Patent Application: US-55327196-A

Abstract:
a biologically - active peptide fragment of the nef protein of human immunodeficiency virus is provided , pharmaceutical compositions comprising the peptide , analogs or derivatives of the peptide and therepeutic and screening methods which utilize the peptide and compositions which comprise them . the invention is particularly useful in the suppression of the immnune respons or in the suppression of symptoms of autoimmune disease .

Description:
the invention will now be described in detail by way of reference only to the following non - limiting examples , and to the drawings , in which : fig1 summarises the scheme employed for cloning and expression of hiv1 nef genes in e . coli , yeast , and baculovirus ; fig2 a and 2b show plots of hydrophobic moment against hydrophobicity ; fig3 a and 3b represent helical wheel diagrams illustrating nef and melittin , showing the distribution of polar and hydrophobic residues around the α - helices of the two peptides ; fig4 a and 4b represent energy - minimised structures for a representative nef peptide sequence from hiv ( a ) and for melittin ( b ); fig5 shows light - scattering of small unilamellar phospholipid vesicles ( suv ) in the presence of nef 2 - 19 peptide , nef 27 and nef 25 ; fig6 shows 31 p nmr spectra of suv after addition of nef 2 - 19 peptide , nef 27 and nef 25 ; fig7 shows tryptophan fluorescence spectra of nef 2 - 19 peptide in waters ethanol and suv ; fig8 depicts a molecular model showing monomeric nef peptide with its hydrophobic region inserted in the hydrophobic region of a lipid bilayer , and its polar residues oriented to the exteral aqueous medium ; fig9 a and 9b show immunoblots of recombinant nef 27 ( a ) and nef 25 b reacted with affinity purified anti - nef . sub . ( 15 - 27 ). varying amounts of recombinant nef protein ( lanes a and h : 3 . 0 μmol ; lanes b and g : 1 . 5 μmol ; lanes c and f : 0 . 5 μmol and lanes d and e : 0 . 5 μmol ) were used to determine the amount of protein detectable by the antibodies in western immunoblotting . nef 27 is in lanes a - d and nef 25 is in lanes e - h . strong bands specific for nef 27 or nef 25 were still observed when 0 . 15 μmol of protein was tested ; fig1 illustrates detection of nef protein in electroporated mt - 2 cells by immunoblotting . immunoblots of cell lysates prepared from mt - 2 cells were electroporated as follows : fig1 a - d show expression of cell surface - located cd4 in mt - 2 cells ( a ) and pha - stimulated peripheral blood mononuclear cells ( b ), and of il - 2 r in mt - 2 cells ( c ) and pha - stimulated pbmc ( d ). cells were mock - electroporated ( 1 ) or electroporated with either bsa ; 1 . 0 μmol ( 2 ), nef 27 ; 1 . 0 μmol ( 3 ), or nef 25 ; 1 . 0 μmol ( 4 ), and incubated at 37 ° c . for 24 h . cells were then reacted with anti - cd4 , anti - cd25 , denoted by the striped bars , or the appropriate isotype control , denoted by the black bars , and analysed by flow cytometry . mean fluorescence intensities of positive cells are indicated ; fig1 a and 12b show expression of cell surface - located transferrin receptor ( tfr ) ( a ) or il - 2 r ( b ) in mt2 cells . cells were electroporated with either nef 27 ; 1 . 0 μmol (), nef 25 ; 1 . 0 μmol (), bsa ; 1 . 0 μmol () or mock eectroporated (▪), and incubated at 37 ° c . for 24 h . cell surface expression of tfr on mt - 2 cells was stable , even after electroporation of cells with hiv - 1 nef 27 ; fig1 shows silver staining of polyacrylamide gel electrophoresed cellular protein precipitstes a ) precipitates prepared from m - 2 cell lysates after reaction with tbs ( track 1 ), gst ( track 2 ) or gst - nef 27 ( track 3 ) followed by glutathone - sepharose . b ) precipitates prepared from pha - activated pbmc lysates after reaction with tbs ( track 3 ), gst ( track 4 ) or gst - nef 27 ( track 5 ) followed by glutatione - sepharose . track 1 represents gst protein ( 2 μg ), track 2 represents gst - nef 27 protein ( 2 μg ). c ) precipitates prepared from pha - activated pbmc lysates after reaction with tbs ( track 5 ), gst ( track 6 ) or gst - nef 27 ( track 7 ) followed by glutathione - sepharose . track 1 represents low molecular weight markers , track 2 represents gst protein ( 2 μg ), track 3 represents gst - nef 25 ( 2 μg ). a ) precipitates prepared from mt - 2 cells after reaction with tbs ( track 1 ), gst ( track 2 ) and gst - nef 27 ( track 3 ). anti - p56 lck was used as the primary antibody . b ) precipitates prepared from pha - activated pbmc after reaction with tbs ( track 4 ), gst ( track 3 ) and gst - nef 27 ( track 2 ). anti - p56 lck was used as the primary antibody . track 1 represents low molecular weight markers . fig1 shows western immunoblotting of cellular protein precipitates prepared from pha - activated mt - 2 cells after reaction with tbs ( track 1 ), gst ( track 2 ) and gst - nef 27 ( track 3 ) or pha - activated pbmc after reaction with tbs ( track 4 ), gst ( track 5 ) and gst - nef 27 ( track 6 ) as described below . anti - cd4 was used as the primary antibody . fig1 illustrates the proliferative response to il - 2 ( 0 , 10 , 30 and 100 iu / ml ) of pha - activated pbmc mock - electroporated or electroporated with nef 25 , nef 27 or bsa , using 3 h !- thymidine incorporation as the index . pha - activated pbmc which did not undergo the electroporation process were included as controls . results represent mean ± s . d . for three experiments . fig1 shows western immunoblotting of nef - treated pbmc lysate with anti - p56 lck . pha - activated pbmc mock electroporated ( tracks 1 , 2 and 3 ), electroporated with nef 27 ( tracks 4 , 5 and 6 ), or electroporated with nef 25 ( tracks 7 , 8 and 9 ) were treated with il - 2 ( 1000 u ) for 0 min ( tracks 1 , 4 and 7 ), 15 min ( tracks 2 , 5 and 8 ) or 30 min ( tracks 3 , 6 and 9 ), and electrophoresed in a 13 % polyacrylamide gel . after transfer to hybond - c super nitrocellulose the filters were reacted with anti - p56 lck as described below . fig1 shows western immunoblotting of nef - treated pbmc lyzates with anti - p56 lck before and after pma stimulation . pbmc mock electroporated ( tracks 1 and 2 ), electroporated with nef 27 ( tracks 3 and 4 ) or electroporated with nef 25 ( tracks 5 and 6 ) were treated with pma ( 2ng / ml ; tracks 1 , 3 and 5 ) or incubated in medium alone for 2 h at 37 ° c . cell lysates were electrophoresed , transferred to hybond - c super nitrocellulose and subsequently probed with anti - p56 lck . fig1 shows levels of nucleoprotein c - myb in nef - treated mt - 2 cells as detected by western immunoblotting . mt - 2 cells electroporated with nef 27 ( tracks 4 and 5 ), nef 25 ( track 3 ), mock - electroporated cells ( track 2 ) or control mt - 2 cells ( track 1 ) were lysed , electrophoresed , transferred to hybond - c super nitrocellulose and reacted with anti - c - myb . reagents : dipalmitoyl phosphatidyl choline ( dppc ) was purchased from sigma , st louis mo . and the spin - labelled phospholipids ( 1 - palmitoyl - 2 -( 16 - doxyl stearoyl ) phosphatidyl choline ( 16 pc - sl ), 1 - palmitoyl - 2 -( 12 - doxyl stearoyl ) phosphatidyl choline ( 12 pc - sl ), and 1 - palmitoyl - 2 -( 5 - doxyl stearoyl ) phosphatidyl choline ( 5 pc - sl ) were purchased from avanti polarlipids , pelham ala . tempo choline chloride ( tcc ), the fluorochromes octadecyl rhodamine , 1aminonaphthaline - 3 , 6 , 8 - trisulphonic acid ( ants ) and the fluzorescence quencher n , n &# 39 ;- p - xylenebis -( pyridinium bromide ) ( dpx ) were purchased from molecular probes , junction city oreg . nef 27and nef 25 : these proteins were exressed in e . coli and purified as described in azad et al ( 54 ). this method enables the production of tens of milligrams of nef 27 and nef 25 , amounts which permit biological and structural studies to be undertaken . seqences encoding nef 27 and nef 25 were amplified by pcr from the hiv - 1 infectious clone pnl4 . 3 ( adachi et al , j . virol . 1986 59 284 - 291 ) and subcloned directly into e . coli , yeast and baculovirus epression vectors . the scheme employed is summarised in fig1 . the yeast and baculovirus - derived nef had native n - termini , but the expression levels were low . the expression level of the e . coli - derived glutathione ( gst )- nef fusion proteins was very high , and a major portion of the expression products was soluble . large - scale production of e . coli - derived nef 27 and nef 25 was carried out by growing recombinant cells in a fermenter under fed - batch conditions . the soluble gst - nef fusion proteins were affinity purified on glutathione - sepharose . after thrombin cleavage at the fusion junction , the gst was selectively removed by binding it to glutathione - sepharose . under conditions of large - scale production nef 27 was always contaminated with a slightly smaller n - terminal cleavage product , and the two could be resolved by selective binding of intact nef 27 to a reactive - red 120 dye ligand . a final gel filtration step was sometimes necessary to remove traces of contaminating bacterial proteins . after purification nef 27 and nef 25 had the expected n - terminal sequences , and appeared as single monomeric bands on reducing sds - page and on gel filtration . under non - reducing conditions both nef 27 and nef 25 , existed as a mixture of monomers and dimers . the highly purified nef proteins had no g - protein activities ; neither nef 27 nor nef 25 had detectable gtp or autophosphorylation activity . although both proteins showed appreciable gtp binding when compared to bovine serum albumin , chymotypsin or lysozyme , the level of binding was insignificant compared to that shown by p21 ras . peptide synthesis : peptides corresponding to the 2nd to 19th and 2nd to 22nd residues of the amino - terminal sequence of nef were synthesised on an applied biosystems peptide synthesiser , using the standard merrifield solid - phase technique in which the α - amino groups of the amino acids were protected by acid - labile boc groups . the sequences from the nl4 . 3 straain of hiv - 1 were : ## str1 ## amino acid 1 is met , which is cleaved in vivo in eukaryotic cells . the serine side chains were protected by benzyl groups and arginine side chains by mesitylene - 2 - sulphonyl groups . couplings were achieved by using a 2 - molar excess of the asymmetric anhydride of each amino acid , except for arginine , which was coupled using a 4 - molar excess of the butanol ester . the completed peptide was cleaved from the resin using the low trifluoromethane sulphonic acid / high hydrofluoric acid technique which yielded the peptide from the 4 - methylbenzylbenzhydryl amine resin . the peptide was purified by hplc on a c4 vydac column . small unilamellar phospholipid vesicles ( suv ) were prepared by hydrating dried phospholipids in 0 . 05m tris - hcl buffer ; ph 7 . 3 , to a final concentration of 50 mm and sonicating under nitrogen at 35 ° c . until the suspension was no longer turbid , followed by ultracentrifugation to remove larger vesicles and any titanium particles from the sonicator tip . the spin - labelled phospholipids were incorporated into the vesicles by mixing them with the phospholipid in excess 70 / 30 chloroform - methanol at the desired spin label / lipid ratio . the mixture was dried under a stream of nitrogen and then kept under vacuum for 24 hr to ensure thorough removal of solvent . the mixture was them hydrated in the desired buffer and sonicated as above . ultrarviolet fluorescence studies : fluorescence spectra of the tryptophan residues in the nef peptide were recorded with a perkin elmer mpf3 fluorescence spectrophotometer using an excitation wavelength of 290 nm . light - scattering of suv in the presence and absence of nef peptide , nef 27 and nef 25 was measured using an hitachi - perkin elmer m35 spectrofluorimeter with the excitation and emission wavelengths both set at 560 nm . a relative value of scattering s was obtained from the equation s = e c / e t , where e c is the emission due to right - angle scattering of incident radiation by the control suv and e t is the mission from suv to which the proteins and peptides had been added . the concentration of suv corresponded to 35 - 50 μg of lipid / ml . nmr -- 31 p nmr spectra of suv were acquired on a bruker amx600 spectrometer at 81 mhz , using standard pulse sequences . prediction of peptide secondary structure was carried out using the chou - fasman algorithm using an extended data base of 64 proteins , the schiffer / edmundson α - helical wheel and the hydrophobic moment program of eisenberg and wesson . molecular modeling was carried out using the biosym insight ii and discover packages on a silicon graphics iris workstation . cells and cell culture : peripheral blood was obtained from five hiv - 1 sero - negative volunteers and mononuclear cells prepared by centrifugation an a ficoll / hypaque density gradient using a conventional method ( 24 ). peripheral blood mononuclear cells ( pbmc ) were stimulated with phytohemagglutinin ( pha ; 10 μg / 10 6 cells ) for up to 72 h at 37 ° c ., and cultured in rpmi 1640 medium supplemented as described below . the cd4 + t - cell lines mt - 2 ( kindly provided by dr . y . hinuma , institute for virus research , kyoto university , kyoto , japan ), cem and jurkat ( both from atcc ) were grown in rpmi 1640 medium supplemented with 10 % ( vol / vol ) foetal calf serum , 25 μg / ml glutamine , penicillin ( 100 iu / ml ) and streptomycin ( 100 μg / ml ) in a 5 % co 2 atmosphere at 37 ° c . mt - 2 cells were harvested from culture during mid - exponential growth and adsorbed with hiv - 1 isolate 228200 or htlv - iiib for 2 h at 37 ° c . with agitation . after this incubation period the cells were resuspended in supplemented rpmi 1640 , and incubated at 37 ° c . for 3 to 4 days . mt - 2 cells and jurkat cells were also harvested from culture during mid - exponential growth and prepared for electroporation . antipeptide antisera : antibodies specific for hiv - 1 nef were raised against a peptidee synthesised as described previously ( 25 ), corresponding to the amino acid residues 15 - 27 ( avrermrraepaa ) of nef encoded by the hiv - 1 clone pnl4 . 3 . the peptide was conjugated to keyhole limpet haemocyanin ( klh ; calbiochem , bebring diagnostics , ca ) via glutaraldehyde and this complex was used to immunise sheep ( 0 . 5 mg peptide conjugate / sheep ). antibodies to the peptide were purified by affinity chromatography . reactivity of the antibodies with recombinant hiv - 1 nef 25 and 27 was demonstrated by immunoblotting . monoclonal antibodies ( mabs ) reactive against an epitope in the carboxyl terminus of the full length nef protein ( ae6 and ag11 ; national institute of allergy and infectious disease aids research and reference reagent programme and anti - nef mab ; abt , maryland , u . s . a .) or reactive with an epitope in the amino terminal part of nef ( nef2 - b2 ; national institute of allergy and infectious disease aids research and reference reagent programme ) and anti - nef mab raised against a synthetic peptide corresponding to amino acid residues 15 - 27 of hiv - 1 , htlv - iiib nef ; ( new england nuclear division , du pont , u . s . a .) were used in the various assays described below . theoretical calculations were carried out to assess the ability of the nef peptide to assume a defined secondary structure , such as α - helix or β - strand . chou - fasman analysis suggested a low probability of α - helix formation in aqueous solution , and hydrophobic moment plots suggested that the peptide was close to the boundary between surface seeking and &# 34 ; globular &# 34 ; behaviour . plots of hydrophobic moment against hydrophobicity are given in fig2 . fig2 a was plotted using a 6 residue ` window ` and 2b was plotted using a 4 residue ` window `. it was considered possible that , like melittin , the nef peptide might form an α - helix in the appropriate environment , such as the interface between a lipid bilayer and aqueous medium . schiffer - edmundson diagrams for both peptides are given in fig3 . it can be seen that the nef peptide has a considerably smaller arc than does melittin of hydrophobic residues which could interact with the hydrocarbon region of a lipid bilayer . the n - terminal sequences of nef and of melittin are compared in table 1 . table 1__________________________________________________________________________sequence similarity between n - terminus of nef and bee venom__________________________________________________________________________mellitin1 # str2 ## __________________________________________________________________________ an energy - minimised structure of a representative nef peptide representing several different strains of nef is compared with the structure of melittin in fig4 . a set of coordinates for the structure of the latter is available from nmr studies . the nef structure was assembled applying α - helical constraints , and energy minised using the discover program . both the structures are presented with the hydrophobic residues facing downwards . like melittin , the nef peptide has a proline residue , approximately half - way along the sequence , which produces a characteristic kink in the helix . a striking difference between melittin and nef is the fact that while the hydrophobic residues of the former lie along the concave face of the helix , those of the latter lie along its convex face . light - scattering studies on the interaction of nef 27 , nef 25 and nef peptide with suv nef 27 and nef peptide caused a rapid increase in light - scattering of suv , as shown in fig5 . nef 25 increased scattering to a considerably lesser extent . the increase in light scattering indicates aggregation of the vesicles , probably due to fusion . nef 27 and nef peptide both induce isotropic peaks in 31 p nmr spectra of suv , whereas nef 25 does not do this . this is illustrated in fig6 . the sharp line of the phospholipid vesicles alone is characteristic of a highly - curved lipid bilayer above its transition temperature . this is also seen for nef 25 . the broadening and appearance of an additional peak seen with nef 27 and the nef peptide are both characteristic of bilayer disruption , which is a necessary precondition for vesicle fusion . the fluorescence spectra of nef peptide in buffer and after the addition of 35 mg / ml suv , giving a peptide / lipid ratio ( p / l ) of 1 / 1100 , is shown in fig7 . the blue shift of the tryptophan emission is characteristic of the change in enviroment from a aqueous to a hydrocarbon medium . the position of the trytophan in the bilayer was probed by adding the peptide to systems containing various nitroxide spin probes which quench its fluorescence . the results are set out in table 2 . table 2______________________________________quenching of nef peptide tryptophan emissionby nitroxides in different environments buffer + suv + suv + suv + p / l control tcc 5 pc - sl 12 pc - sl 16 pc - sl______________________________________1 / 200 1 . 00 0 . 347 0 . 269 0 . 967 0 . 9411 / 100 1 . 00 -- 0 . 286 0 . 835 0 . 9081 / 50 1 . 00 -- 0 . 274 0 . 617 0 . 6291 / 25 1 . 00 -- 0 . 218 0 . 338 0 . 413______________________________________ it can be seen that tcc , which does not penetrate the bilayer , has no effect on the tryptophan fluorescence in the presence of suv , although it does quench the fluorescence of the peptide in buffer . at peptide / lipid ratios & lt ; 1 / 100 the tryptophan is quenched mainly by the 5pc - sl , while at a peptide / lipid & gt ; 1 / 50 there is quenching also by the 12 and 16 pc - sl . the nef peptide appears to interact with bilayer lipid membranes in a number of ways . the blue shift in the fluorescence emission of trptophan which occurs after addition of suv shows that these residues are buried in the hydrocarbon region of the lipid bilayer . this observation is confirmed by the lack of quenching of the fluorescence by the water - soluble tcc when suv are preset in the system . since this effect was present at a p / l of 1 / 50 and 1 / 25 it appears that the suv took up the peptide quantitatively at guite high p / l . on the other hand , at p / l & lt ; 1 / 100 the tryptoph fluorescence was quenched by 5 pc - sl , while at p / l & gt ; 1 / 50 it was also quenched by 12 - and 16 pc - sl . these results suggest that at low p / l the peptide is associated with the surface region of the bilayer , with the trytophs located in the upper region of the bilayer hydrocarbon , possibly as shown in fig8 . in contrast , at high p / l the peptide is located so that at least one of the try tophans is located in the 12 - 16 carbon region of the bilayer , possibly assuming a multimeric trans - membrane conformation . the increase in light scattering on addition of the peptide to suv suggests that it aggregates phospholipid vesicles . furthermore , the occurrence of a marked isotropic line in the 31 p nmr spectra of suv after the addition of the nef peptide to a p / l of 1 / 100 suggests that the association of the peptide with the bilayer leads to the formation of non - lamellar lipid phases , an event which precedes membrane fusion . the nef 27 protein also increases light - scattering rapidly when added to suv , and causes the appearance of an isotropic line in the 31 p nmr spectra of the suv . because nef 25 causes none of these effects , it appears that the n - terminal region of nef 27 contains a membrane active domain . while fusion events inside normal cells occur physiologically , unscheduled fusion brought about by the presence of a foreign membrane - active molecule such as nef may be very disruptive as a result of breakdown of intracellular compartmentation . for example , the membrane - fusing properties of nef may be relevant to its actions in dowuregulation of cd4 by hindering transport of the antigen to the cell surface . the n - terminal stretch of the protein may , therefore , be a useful therapeutic target . different allelic forms of the nef protein are produced in the course of hiv - 1 virus infection , depending on the isolate of the virus being studied . in our laboratory we have two such isolates , one of which produces marked syncytium formation and cytopathic effect and is designated syncytium - inducing ( si ), and a second which does not induce syncytium , and which causes a lesser degree of cell death . this latter isolate is designated non - syncytium inducing ( nsi ) characterisation of cells infected with these isolates shows that the si isolate was able to down - regulate surface cd4 , expression , whereas the nsi isolate did not . we have shown that nef protein of the is hiv - 1 strain designated pnl4 . 3 is able to down - regulate the surface cd4 , expression . a series of monoclonal antibodies was able to recognise different allelic forms of expressed nef protein , thus demonstrating structural differences between these allelic forms . the course of infection induced by the nsi strain was altered by addition of recombinat nef 27 of the pnl4 . 3 allelic form . this indicates that the recombinant nef 27 is able to restore the ability of the virus to for syncytia , thereby allowing increased clumping of the cells . in contrast to the effect of nef 27 , the n - terinal truncated nef 25 protein was not able to induce this restoration of function . reactivity of anti - nef . sub . ( 15 - 27 ) with recombinant nef 27 and nef 25 in immunoblotting the reactivity of sheep anti - nef . sub . ( 15 - 27 ) with recombinant nef 27 and nef 25 protein was determined by immunoblotting . antibodies to the immunogenic peptide ( avrermrraepaa ) were purified by affinity chromatography . the antibody preparation used in the immunoblotting experiments was essentially free of antibodies to the immunogenic carrier protein and coupling agent . purified recombinant nef 27 and nef 25 ( 012 - 1 . 0 μm ) were electrophoresed in a sds - 13 % polyacrylamide gel ( sds - page ) and subsequently tran farred to hybond - c nitrocellulose ( amersham , buckinghamshire , england ) for 1 h at 100 v using a bio - rad protein transfer cell ( bio - rad , richmond , calif .). membranes were pre - incubated with 1 % bovine serum albumin ( bsa ) for 2 h at room temperature and then reacted with affinity - purified sheep anti - nef . sub . ( 15 - 27 ), diluted 1 : 100 , overnight at 4 ° c . after three washes in pbs / 0 . 05 % tween 20 , the blots were incubated with donkey anti - sheep immunoglobulin conjugated to biotimn diluted 1 : 500 , for 1 h at room temperature . after extensive washing as described above the membranes were incubated with streptavidin - conjugated horse radish pexoxidase , diluted 1 : 500 , ( amersham ) for 1 h at room temperature . all dilutions were made with 1 % bsa in pbs . after further washing the membrane was developed with phenylenediamine substrate ( dako , dakopatts , denmark ). results of immunoblotting reactions in which e . coli - derived nef 27 and nef 25 were reacted with anti - nef . sub . ( 15 - 27 ) are shown in fig9 a and 9b . strongly reactive bands of approximately 27 kda and 25 kda were observed with nef 27 and nef 25 respectively . the antibodies could detect at least 0 . 15 μmol of either nef 27 or nef 25 by immunoblotting , indicating equivalent recognition of both proteins by this polyclonal antibody . mt - 2 cells ( 3 × 10 7 cells ) grown to confluence were harvested from culture , placed in a 50 ml polypropylene test tube ( nunc , roskilde , denmark ) and adsorbed with hiv - 1 ( isolate 228200 ; 27 ) for 2 h at 37 ° c . with agitation . after this incubation period the cells were resuspended in supplemented rpmi 1640 medium and incubated at 37 ° c . for 4 days . mt - 2 cells incubated in medium alone were included as a control . following the 4 day incubation period , hiv - 1 infected mt - 2 cells and control cells were harvested from culture , resuspended in lysis buffer ( 0 . 5 % nonidet p40 , 0 . 5 % sodium deoxycholate , 50 mm nacl , 25 mm tris / hcl . 5 mm benzamidine / hcl , 10 mm edta , 0 . 1m phenylmethyl sulfonyl fluoride in n - propanol and 0 . 01 % sodium azide , ph 8 ) and the cell lysates electrophoresed , transferred to hybond - c nitrocellulose paper ( amersham ) and immunoblotted with anti - nef . sub . ( 15 - 27 ) as described above . nef protein was detected in amounts similar to those seen on electroporation of cells with recombinant protein . electroporation of e . coli - derived hiv - 1 nef 25 or 27 into t - cell lines and pha - activated pbmc an efficient electroporation technique termed baekonization was used to transfer bsa , recombinant nef 27 or recombinant nef 25 protein into pbmc an the cd4 + t - cell lines m - 2 , cem 9 and jurkat cells . this form of electroporation uses electric field rather than electric charge to transfer macromolecules across the cell membrane and results in greater cell viability . the baekon 2000 advanced macromolecule transfer system ( baekon , inc . fremont , calif .) was chosen because of its ease of operation and its electrode configuration , which allows rapid macromolecule transfer . optimal conditions for electroporation were standardised using cem cells at a concentration of 5 × 10 5 cells / 100 μl , 1 μm of recombinant hiv - 1 nef 27 or nef 25 and electroporation medium ( 1 . 5 mm na 2 hpo 4 , 0 . 5 mm kh2po 4 , and 0 . 27m sucrose , ph 7 . 0 ) ( 26 ). pbmc or t - cells harvested from culture during the middle of their exponential growth phase were washed once with pbs and resuspended in electroporation buffer at a concentration of 5 × 10 6 cells / ml . cells were then mixed with 0 . 5 - 6 . 0 μg of nef 27 or nef 25 in a final volume of 80 μl containing 8 × 10 5 cells and incubated for 10 min on ice . cells were then electroporated , washed twice with ice - cold pbs , and incubated with supplemented rpmi1640 medium . control cells included those that were electroporated without any nef 27 , nef 25 or bsa , and cells that were electroporated with bsa at an equivalent protein concentration . after testing various parameters , the settings which gave optimal electroporation with the baekon apparatus were chosen as follows : amplitude , 5 kv ; pulse frequency , 2 8 ; burst time , 0 . 8 ; cycle number , 10 , pulse width 160μ and distance of electrode from surface of buffer , 1 . 32 mm . immediately after electroporation pbmc were stimulated with pha as described above . fig1 shows a nef - specific 27 kda or 25 kda band detected in cem cells electroporated with nef 27 or nef 25 , respectively . the band intensities obtained after reaction of anti - nef . sub . ( 15 - 27 ) with recombinant protein ( 0 . 75 - 1 . 0 μmol ) were similar to those obtained after reaction of the antibodies with lysates prepared from cells electroporated with the recombinant protein . this indicates that most of the protein placed in contact with the cells during electroporation was taken up by the cells . similarly , the level of nef protein incorporated into cells after electroporation corresponded to the amount detected in mt - 2 cells 48 h post - infection with hiv - 1 , isolate 228200 ( data not shown ). this indicates that the molar concentration of nef produced in naturally infected cells is similar to that in electroporated cells . detection of electroporated hiv - 1 nef 25 or 27 by indirect immunofluorescence immediately after electroporation , cells were washed twice with pbs and prepared for detection of hiv - 1 nef by indirect immunofluorescence staining . for detection by immunofluorescence a drop of cell suspension was air dried onto glass slides and fixed with freshly prepared 3 . 5 % paraformaldehyde for 10 min at room temperature . the fixed cells were washed in pbs and either rendered permeable by treatment with 0 . 05 % nonidet p40 ( bdh chemicals ltd , poole , dorset ) in pbs for 5 min at room temperature , or incubated for 5 min in pbs alone . the cells were incubated with 1 % bsa pbs for 30 min at room temperature in a humidified chamber prior to staining to reduce non - specific binding . the cells were them incubated with sheep anti - nef . sub . ( 15 - 27 ) ( diluted 1 : 100 ), pre - immune sheep serum ( diluted to the same protein concentration as the specific antibodies ), mab nf2b2 , ac6 , ag11 , anti - nef mab ( nen ) or anti - nef mab ( abt ) or mouse monoclonal isotype control in a humidified c sr overnight at 4 ° c . following extensive washing with pbs , the slides were incubated for 30 min at room temperature in a humidified chamber with fluorescein isothiocyanate ( fitc )- conjugated donkey anti - sheep igg ( diluted 1 : 50 ; amersham ) or fitc conjugated sheep anti - mouse igg ( diluted 1 : 50 ; amersham ). all dilutions were carried out in pbs containing 1 % bsa . specimens were examined by fluorescence microscopy using a narrow band blue filter of 488 nm . examination of mt2 cells and pbmc electroporated with nef 27 by indirect immunofluorescemce using anti - nef . sub . ( 15 - 27 ) showed that after electroporation virtually the entire population of cells contained nef 27 . similarly , 100 % of mt - 2 cells and pbmc electroporated with nef 25 showed intense fluorescence when reacted with anti - nef . sub . ( 15 - 27 ) indicating that after electroporation 100 % of cells contained nef 25 . similar results were obtained with cem and jurkat cells . staining of both permeabilised and non - permeabilised cells using several antibodies specific for nef was performed , and the results confirmed that the protein was inside the cell rather than located on the cell surface . reaction of mock - electroporated cells with anti - nef . sub . ( 15 - 27 ) produced only very low fluorescence , considered to be background staining , thus confirming the specificity of the antibodies for hiv - 1 nef protein . the pattern of fluorescence observed after reaction of anti - nef . sub . ( 15 - 27 ) with nef 27 - or nef 25 - containing cells showed both proteins to be predominantly located at the plasma membrane , and to a much lesser extent elsewhere in the cell , in all cell types analysed . the stability of the nef protein incorporated in cem cells was examined by taking samples at various times after electroporation . although both nef 27 and nef 25 protein were still present in electroporated cells at 48 h post electroporation , less protein was detected . examination of permeabilised pbmc and mt - 2 cells electroporated with nef 27 or nef 25 using mabs ag11 , ae6 and anti - nef mab abt ( all directed against the c - terminus of nef ) showed intense fluoresecence staining , located predominantly at the plasma membrane and to a much lesser extent in the cytoplasm in almost 100 % of cells electroporated , when these antibodies were reacted with permeabilised cells . this indicates that virtually all cells in these populations incorporated nef 27 or nef 25 during electroporation . the same observations were made with the other cd4 + ve cell lines tested . similar fluorescence staining , occurring predominantly at the plasma membrane , was also observed when antibodies directed at the n - terminus of nef ( nf2b2 and anti - nef . sub . ( 15 - 27 ) polyclonal and monoclonal antibodies ) were reacted with pereabilised m - 2 cells or pbmc electroporated with nef 27 . intense membrane - located fluorescence was also observed when anti - nef . sub . ( 15 - 27 ) ( polyclonal or monoclonal ) was reacted with nef 25 electroporated mt - 2 cells and pbmc ; however , no significant fluorescence staining was observed when nf2b2 was reacted with the cells . only low background fluorescence was observed when either ae6 , ag11 , abt , nf2b2 or anti - nef . sub . ( 15 - 27 ) were reacted with mock - electroporated cells indicating the specificity of the staining reactions for nef . reaction of non - permeabilised mt - 2 cells or pbmc electroporated with either nef 27 or nef 25 with any of the c - terminal monoclonal antibodies gave excellent low background staining , confirming that at least the c - terminal regions of the nef proteins were inside the treated cells rather than bound to the cell surface . however , reaction of non - permeabilised cells which had been electroporated with nef 27 or nef 25 with antibodies directed at the n - terminus ( anti - nef . sub . ( 15 - 27 ), both polyclonal and monoclonal antibodies ) of nef showed intense membrane - located fluorescence , indicating exposure at the cell surface of the n - terminal region of the nef proteins . detectable fluorescence was observed whem non - parmeabilised , mock - electroporated cells were reacted with antibodies directed against the c - terminus of nef . as a control for ability of antibodies to traverse cell membranes prior to permeabilisation , antibodies specific for mitochondrial protein were included in the staining procedures . fluorescence staining specific for mitochondrial proteins was only observed when mt - 2 cells were permeabilised with np40 treatment . only low background fluorescence staining was observed when the mitochondrial - protein antibody was reacted with non - permeabilised cells ( data not shown ). thus for the procedure used in this study , pereabilisation treatment is required for antibodies to traverse the plasma membrane , whilst fixation with paraformaldehyde as performed in this study , does not lead to cell membrane fenestration . mt - 2 cells ( 5 × 10 6 / sample ) electroporated with nef 27 , nef 25 or mock - electroporated as described above were resuspended in a 1 ml solution of trypsin / versene for two minutes at 37 ° c . after this incubation period the cells were extensively washed three times in 50 ml of pbs . after washing , the cells were resuspended at a concentration of 5 × 10 6 cells / ml in pbs and a drop air - dried onto glass slides and fixed as described above . after fixation the cells were extensively washed in pbs , and either permeabilised by treatment with 0 . 05 % ( v / v ) np40 as described above or incubated in pbs for the same period of time . after incubation in 1 % ( w / v ) bsa / pbs as described above , the cells were incubated with sheep anti - nef . sub . ( 15 - 27 ) or a mab directed at the c - terminus ( each diluted 1 : 100 ) overnight at 4 ° c . the cells were then washed , reacted with fitc - conjugated donkey anti - sheep ig or fitc - conjugated sheep anti - mouse ig ( both diluted 1 : 50 ) and viewed using fluorescence microscopy as described above . treatment of non - permeabilised and electroporated cells with trypsin abolished the ability of the n - terminal nef antibodies to react with electroporated cells . however , reaction of antibodies directed against the c - termimal region of nef with cells which were permeabilised following tyain treatment still showed fluorescence staining associated with the membrane . analysis of cell surface markers on cells containing e . coli - derived nef 27 or nef 25 flow cytometry was used to examine whether the levels of cd4 , cd25 , cd2 , cd7 and transferrin receptor ( tfr ) varied as a consequence of the incorporation of nef 27 or nef 25 protein into the cd4 + - t cells , mt - 2 , cem and jurkat and pbmc . cells which underwent a mock - electroporation and cells electroporated with bsa were also used for comparison . for quantitation of cell surface cd4 and il - 2 r , mock electroporated cells or cells containing electroporated recombinant nef 27 , nef 25 or bsa were incubated in supplemented rpmi 1640 medium in 24 well costar plates ( nunc ) for 48 h at 37 ° c . samples of cells were taken every two hours until 10 h post - electroporation , and again at 24 h post - electroporation . upon harvesting from culture , the cells were washed once with pbs and incubated with either fluorescein isothiocyanate - conjugated - anti - cd4 ( leu 3a + leu3b ), - anti - cd25 , - anti - transferrin receptor ( tfr ), - anti - cd2 , or - anti - cd7 antibody ( all from becton dickinson , san jose , calif .) and the appropriate isotype controls , for 1 h on ice . after this incubation period , cells were washed twice in pbs and fixed with 3 . 5 % parafosaldehyde for 10 min at room temperature . cells were subsequently analysed by flow cytometer using the facstar plus ( becton dickinson , san jose , calif .). strong specific : fluorescence staining was obtained after reaction of anti - cd4 , anti - tfr and anti - cd7 with mt - 2 , cem and jurkat cells which were ocak electroporated or electroporated with bsa . representative results showing mt - 2 cells after reaction with anti - cd4 , or the appropriate isotype ccntrol , are shown in fig1 a . strong fluorescence staininig was also obtained after reaction of pbmc which had been mock - electroporated , or electroporated with bsa , with anti - cd4 or anti - c2 results showing pbmc after reaction with anti - cd4 or isotype control monoclonal antibodies are depicted in fig1 b . the levels of expression of these surface molecules were comparable with those in control cells which did not undergo electroporation , indicating that the electroporation process did not alter the expression of these cell surface antigens . similarly strong surface - located fluorescence was obtained after reaction of mt - 2 cells or pha - activated pbmc which bad been mock - electroporated or electroporated with bsa with anti - cd25 ( fig1 c and 11d ). in contrast to control cells mt - 2 , cem and jurkat cells and pbmc containing hiv - 1 nef 27 displayed reduced expression of cell surface cd4 . the level of surface cd4 in each of the cd4 + cell lines and pbmc was reduced by 30 - 50 % at 24 h post - electroporation ( fig1 a and 11b ). all cells in the t - cell line populations and all cd4 + cells in the pbmc population showed significantly reduced cd4 surface expression ( p & lt ; 0001 , student &# 39 ; s t - test ). thus , all t - cells containing nef 27 displayed significantly reduced levels of surface cd4 . in a typical experiment , flow cytometric analysis of pbmc mock - electroporated or electroporated with bsa and subsequently reacted with anti - cd4 24 h post - electroporation showed 40 % of cd4 + t - cells distributed around a mean fluorescence intensity of 42 . in contrast , cd4 + pbmc which contained nef 27 after electroporation showed a single peak of fluorescence with a mean fluorescence intensity of 21 after reaction with anti - cd4 . by way of comparison , fluorescence staining obtained after reaction of pbmc electroporated with nef 27 , bsa or mock electroporated with am isotype control was distributed around a mean fluorescence intensity of 7 . levels of cd2 , tfr and cd7 on the surface of these cells were not affected by the presence of hiv - 1 nef 27 . expression of cell surface tfr and il - 2 r in mt - 2 cells slactroporated with hiv - 1 nef 27 , hiv - 1 nef 25 , bsa or mock electroporated cells is shown in fig1 . mt - 2 cells and pha - activated pbmc containing nef 27 protein also showed significantly reduced expression of il - 2 r . since the other t4 - cell lines used in this study constitutively epress only very low levels of il - 2 r , the effect of hiv - 1 nef 27 on the expression of il - 2 r in these cells was not examined . in the case of mt - 2 cells , mock - electroporated or electroporated with bsa , a single peak of fluorescence distributed around a mean of 90 was obtained after reaction of the cells with anti - cd25 . in comparison , the peak of fluorescence obtained after reaction of mt - 2 cells which contained nef 27 with anti - cd25 , 24 h post - electroporation , was distributed around a mean fluorescence of 10 ( fig1 c and 11d ). this level of fluorescence staining was similar to that produced after reaction of the isotype control with the cells , indicating that il - 2 r expression was reduced to background levels by the presence of nef 27 . all cells in the mt - 2 population demonstrated reduced expression of il - 2 r ( fig1 c ), in the case of pha - activated pbmc , the percentage of il - 2 r positive cells was reduced from 19 % to 4 % at 24 h post - electroporation of nef 27 into these cells ( fig1 d ). again , the expression of tfr , cd2 and cd7 was not affected by the incorporation of nef 27 in these cells . nef 25 does not down - regulate expression of cd4 or il - 2 r nef 25 was also electroporated into the various t - cell lines and pbmc to investigate its effect on the expression of surface cd4 and il - 2 r . suprisingly , cem , mt - 2 and jurkat cells and pbmc electroporated nef 25 showed normal expression of surface cd4 ( fig1 a and 11b ). the fluorescence staining obtained after reaction of anti - cd4 with the nef 25 containing cells was similar in fluorescence intensity to that obtained after reaction of the antibody with control cells ( mock electroporatsd or bsa electroporated cells ) ( fig1 a and 11b ). similarly , nef 25 had no effect on the expression of il - 2 r in mt - 2 cells or pha - stimulated pbmc ( fig1 c and 11d ). the fluorescence staining after reaction of mt - 2 cells or pha - activated pbmc which contain nef 25 with anti - cd25 was similar to that obtained with control cells . thus , hiv - 1 nef 27 , but not nef 25 , causes a down - regulation in expression of cd4 and il - 2 r in various t - cell lines and in pbmc . time course of cd4 and il - 2 r down - regulation by hiv - 1 nef 27 harvesting of mt - 2 cells containing nef 27 , nef 25 or mock - electroporated mt - 2 cells at various times post - electroporation showed that the reduction in expression of surface cd4 and il - 2 r as a result of nef 27 incorporation into the cells begins to occur 8 - 10 h after electroporation . samples of cells containing nef 27 harvested at 0 , 2 , 4 and 6 h post - electroporation showed that the levels of surface cd4 and il - 2 r were similar to those expressed in control cells . however , between 8 to 10 h post - electroporation levels of cd4 and il - 2 r were reduced by approximately 10 % and 30 %, respectively . no further reduction in expression of surface cd4 or il - 2 r was seen 24 h post - electroporation . down - regulation of cd4 and il - 2 r by hiv - 1 nef 27 is dose - dependent to investigate whether the effect of nef 27 on the expression of surface cd4 and il - 2 r is dependent upon the amount of nef 27 protein , cem and mt - 2 cells were electroporated with various concentrations of nef 27 or nef 25 protein . the effec t of these proteins on cd4 and il - 2 r cell surface expression was subsequently measured at 24 h post - electroporation . electroporation of cem an mt - 2 cells with 0 . 1 - 3 . 0 μmol of nef 27 showed a dose - dependent effect on the expression of cd4 in the case of cem cells and of both cd4 and il - 2 r in the case of mt - 2 cells . the level of down - regulation of cd4 and il - 2 r was highest when 3 . 0 μmol of nef 27 was used for electroporation . this resulted in 50 % reduction in surface cd4 in cem cells , and reduction of il - 2 r to near background levels in mt - 2 cells . the negative regulatory effect of hiv - 1 nef 27 upon cell surface cd4 and il - 2 r expression in cem and mt - 2 cells was still observed when 0 . 1 μmol of nef 27 protein was used for electroporation . in this case reduction of surface cd4 and il - 2 r expression in cem and mt - 2 cells by approximately 6 % ( cd4 in cem cells ) and 13 % ( il - 2 r in mt - 2 cells ) was observed . mt - 2 cells or pha - activated pbmc ( 3 × 10 7 cells / sample ) were washed twice in pbs , resuspended in 300 μl of lysis buffer ( 0 . 5 % v / v nonidet - p40 , 0 . 5 % w / v sodium deoxycholate , 50 mm nacl , 25 mm tris / hcl , 10 mm edta , 5 mm benzamidine / hcl , 1 10 mm phenylmethylsulfonyl fluoride in n - propanol and 0 . 01 % w / v sodium azide , ph 8 . 0 ) and incubated on ice for 5 min . after this incubation period the cell lysates were centrifuged at 12 , 000 g for 10 min and the cytoplasmic extract recovered . the cytoplasmic fraction was then pre - cleared by incubation with 50 μl of a 50 % slurry of glutathione sepharose 4b ( pharmacia , uppsala , sweden ) in tris buffered saline , ph 7 . 5 ( tbs ) at 4 ° c . for 10 min . after centrifugation at 12 , 000 g for 2 min the supernatant was removed , divided into three 1 . 5 ml conical tubes ( eppendorf , germany ) and glutathione ( gst , 5 μg / sample ), gst - nef 27 or gst - nef 25 ( 5 μg / sample ) or tbs added . the cell supernatants and added proteins were then incubated together overnight at 4 ° c . next day , 50 gl of a 50 % slurry of glutathione sepharose beads was added to each tube and incubated with continuous agitation for 30 min at room temperature . each suspension was then cooled on ice for 15 min before the sepharose was pelleted by centrifugation for 2 min at 12 , 000 g . the glutathione sepharose beads were washed three times with 1 ml of ice - cold tbs containing 0 . 05 % ( v / v ) np40 . bound proteins were eluted by incubation of the sepharose beads with 20 μl of 10 mm glutathione for 10 min at room temperature . after centrifugation the supernatant was removed and stored , and the elution step repeated twice . aliquots ( 20 μl ) of the eluted material were then electrophoresed on a 13 % polyacrylamide , gel and either the separated material detected by silver staining or transferred to hybond c - super nitrocellulose ( amersham , uk ) and immunoblotted with antibodies reactive with p56 lck or cd4 . multiple proteins were precipitated from lysates prepared from mt - 2 cells and pha - activated pbmc when the gst - nef fusion protein was incubated with the cell lysates . the proteins precipitated from mt - 2 cell lysates were approximately 24 , 27 , 32 , 36 , 45 , 50 , 56 and 75 kda in size whilst proteins prepared from pha - activated pbmc found to interact with gst - nef 27 were of 27 , 28 , 30 , 32 , 36 , 56 , 60 and 75 kda ( fig1 a and 13b , respectively ). when gst - nef 25 was used as the binding antigen , proteins of approximately 27 , 28 and 56 kda were precipitated from lysates prepared from pha - activated pbmc ( fig1 c ). no detectable proteins were precipitated in any case when gst was incubated with the pre - cleared lysates . similarly , no detectable proteins were precipitated by interaction with the glutathione - sepharose beads alone , indicating that the proteins precipitated with gst - nef 27 and gst - nef 25 formed specific interactions with the nef proteins . for further confirmation of the specificity of the interaction of these cellular proteins with nefe recombinant nef 27 or nef 25 was added to aliqouts of pre - cleared pbmc and mt - 2 cell lysates . addition of exogenous nef was able to significantly inhibit the binding of each of the proteins to gst - nef 27 or gst - nef 25 ( data not shown ). to identify several of the cellular proteins interacting with gst - nef 27 and gst - nef 25 , the gst - nef 27 and gst - nef 25 precipitates prepared from mt - 2 cells and pha - activated pbmc were electrophoresed in a 13 % polyacrylamide gel , and subsequently transferred to hybond - c super nitrocellulose and probed with anti - p56 lck or anti - cd4 . reaction of anti - p56 lck with the mt - 2 and pha - activated pbmc cellular proteins precipitated with gst - nef 27 showed that p56 lck was a constituent in the precipitates prepared from the mt - 2 - cell - and pbmc - gst - nef 27 precipitates ( fig1 a and 14b , respectively ). p56 lck most likely represents the protein identified as 56 kda . furthermore , when the pha - activated pbmc cellular protein precipitate was probed with anti - p56 lck the 60 kd phosphorylated isoform of p56 lck was also identifed to be present ( fig1 b ) the 60 kda isoform of p56 lck was not identified in the cellular proteins precipitated from the mt - 2 cell lysate , possibly due to lower amounts of the phosphorylated form present in mt - 2 cells as compared with levels present in pbmc ( data not shown ). the specificity of the reaction with anti - p56 lck was verified by using two other antibodies which recognise p 56 lck . reaction with anti - cd4 of mt - 2 and pbmc cellular proteins precipitated with gst - nef 27 also showed cd4 to be present in precipitates prepared from both mt - 2 cells and pbmc ( fig1 ). when cellular proteins precipitated from mt - 2 cells or pbmc with gst - nef 25 were reacted with either anti - p56 lck or acti - cd4 in western immunoblotting , neither protein was shown to be present in detectable levels ( data not shown ). growth factor - dependent dna synthesis was determined by measurement of 3 h !- thymidine incorporation into cellular dna as previously described ( 45 ). briefly , pha - activated pbmc ( 1 × 10 6 / ml ) electroporated with nef 27 , nef 25 or mock - electroporated as described above were incubated in medium for 24 h and then stimulated with il - 2 ( boebringer mannheim , germany ) at 10 , 30 or 100 iu / ml ( triplicate samples of 10 6 cells / sample ). after 24 h in culture , cells were pulsed with 3 h !- thymidine ( 5 μci / ml ) for 16 h , and incorporation of radioactivity into dna was measured by liquid scintillation counting . each sample was assayed in duplicate . the values presented are the mean ± s . d . for three experiments . the il - 2 proliferative response of pha - activated pbmc which had been mock - electroporated or electroporated with bsa increased in parallel with the concentration of il - 2 . the results are shown in fig1 . proliferation was not significantly altered by electroporation , as control cells treated with il - 2 incorporated similar amounts of 3 h !- thymidine to pbmc mock - electroporated or electroporated with bsa . in contrast , incorporation of nef 27 into pha - activated pbmc prior to stimulation with il - 2 dramatically reduced the proliferative response of these cells to il - 2 . similarly , nef 25 treated cells also showed a reduced proliferative response to il - 2 ; however the effect was not as large as that observed with nef 27 . p56 lck has been shown to be associated with the cytoplasmic domains of both cd4 and il - 2 receptor through its n - terminus and c - terminus regions , respectively ( 46 , 47 ). the inhibitory effect of nef 27 on cell proliferation and on phosphorylation of p56 lck response to il - 2 stimulation may be a direct consequence of the interaction of nef with p56 lck . to investigate the possible effect of nef on cellular distribution of lck , indirect immnofluorescence studies using anti - p56 lck were performed on nef 27 -, nef 25 -, bsa - and mock - electroporated jurkat cells immediately after electroporation , and again at 24 h post - electroporation . jurkat cells were chosen for these localisation studies since these cells express relatively high levels of p56 lck . jurkat cells were electroporated with recombinant nef 27 , nef 25 or mock - electroporated , and incubated in supplemented rpmi 1640 medium for up to 24 h at 37 ° c . cells were harvested at 0 , 10 and 24 h post - electroporation , washed twice in pbs and a drop of cell suspension ( 5 × 10 6 cells / ml ) air dried onto glass slides , and fixed in 3 . 5 % paraforaldehyde for 10 min at room temperature . after blocking with 1 % fetal calf serum the cells were incubated with anti - p56 lck , diluted 1 : 100 , at 4 ° c . overnight . as a control , cells were incubated with normal rabbit serum for the same period of time . next day the slides were washed three times in pbs and incubated with anti - rabbit conjugated tritc ig secondary antibody ( silenus , diluted 1 : 50 ) for 1 h at room temperature . after washing in pbs , the slides were viewed using conventional fluorescence microscopy . intense , predominantly membrane - associated fluorescence specific for p 56 lck was observed in mock - electroporated and bsa - electroporated jurkat cells when cells were reacted with anti - p56 lck immediately after electroporation . similarly , cells electroporated with nef 27 or nef 25 also displayed intense predominantly membrane associated fluorescence when reacted with anti - p56 lck immediately after electroporation . when the electotrporated cells waos analysed 24 h after electroporation mock and bsa - electroporated cells still displayed intense , membrane located fluorescence . in direct contrast , jurkat cells electroporated with nef 27 showed diffuse fluorescence staining which was of lower intensity compared with that of control cells . nef 25 - treated jurkat cells also displayed reduced membrane associated fluorescence when reacted with anti - p56 lck ; however the effect was not as marked as observed in nef 27 treated jurkat cells . il - 2 dependent and pma - induced phosphorylation of p56 lck is inhibited in nef 27 treated pbmc addition of il - 2 to t - cells expressing the high affinity il - 2 r complex results in the transmission of a proliferative stimulus . increasing evidence suggests that tyrosine protein kinases and serine / threonine protein kinases are involved in il - 2 dependent proliferative signals ( 48 ). indeed , p56 lck tyrosine kinase activity is rapidly stimulated following il - 2 addition , and undergoes subsequent il - 2 dependent serine / threonine phosphorylation modifications which result in the retardation in mobility of the p56 lck protein by polyacrylamide gel electrophoresis ( 18 ). alterations in phosphorylation of p56 lck in response to il - 2 were evaluated in pbmc electroporated with nef 27 , nef 25 or mock - electroporated . electroporated pbmc or control pbmc were treated with il - 2 for increasing periods of time . human pbmc were isolated from normal adults by ficoll - paque density centrifugation . cells were cultured in rpmi 1640 medium supplemented with 10 % fetal calf serum in the presence of pha ( 10 μg / 10 6 cells ) for 3 days . the pha - activated pbmc were then incubated in supplemented rpmi 1640 medium for 24 h , and electroporated with nef 27 , nef 25 or mock electroporated , as described above . after incubation in medium for a further 24 h to reduce low level production of il - 2 , the cells were harvested , washed three times and then incubated with 1000 iu of recombinant il - 2 ( boehringer mannheim ) for 0 , 15 and 30 min after incubation , cells were lysed and analysed for the presence of phosphorylated forms of p56 lck by immunoblotting with anti - p56 lck . for activation of pbmc by phorbol 12 - myristate 13 - acetate ( pma ), cells were mock electroporated or electroporated with nef 27 , nef 25 , bsa as described above , incubated at 37 ° c . for 24 h and then stimulated with pma ( 20 ng / m ) for 4 h at 37 ° c ., after which time the cells were harvested and prepared for immunoblotting with anti - p56 lck . the results are presented in fig1 . addition of il - 2 to control pbmc resulted in reduced electrophoretic mobility of a portion of p56 lck . the slower migrating species of p56 lck was found to co - migrate with the altered mobility species of p56 lck induced after pma treatment of pbmc ( data not shown ). similarly , addition of il - 2 to mock - electroporated pbmc also caused the conversion of a portion of p56 lck to its p60 isoform . however , addition of il - 2 to pbmc electroporated with nef 27 did not result im reduced electrophoretic mobility of p56 lck . even after incubation of the nef 27 treated cells with il - 2 for 30 min , no detectable shift in electrophoretic mobility of p56 lck was observed , indicating that nef 27 inhibited il - 2 dependent signalling , possibly by inhibition of serine / threonine kinases responsible for phosphorylation of the p56 lck gene product , or directly by physical association with p56 lck ( see fig1 ). in contrast , nef 25 had no detectable effect on alteration of p56 lck in response to il - 2 treatment , as both isoforms , p56 lck and p60 lck were detected in nef 25 - containing pbmc after treatment with il - 2 ( fig1 ). the phorbol ester pma induces the activation of protein kinase c ( pkc ) in t - cells . a number of cellular proteins , including p56 lck , are phosphorylated on serine / threonine residues as a result of pkc activation . treatment of pbmc with nef 27 also inhibited the phosphorylation of p56 lck in response to pma treatment , as shown in fig1 . no inhibitory effeact was observed in pbmc treated with nef 25 . similarly , electroporation of pbmc with bsa or mock - electroporation of cells had no effect on the phosphorylation of p56 lck in response to pma treatment . the nuclear oncoprotein myb is believed to be an important regulator of cell growth and differentiation in hematopoietic cells , being required for transition of cells from the g 0 / g 1 phase of the cell cycle to early s - phase . furthermore , expresion of the cd4 gene requires a myb transcription factor . the levels of c - myb protein were investigated in nef 27 , hof 25 , or mock - electroporated mt - 2 cells by western immunoblotting using anti - c - myb . mt - 2 cells , harvested during mid - exponential growth phase , were washed twice in pbs and electroporated with recombinant nef 27 , nef 25 , bsa or mock - electroporated as described above . after electroporation the cells were washed twice in pbs , resuspended at a concentration of 1 × 10 6 cells in supplemented rpmi and incubated for 24 h at 37 ° c . following the incubation period the cells were washed in pbs again and then resuspended in lysis buffer at a concentration of 10 6 cells / ml and incubated on ice for 10 min . the cytoplasmic extract was recovered by centrifugation for 10 min at 12 , 000 g . cell lysate corresponding to 2 × 10 6 cells was electrophoresed in a 13 % polyacrylamide gel , transferred to hybond c - super nitrocellulose and immunoblotted with anti - c - myb , diluted 1 : 1000 ( 49 ). following washing in pbs containing 0 . 05 % ( v / v ) tween 20 the filters were incubated with anti - rabbit - biotinylated ig ( amersham ), diluted 1 : 1000 for 1 h at room temperature . the filters were then incubated with streptavidin - horse radish peroxidase ( diluted 1 ; 1000 ; amersham ) following further washing , and the signal detected using 1 , 4 - dichloronapthol as substrate . the results are shown in fig1 . levels of c - myb were not affected by electroporation of cells . however , introduction of nef 27 and also nef 25 in mt - 2 cells significantly reduced the levels of c - myb as determined by western immunoblotting . human cd4 + t - lymphocytes form the primary target for infection by hiv - 1 in vivo ( 16 ). cells from hiv - infected patients demonstrate aberrant immune responsiveness which may be relevant to the progression from asymptomatic to aids ( 28 ). the nef gene is present in most strains of hiv - 1 , hiv - 2 and siv ( 29 ). to test whether the hiv - 1 nef gene product affects normal cell functioning , we have introduced recombinant nef 27 and nef 25 protein , translated from the first and second initiation codons respectively , into various cd4 + t - cells . cells containing either nef 27 or nef 25 were compared with control cells for the effect of nef on cell surface molecules involved in antigen recognition , signal transduction and development of normal immune response . for the first time , using immunohistochemical techniques , we have shown that hiv - 1 nef 27 , but not nef 25 , causes the specific down - regulation of cell surface cd4 and il - 2 r in various t - cell lines and pbmc in a dose - dependent manner . no effect on other cell surface antigens was detected . the novel approach of transferring highly purified recombinant hiv - 1 nef protein across cell membranes by electric field electroporation in appropriately controlled experiments ensures that the properties measured are attributable only to the nef protein . the dose - dependent effect of nef 27 suggests that the amount of nef 27 may be important for full inhibitory activity . the amounts of nef protein used in this study correspond to those produced during the course of hiv - 1 infection in vitro , while localisation of electroporated recombinant nef protein predominantly at the plasma membrane is in agreement with studies showing naturally occurring nef to be localised at the cell membrane during infection . our results showing the down - regulation of cd4 surface expression by nef 27 conclusively confirm earlier reports ( 20 , 21 ) of the down - regulation of cd4 in cells transfected with nef expression vectors . guy et al . ( 20 ), observed down - regulation of cd4 in cem cells expressing the hiv - 1 bru nef protein . similarly garcia and miller ( 21 ), reported cd4 down - regulation in t -, b - and macrophagic - t cells transfected with hiv sf2 nef . however , it was essential to substantiate these findings , since gama sosa et al . ( 23 ), reported the down - regulation of cd4 by transfection of cells with plasmid alone . progression of in vivo hiv - 1 infection to immunological disease is characterised by the depletion of cd4 + t - cell population from the peripheral blood system , and by reduced expression of cellular il - 2 r by pbmc even after stimulation with pha ( 28 , 30 , 31 , 32 ). correspondingly , our laboratory infection of mt - 2 cells with hiv - 1 isolate 228200 also results in the reduced expression of surface located cd4 and il - 2 r in these cells . besides its role as the major receptor for hiv - 1 , cd4 , through binding of antigen in the context of mhc class proteins , is intimately involved in the signal transduction pathway which culminates in the clonal expansion of antigen - reactive t - cells and the efficient recruitment of other hematopoietic cells involved in development of the immune response ( 33 ). the down - regulation by nef protein of surface cd4 and il - 2 r in t - cell lines , but most importantly in pbmc , may be particularly relevant to the progression of hiv - 1 infection to disease . the formation of high affinity il - 2 rs is a well - documented response following stimulation through t - cell receptor ( 34 ). antigen presentation to the t - cell receptor triggers intracellular signalling that subsequently leads to the transcriptional activation of various genes including il - 2 . binding of il - 2 to the high affinity il - 2 r is necessary for cell division and clonal expansion of antigen reactive cells ( 34 , 35 ). reduced expression of il - 2 r by hiv - 1 nef 27 may be linked to the inhibition by nef of il - 2 gene expression through disturbance of the il - 2 / il - 2 r autocrine pathway . alteratsively , reduced il - 2 r expression in t - cells may be a result of reduced levels of nfkb protein - binding activity . regulation of il - 2 r gene expression by the htlv - 1 tax gene product has been reported , in which activation of il - 2 r - alpha gene expression by tax protein occurs through an interaction with , or activation of , a host transcription factor with properties similar if not identical to nfkb ( 36 , 37 ). in addition , reduced nfkb protein binding capabilities have been observed in cells transfected with nef ( 14 ). the down - regulation in expression of cell surface cd4 and il - 2 r by nef may represent mutually exclusive events . alternatively , the present study may implicate nef 27 in the modulation of expression or activation of the src protein tyrosine kinase p56 lck . cd4 is known to be intimately associated with p56 lck ( 19 ). phorbol ester - induced modulation of h cd4 is accompanied by the dissociation of p 56 lck from the receptor , whilst accumulating evidence suggests that p 56 lck is involved in signal transduction following the interaction of il - 2 with the il - 2 r ( 18 , 38 ). expression of nef during hiv - 1 infection may modulate intracellular signals via p 56 lck . perturbation of this signal transduction pathway may be advantageous for the survival and spread of virus both in vitro and in vivo , resulting in the demise of the t - cell population . our results have also highlighted fundamental differences in activity between hiv - 1 nef 27 and nef 25 . whereas nef 27 causes the specific down - regulation of both cd4 and il - 2 r , nef 25 does not affect the cell surface expression of either of these surface antigens . immunofluorescence detection of electroporated nef 27 and nef 25 showed localisation to be predominantly at the plasma membrane for both proteins . this indicates that differences in activity are not a conseqence of variation in localisation of the two nef species . however , the differences in biological activity observed for nef 27 and nef 25 suggest that the functional domain of nef resides at the n - terminus of the protein . alteratively , deletion of the first 19 amino acids may alter the conformation of the protein such that normal activity is abolished . in either case , this 19 amino acid sequence appears to be essential for the biological function of nef . it has previously been shown that during hiv - 1 infection there is variation in the level of expression of two species of nef of approximate mr 27 and 25 ( 13 , 40 ), but it is not known whether the smaller species results from interal initiation , premature termination or proteolytic cleavage . if nef 25 arises in vivo by proteolytic cleavage at the c - teiminus of nef 27 , or deletion of the 3 &# 39 ; end of the nef gene , them it would be a different molecule from the nef 25 described in this paper . in any case it is possible that there is a selection for a sub - population of viruses that produce high levels of nef 25 , because this protein acts as an inhibitor of a viral protein that interferes with cell propagation . precedents for viruses encoding antagonistic regulatory proteins exist . both the bovine papilloma virus e2 open reading frame and the adenovirus eia region encode two antagonistic regulatory proteins ( 41 , 42 ). in each case the antagonistic proteins share carboxyl terminal sequences . hence it is plausible that nef 25 may be a natural competitor of nef 27 , regulating the negative effect of this protein . this idea is currently being pursued . our results demonstrate unequivocally that nef 27 , but not nef 25 , causes reduced expression of cell surface located cd4 and il - 2 r in various cd4 + t - cell lines and in pha - activated pbmc . we have also demonstrated that hiv - 1 nef 27 , and to a lesser extent hiv - 1 nef 25 , inhibits the proliferative response of pbmc to il - 2 and the mitogen pha . treatment of pbmc with nef resulted in the inhibition of il - 2 dependent and pha activation / phosphorylation of the src family kinase p56 lck . membrane localisation of p56 lck in jurkat cells was disrupted by treatment with nef 27 and to a lesser extent by treatment with nef 25 , whilst electroporation of nef 27 and nef 25 into the htlv - 1 transformed cell line mt - 2 dramatically reduced the expression of the protooncogene c - myb . precipitation studies using gst - nef 27 as the binding antigen showed nef to interact specifically with a number of cellular proteins , two of which have been identified as p56 / p60 lck and cd4 . the localisation of nef in both electroporated cells and hiv - 1 infected mt - 2 cells shows nef to be intimately associated with the plasma membrane , such that a portion of the n - terminus of nef is exposed to the extracellular matrix . the orientation of nef , together with its interaction with numerous proteins of which several are important for antigen recognition and cellular signalling , suggests that nef plays an intricate role in disturbing normal cellular activation / proliferation pathways . the difference in activity between nef 27 and nef 25 may indicate that the functional domain of nef resides at the n - terminus of the protein . alternatively , deletion of the first 19 amino acid residues may affect overall conformation of the protein such that detectable biological activity ( defined as the biological activities described herein ) is affected . further investigation of nef activity in this study shows that while nef 25 possesses similar activity to nef 27 , the effect of nef 25 is not as significant as that observed for nef 27 . hence the first 20 amino acid residues of the nef protein are required for full activity , as defined in our experiments . a recent report ( 50 ) showed that the first 35 amino acid residues of nef are required for transactivation of the hiv - 1 ltr , supporting the concept that the n - terminus of nef is essential for full biological activity . we have used an e . coli expressed gst - nef 27 and gst - nef 25 fusion protein as affinity reagents to identify cellular proteins that specifically interact with nef . a number of proteins from cytoplasmic extracts prepared from pbmc ( pha - activated ) and mt - 2 cells were identified to interact specifically with nef . the proteins precipitated from mt - 2 cells when gst - nef 27 was used as the affinity reagent were of 24 , 27 , 32 , 36 , 45 , 50 , 56 and 75 kda . corresponding proteins were al so identified in the lysates of pbmc , however the 24 kda species was absent , whilst 28 and 60 kda species were present in detectable levels in the pbmc preparation only . two proteins were identified from the lysates as being p56 / 60 lck and cd4 . the precipitation of a number of proteins with gst - nef indicates an indirect interaction for some of these proteins . it is not known whether the interaction of nef with cd4 and lck is a result of nef binding directly to either of these proteins or is a result of interaction with other unidentified proteins . a recent study using a baculovirus - expressed nefgst fusion protein as affinity reagent identified a numbesr of proteins from cytoplasmic extracts of jurkat cells ( 27 ). several of these proteins , p280 , p32 and p28 , bound only to a myristoylated form of nef . in our study p32 and p28 proteins were also identified , but bound to a non - myristoylated form of nef . precipitation of p56 lck and cd4 with gst - nef 27 suggests that nef may disturb signalling pathways by binding directly or indirectly with these proteins . indeed , nef may disrupt normal localisation of these proteins resulting in disturbance of cell activation . we have shown that membrane localisation of lck is disrupted following treatment of jurkat cells with nef 27 , and to a lesser extent following treatment with nef 25 . fluorescence staining for lck in nef 27 / nef 25 treated cells was more diffuse in nature , suggesting movement of the protein away from the membrane . it has been extensively shown that cross - linking cd4 or cd8 with the tcr / cd3 complex markedly enhances stimulation of t - cells compared to cross - linking of the tcr / cd3 complex alone ( 51 ). p56 lck association with cd4 is critical for the colocalisation of cd4 with the tc / cd3 complex after t - cell stimulation , whilst the p56 lck - dependent loss of association between cd4 and the tcr / cd3 complex results in diminished cd4 - depeandent antigen responsivemess . thus p56 lck appears to play multiple roles in t - cell stimulation ( 52 ), and disturbance of p56 lck membrane association and or binding of nef to lck , either directly or indirectly , may greatly impair t - cell responsiveness . il - 2 has a pivotal role in regulating the proliferation and differentiation of hematopoietic cells . il - 2 , exerts its biological effects through binding of specific receptors on the cell surface . treatment of pbmc with nef 27 inhibited their proliferative response to il - 2 as judged by 3 h ! thymidine incorporation . treatment of pbmc with nef 25 also reduced their capacity to respond to il - 2 ; however the negative effect of nef 25 was not as great as that observed with nef 27 . induction of t - lymphocyte proliferation is dependent on a multiplicity of events , including the activation of numerous genes including the protooncogene c - myb . several studies provide evidence that c - myb gene function is required for t - lymphocyte proliferation , and is specifically required for transition through late g 1 or early s - phase of the cell cycle . we have found that nef affects the levels of c - myb in mt - 2 cells , and may be responsible in part for decreased proliferation in response to il - 2 . furthermore , a transcription factor from the myb family plays a critical role in cd4 promoter function , and therefore contributes to expression of the cd4 gene ( 50 ). thus decreased cell surface expression of cd4 in pha - activated pbmc and various cd4 + t - cell lines may be a consequence of reduced levels of c - myb . we have shown that nef 27 inhibits phosphorylation and thus ( presumably ) activation of p56 lck in response to il - 2 or pha stimulation . this may be a direct result of interaction of nef with lck , or may indicate inhibition of serine / threonine kinases such as protein kinase c . pma is a potent activator of protein kinase c . treatment of pbmc with nef 27 also inhibited pma - induced phosphorylation of p56 lck . nef 25 did not appear to affect phosphorylation of p56 lck , but may reflect the more qualitative nature of western blot assessment . our immunofluorescence studies of nef localisation in electroporated cells and hiv - 2 infected mt - 2 cells have shown that the protein is intimately associated with the plasma membrane , whilst a portion of the n - terminus of nef is exposed to the extracellular matrix . comparison of permeabilised and nonpermeabilised cells showed that antibodies directed against the c - terminus of nef detected nef only in permeabilised cells , whilst antibodies directed against the n - terinal region of nef reacted with both permeabilised and nompermeabilised cells containing nef . hiv1bru nef has been reported to contain a two - peptide domain sequence which as striking similarity to the structure of neuroactive scorpion peptides ( 53 ). nef was also shown to reversibly increase the total k + current after membrane depolarisation in patch clamp experiments . such an effect on k + channels of neuronal cells requires the extracellular presence of nef . this may be achieved by exposure of part of the molecule to the cell surface . further characterisation of the biological activity of nef and investigation into the mechanism of action is currently being pursued , and will provide useful information for the rational design of antiviral agents able to abrogate the function of nef . the partial homology between the nef n - terminal sequence and melittin suggests that the latter is a suitable candidate molecule . in particular , we are determining the minimum sequence within nef 2 - 19 which can effect the biological function of nef 27 in the biophysical systems and antigen expression systems disclosed herein . peptide analogues of nef 2 - 19 are being synthesised . in addition , anti - sense peptides and anti - sense oligonucleotides as well as antibodies and their fragments are being investigated . a variety of means for introducing analogues or antagonists of nef 2 - 19 peptide into cells are contemplated . following such introduction , the cells may be challenged with hiv , and parameters such as syncytium formation , down - regulation of cd4a and down - regulation of il - 2 r investigated . such means include , but are not limited to , lipofection with liposomes , peptide - fat conjugation , electroporation , and transfection of cells with genes expressing anti - sense oligonucleotides directed against the n - terminus of the nef gene , which encodes the nef 2 - 19 peptide . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . 1 . cullen , b . r . &# 34 ; trans - activation of human immunodeficiency virus occurs via a bimodal mechanism &# 34 ; cell , 1986 46 973 - 982 . 2 . terwilliger , e . f ., sodroski , j . g ., rosen , c . a ., and hazeltine , w . a . &# 34 ; effects of mutations within the 3 &# 39 ; orf open reading frame region down - regulates virus replication &# 34 ; j . virol ., 1986 60 754 - 760 . 3 . arya s . k . beaver , k ., yagodzinski , l ., ensoli , b ., kanki , p . j ., albert , j ., fenyo , e . m ., biberfeld , g ., zagury , j . f ., laure , f ., essex , m ., norrby , e ., wong - 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662 . 11 . cheng - mayer , c ., iannello , p ., shaw , k ., luciw , p . a ., and levy , j . a . &# 34 ; differential effects of nef on hiv replication : implications for viral pathogenesis in the host &# 34 ; science , 1989 246 1629 - 1632 . 12 . luciw , p . a ., cheng - mayer , c . and levy , j . a . &# 34 ; mutational analysis of the human immunodeficiency virus the orf - b region down - regulates virus replication &# 34 ; proc . natl . acad . sci . usa ., 1987 84 , 1434 - 1438 . 13 . ahmad , n ., and venkatesan s . &# 34 ; nef protein of hiv - 1 is a transcriptional repressor of hiv - 1 ltr &# 34 ; science , 1988 241 1481 - 1485 . 14 . niederman , t . m ., garcia , j . v ., hastings , r . w ., luria , s ., and ratner , l . &# 34 ; human immnodeficiency virus type 1 nef protein inhibits nf - kb induction in human t cells &# 34 ; j . virology , 1992 66 6213 - 6219 . 15 . kim , s ., ikeuchi , k ., byrn , r ., groopman , j ., and baltimore , d . &# 34 ; lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1 &# 34 ; proc . natl . acad . sci . usa , 1989 86 9544 - 9548 . 16 . dalgleish , a . g ., beverley , p . c . i ., clapham , p . r ., crawford , d . h ., greaves , m . f ., and weiss , r . a . &# 34 ; the cd4 ( t4 ) antigen is an essential component of the receptor for the aids retrovirus &# 34 ; nature , 1984 312 763 - 766 . 17 . mcdougal , j . s ., mawle , a ., cort , s . p ., nicholson , j . k . a ., cross , g . d ., scheppler - capbell , j . a ., hicks , d . and sligh , j . &# 34 ; cell tropism of the human retrovirus , htlv - iii / lav 1 . role of t cell activation and expression of the t4 antigen &# 34 ; j . immunol , 1985 135 3151 - 3162 . 18 . horak , i . d ., gress , r . e ., lucas , p . j ., horak , e . m ., waldman , t . a ., and boleen , j . b . ( 1991 ). &# 34 ; t - lymphocyte interleukin 2 - dependent tyrosine protein kinase signal transduction involves the activation of p56 lck &# 34 ; proc . natl . acad . sci . usa ., 1991 88 1996 - 2000 . 19 . rudd , c . e . &# 34 ; cd4 , cd8 and the tcr - cd3 complex : a novel class of protein - tyrosine kinase receptor &# 34 ;. immunol . today , 1990 11 400 - 407 . 20 . guy , b ., riviere , y ., dott , k ., regnault , a . and kieny , m . p . &# 34 ; mutational analysis of the hiv nef protein &# 34 ; virology , 1990 176 413 - 425 . 21 . garcia , j . v . and miller , a . d . &# 34 ; serine phosphorylation - independent downregulation of cell - surface cd4 by nef &# 34 ; nature , 1991 350 508 - 511 . 22 . luria , s ., chambers , i ., and berg , p . &# 34 ; expression of the type 1 human immunodeficiency virus nef protein in t cells prevents antigen receptor - mediated induction of interleukin 2 mrna &# 34 ; proc . natl . acad . sci . usa . 1991 88 5326 - 5330 . 23 . gama sosa , m . a ., degasperi , r ., kim , y - s ., fazely , f ., sharma , p . and ruprecht , r . m . &# 34 ; serine phosphorylation - independent downregulation of cell - surface cd4 by nef &# 34 ;. aids res . human retroviruses , 1991 7 859 - 860 . 24 . peper , r . j ., tina , w . z ., and mickelson , m . m . ( 1968 ). &# 34 ; purification of lymphocytes and platelets by gradient centrifugation &# 34 ; j . lab . clin . med , 1968 72 842 - 846 . 25 . kemp , b . e ., rylatt , d . b ., bundensen , p . g ., doherty , r . r ., mcphee , d . a ., stapleton , d ., cottis , l . e ., wilson , k ., john , m . a ., khan , j . m ., dinh , d . p ., miles , s ., and hillyard , c . j . &# 34 ; autologous red cell agglutination assay for hiv - 1 antibodies : simplified test with whole blood &# 34 ; science , 1988 241 1352 - 1354 . 26 . zhao , x ., wong , t . k ., and batten , b . &# 34 ; electric mediated protein transfer into mouse oocytes &# 34 ; techniques , 1989 1 37 - 42 . 27 . kiernan , r ., marshall , j ., bowers , r ., doherty , r ., and mcphee , d . a . &# 34 ; kinetics of hiv - 1 replication and intracellular accumlation of pathology in htlv - 1 transformed cells &# 34 ; aids res . human retroviruses , 1990 6 743 - 752 . 28 . nicholson , j . k ., spira , j . j ., aloisio , c . h ., jones b . m ., kennedy , s ., holman , r . c ., and mcdougal , j . s . &# 34 ; serial determination of hiv - 1 titers in hiv - 1 infected homosexual men : association of rising titers with cd4 cell depletion &# 34 ; aids res . human retroviruses , 1989 5 205 - 215 . 29 . franchinie g ., and bosch , m . l . &# 34 ; genetic relatedness of the human immunodeficiency viruses type 1and 2 ( hiv - 1 , hiv - 2 ) and the simian immunodeficiency virus ( siv )&# 34 ; ann , n . y . acad . sci ., 1989 554 81 - 87 . 30 . honda , m ., kitamura , k ., matsuda , e ., yokota , y ., yamamoto , n ., mitsuyasu , r ., chermann , j - c ., and tokunaga t . ( 1989 ). &# 34 ; soluble il - 2 r in aids . correlation of its serum level with the classification of hiv - induced diseases and its characterisation &# 34 ; j . immunol ., 1989 12 4248 - 55 . 31 . hattori , n ., michaels , f ., fargnoli , k ., marion , l ., gallo , r . c ., and franchini , g . &# 34 ; the human immunodeficiency virus type 2 vpr gene is essential for productive infection of macrophages &# 34 ; proc . natl . acad . sci . usa ., 1990 87 8078 - 8084 . 32 . prince , h . e ., kermani - arab , v ., and fahey , j . l . &# 34 ; depressed interleukin 2 receptor expression in acquired immunodeficiency and lymphadenopathy syndromes &# 34 ; j . immunol ., 1984 133 1313 - 1317 . 33 . gay , d ., maddon , p ., sekaly , r ., talle , m . a ., godfrey , m ., long , e ., goldstein , g ., chess , l ., axel , r ., kappler , j ., and marrack , p . ( 1987 ). &# 34 ; fuctional interaction between t - cell protein cd4 and the major histocompatibility compleg hla - dr antigen &# 34 ; nature , 1987 328 626 - 630 . 34 . greene , w . c ., bohnlein , e ., siekewitz , m ., and ballard , w . b . &# 34 ; structure and regulation of the human il - 2 r &# 34 ; adv . exp . med . biol ., 1989 245 55 - 60 . 35 . robb , r . j ., munck , a ., and smith , k . a . &# 34 ; t cell gowth factors : quantitation , specificity and biological relevance &# 34 ; j . exp . med ., 1981 154 1455 - 1474 . 36 . ruben , s ., poteat , h ., tan , t - h ., kawakami , k ., roeder , r ., haseltine , w ., rosen , c . a . &# 34 ; cellular transcription factors and regulation of il - 2 r gene expression by htlv - 1 tax gene products &# 34 ; science , 1988 241 89 - 92 . 37 . arima , n ., kuziel , w . a ., grdina , t . a ., and greene , w . c . &# 34 ; il - 2 - induced signal transduction involves the activation of nuclear nf - kb expression &# 34 ; j . immnunol ., 1992 149 83 - 91 . 38 . hurley , t . r ., luo , k ., and sefron , b . &# 34 ; activators of protein kinase c induce dissociation of cd4 , but not cd8 , from p56 lck &# 34 ; science , 1989 245 407 - 409 . 39 . allan , j . s ., coligan , j . e ., lee , t - h ., mclane , m . f ., kanki , p . j ., groopman , j . e ., and essex , m . &# 34 ; a new htlv - iii / lav encoded antigen detected by antibodies from aids patients &# 34 ; science , 1985 230 810 - 814 . 40 . zweig , m ., samuel , k . p ., showalter , s . d ., bladen , s . v ., dubois , g . c ., lautenberger , j . a ., hodge , d . r ., and papas , t . s . &# 34 ; heterogeneity of nef proteins in cells infected with human immunodeficieny virus type 1 &# 34 ; virology , 1990 179 504 - 507 . 41 . lambert , p . f ., spulholz , b . a ., and howley , p . m . &# 34 ; a transcriptional repressor encoded by bfv - 1 shares a common carboxy - terminal domain with the e2 transactivator &# 34 ; cell 1987 50 69 - 73 . 42 . lillie , j . w ., loewenstein , p . m ., green , m . r ., and green , m . &# 34 ; functional domains of adenovirus type 5 e1a proteins &# 34 ; cell , 1987 46 1043 - 1046 . 43 . einsbahr , k . j ., abraham r . d . and dick , c . j . &# 34 ; protein in tyrosine phosphorilation and p56 lck modification in human natural kilocells &# 34 ; j . immunol ., 1990 145 1490 - 1497 44 . shaw , a . s ., amrein , r . b ., hammond , c ., stern , d . f ., sefton , b . m . and rose , j . k . &# 34 ; the lck tyrosine protein kinase interacts with the cytoplasmic tail of the cd4 glycoprotein through its unique amino - terminal domain &# 34 ;. cell 59 627 - 636 45 . abrahan , r . i ., ho , s . n ., mckean , d . j . &# 34 ; bioassay of interleukins &# 34 ; j . tiss . cult . methods , 1986 10 93 - 99 46 . minami , y ., kono , t ., yamada , k ., kobayashi , n ., kawaliara , a ., preimutter , r . m . and taniguchi , t . &# 34 ; association of p56 lck with il - 2 receptor β chain is critical for the il - 2 induced activation of p56 lck &# 34 ; embo j ., 1993 12 759 - 768 47 . rudd , c . e . &# 34 ; cd4 , cd8 , and the tcr - cd3 complex : a novel class of protein tyrosine kinase receptor &# 34 ; immunol . today , 1990 11 400 - 405 48 . irving , s . g ., june , c . h ., zippel , p . f ., siebenlist , u . and kelly , k . &# 34 ; mitogen - induced genes are subject to multiple pathways of regulation in the initial stages of t - cell activation &# 34 ; mol . cell biol ., 1989 9 1034 - 1040 49 . siu , g ., wurster , a . l ., lipsick , j . s . and hedrick , s . m . &# 34 ; expression of the cd4 gene requires a myb transcription factor &# 34 ; mol . cell biol ., 1992 12 1592 - 1604 50 . harris , m . and coates , k . &# 34 ; identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro : a role for myristoylation &# 34 ; j . gen . virol ., 1993 74 1581 - 1589 51 . barber , e . k ., dasgupta , j . d ., schlossman , s . f ., trevillyan , j . m . and rudd , d . e . &# 34 ; the cd4 and cd8 antigens are coupled to protein - tyrosine kinase ( p56 lck ) that phosphorylates the cd3 complex &# 34 ; proc . natl . acad . sci . usa , 1989 86 3277 - 3281 52 . collins , t . l . et al &# 34 ; p56 lck association with cd4 is required for the interaction between cd4 and the tcr / cd3 complex and for optimal antigen stimulation &# 34 ; j . immunology , 1992 148 2159 - 2162 53 . werner , t ., ferroni , s ., saermark t ., brack - werner , r ., banati , r . b ., mager , r ., steinaa , l ., kreutzberg , g . w . and erfle , v . &# 34 ; hiv - 1 nef protein exibits structural and functional similarity to scorpion peptides interacting with k + channels &# 34 ; aids , 1991 5 1301 - 1308 54 . azad , a . a ., failla , p ., lucantoni , a ., bentley , j ., mardon , c ., wolfe , a ., fuller , k ., hewish , d ., sengupta , s . sandkovich , s ., grgacic , e ., mcphee , d ., macreadie , i . g . &# 34 ; large scale production and characterisation of recombinant human immunodeficiency virus type 1 nef &# 34 ; j . gen . virology , 1994 75 651 - 655 __________________________________________________________________________ # sequence listing - ( 1 ) general information :- ( iii ) number of sequences : 2 - ( 2 ) information for seq id no : 1 :- ( i ) sequence characteristics :# acids ( a ) length : 21 amino ( b ) type : amino acid ( c ) strandedness : not r - # elevant ( d ) topology : not relev - # ant - ( ii ) molecule type : peptide - ( iii ) hypothetical : no - ( iv ) anti - sense : no - ( v ) fragment type : n - terminal - ( vi ) original source : ( a ) organism : human imm - # unodeficiency virus type 1 # nef2 - 22 ( c ) individual isolate :- ( xi ) sequence description : seq id no : 1 :- gly gly lys trp ser lys ser ser - # val ile gly trp pro ala valarg # 15 - glu arg met arg arg 20 - ( 2 ) information for seq id no : 2 :- ( i ) sequence characteristics :# acids ( a ) length : 24 amino ( b ) type : amino acid ( c ) strandedness : not r - # elevant ( d ) topology : not relev - # ant - ( ii ) molecule type : peptide - ( iii ) hypothetical : no - ( iv ) anti - sense : no - ( v ) fragment type : n - terminal - ( vi ) original source : ( a ) organism : apis mell - # ifera - ( xi ) sequence description : seq id no : 2 :- gly ile gly ala val leu lys val - # leu thr thr gly leu pro alaleu # 15 - ile ser trp ile lys arg lys arg 20__________________________________________________________________________