathrservalthrleuleulysalasergluval859095glugluileleuaspglyasnaspglulystyrlysalavalserile100105110serthrgluproprothrtyrleuarggluglnlysalalysargasn115120125serglntrpvalprothrleuserasnserserhishisleuaspala130135140valprocysserthrthrileasnargasnargmetglyargasplys145150155160lysargthrpheproleucyspheaspasphisaspproalavalile165170175hisgluasnalaserglnprogluvalleuvalproileargleuasp180185190metgluileaspglyglnlysleuargaspalaphethrtrpasnmet195200205asnglulysleumetthrproglumetphesergluileleucysasp210215220aspleuaspleuasnproleuthrphevalproalailealaserala225230235240ileargglnglnileglusertyrprothraspserileleugluasp245250255glnseraspglnargvalileilelysleuasnilehisvalglyasn260265270ileserleuvalaspglnpheglutrpaspmetserglulysgluasn275280285serproglulysphealaleulysleucyssergluleuglyleugly290295300glygluphevalthrthrilealatyrserileargglyglnleuser305310315320trphisglnlysthrtyralaphesergluasnproleuprothrval325330335gluilealaileargasnthrglyaspalaaspglntrpcysproleu340345350leugluthrleuthraspalaglumetglulyslysileargaspgln355360365aspargasnthrargargmetargargleualaasnthralaproala370375380trp385 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 204 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( iii ) hypothetical : n ( iv ) anti - sense : n ( v ) fragment type : n - terminal ( xi ) sequence description : seq id no : 3 : alaserglnprogluvalleuvalproileargleuaspmetgluile51015aspglyglnlysleuargaspalaphethrtrpasnmetasnglulys202530leumetthrproglumetphesergluileleucysaspaspleuasp354045leuasnproleuthrphevalproalailealaseralailearggln505560glnileglusertyrprothraspserileleugluaspglnserasp65707580glnargvalileilelysleuasnilehisvalglyasnileserleu859095valaspglnpheglutrpaspmetserglulysgluasnserproglu100105110lysphealaleulysleucyssergluleuglyleuglyglygluphe115120125valthrthrilealatyrserileargglyglnleusertrphisgln130135140lysthrtyralaphesergluasnproleuprothrvalgluileala145150155160ileargasnthrglyaspalaaspglntrpcysproleuleugluthr165170175leuthraspalaglumetglulyslysileargaspglnaspargasn180185190thrargargmetargargleualaasnthralapro195200 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 232 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( iii ) hypothetical : n ( iv ) anti - sense : n ( v ) fragment type : n - terminal ( xi ) sequence description : seq id no : 4 : asngluthrsergluglnleuvalproileargleuglupheaspgln51015aspargaspargphepheleuargaspthrleuleutrpasnlysasn202530asplysleuilelysilegluaspphevalaspaspmetleuargasp354045tyrargphegluaspalathrarggluglnhisileaspthrilecys505560glnserileglngluglnileglnglupheglnglyasnprotyrile65707580gluleuasnglnaspargleuglyglyaspaspleuargileargile859095lysleuaspilevalvalglyglnasnglnleuileaspglnpheglu100105110trpaspileserasnseraspasncysproglugluphealagluser115120125metcysglngluleugluleuproglygluphevalthralaileala130135140hisserilearggluglnvalhismettyrhislysserleualaleu145150155160leuglytyrasnpheaspglyseralailegluaspaspaspilearg165170175serargmetleuprothrilethrleuaspaspvaltyrargproala180185190alagluserlysilephethrproasnleuleuglnileseralaala195200205gluleugluargleuasplysasplysaspargaspthrargarglys210215220argargglnglyargserasnarg225230__________________________________________________________________________