Patent Application: US-54792409-A

Abstract:
the invention relates to the use of the ubiquitous vertebrate glucose transporter glut1 , or of fragments or sequences derived thereof , for the in vitro diagnosis of cancers , when used as a tumor marker , or for the screening of compounds useful for the preparation of drugs for the prevention or the treatment of pathologies linked to an infection of an individual with a ptlv , or pathologies linked to an overexpression of glut1 on cell surfaces , or the in vitro detection of glut1 on cell surfaces . the invention also relates to pharmaceutical compositions containing glut1 , or fragments or sequences derived thereof , and to their uses such as in the frame of the prevention or the treatment of pathologies linked to an infection of an individual with a ptlv .

Description:
the invention is further illustrated with the detailed description hereafter of the determination of glut1 as a specific receptor for ptlv rbd . the human t - cell leukemia virus ( htlv ) type 1 and 2 are present in all areas of the world as endemic or sporadic infectious agents [ slattery , 1999 ]. the etiological role of htlv - 1 in adult t cell leukemia ( atl ) and tropical spastic paraparesis / htlv - associated myelopathy ( tsp / ham ) has been well established [ poiesz , 1980 ; yoshida , 1982 ; gessain , 1985 ; osame , 1986 ]. the apparently restricted tropism of htlv to t lymphocytes in infected patients [ cavrois , 1996 ; hanon , 2000 ] contrasts with the ability of the viral - encoded envelope glycoprotein ( env ) to bind to and direct entry into all vertebrate cell types tested in vitro [ sutton , 1996 ; trejo , 2000 ; kim , 2003 ]. retroviral infections depend on early interactions between env and cellular receptors . identification of cellular receptors and coreceptors for other retroviral envelopes have helped to elucidate certain aspects of retrovirus physiopathology as well as their transmission and spreading within organisms and populations [ berger , 1999 ; clapham , 2001 ; weiss , 2002 ]. however , no clear association between htlv env and htlv - associated diseases has been established and the identity of the receptor ( s ) for htlv - 1 and htlv - 2 env has remained elusive . numerous cell surface components have been shown to play a role in htlv env - mediated syncytia formation [ niyogi , 2001 ; daenke , 1999 ; hildreth , 1997 ]. nevertheless , htlv env - dependent cell membrane fusion and syncytia formation appear to be distinct from receptor binding per se [ denesvre , 1996 ; daenke , 2000 ; kim , 2000 ; kim , 2003 ]. the search for htlv env receptor has been hindered in part by its ubiquitous presence [ sutton , 1996 ; trejo , 2000 ; jassal , 2001 ; kim , 2003 ]. additionally , the induction of rampant syncytium formation in cell culture upon expression of htlv env [ hoshino , 1983 ; nagy , 1983 ] has prevented efficient and persistent env expression . based on our observation that the htlv env amino terminal domain shares striking structural and functional homology with that of murine leukemia viruses ( mlv ), we defined htlv env receptor - binding domain ( rbd ) and derived htlv env - based tools that overcome the problem of syncytia formation [ kim , 2000 ; kim , 2003 ]. we were thus able to follow specific interactions between the env rbd and a primary htlv receptor . using these tools , we have previously shown that the htlv receptor is expressed on the surface on t lymphocytes , the major htlv reservoir in vivo , only following t cell receptor activation [ manel , 2003 ]. here we describe striking metabolic alterations in cell cultures following expression of htlv envelopes as well as htlv receptor binding domains . these alterations are characterized by a defect in the acidification of the cell culture medium associated with a decreased lactate production and a decline in glucose consumption and uptake . these observations as well as the knowledge that env receptors for the related mlv and most of the gammaretrovirus belong to the family of multiple - membrane spanning transporters [ overbaugh , 2001 ] prompted us to test ubiquitous lactate and glucose transport - associated molecules as receptors for htlv env . we show that the ubiquitous glut - 1 glucose transporter , present in all vertebrates , is an essential and specific component of the receptor for htlv . moreover , interaction of glut - 1 with the entire htlv - 1 and htlv - 2 envelopes as well as the truncated htlv - 1 and htlv - 2 rbds alters glucose metabolism . cell proliferation in standard culture media is accompanied by acidification of the milieu that translates into a color change from red to yellow tones in the presence of the phenol - red ph indicator . upon transfection of either highly syncytial htlv - 1 and htlv - 2 envelopes , or a non - syncytial chimeric envelope that harbors the htlv - 1 rbd in a mlv env backbone ( h 183 fenv ), culture medium did not readily acidify , and harbored red tones for several days post - transfection ( fig1 a ). moreover , expression of truncated soluble htlv rbd proteins fused with either gfp , - ha , or - rfc tags also inhibited medium acidification . in contrast , no envelope construct that lacked htlv rbd , including different mlv group envelopes , feline , porcine , lentiviral and jaagsiekte retroviral envs , as well as vsv - g and ebola glycoproteins , had this effect . the lack of acidification associated with htlv - 1 or htlv - 2 env expression was not an indirect consequence of their syncytial activity , since ( i ) medium acidification was observed in cells expressing a syncytial amphotropic - mlv env ( a - mlv devoid of the r peptide ) ( fig1 a ) and ( ii ) medium acidification was blocked when htlv env was expressed in cells that are resistant to htlv - env mediated syncytia formation ( nih3t3 tk − cells )[ kim , 2003 ]. decrease of ph in cell culture is primarily due to extracellular accumulation of lactate [ warburg , 1956 ]. lactate is the major byproduct of anaerobic glycolysis in vitro and its excretion is mediated by an h +/ lactate symporter [ halestrap , 1999 ]. we monitored lactate content in culture supernatants following transfection of various retroviral envelopes and rbd . lactate accumulation was consistently 3 - fold lower in h 183 fenv - and htlv rbd - transfected cells than in control - or mlv env - transfected cells ( fig1 b ). this decrease in extracellular glucose and fructose accumulation after htlv rbd transfection was dna dose - dependent . moreover , we found that the decrease in lactate accumulation following transfection of htlv rbd was apparent as early as 4 hours after the addition of fresh media ( fig1 c ). to examine whether a direct relationship exists between binding of the htlv envelope receptor and diminished extracellular acidification and lactate accumulation , we attempted to generate htlv - 1 rbd ( h1 rbd ) mutants with impaired receptor binding capacities . to this end , mutations resulting in single alanine substitutions were introduced at two different positions in h1 rbd , d106 and y114 which are highly conserved among primate t - lymphotropic viruses . although both d106a and y114a rbd mutants were expressed and secreted as efficiently as the wild - type h1 rbd ( fig3 a ), they exhibited significantly reduced ( d106a ) or non detectable ( y114a ) binding to the htlv receptor as detected by facs analysis ( fig3 b ). moreover , perturbations in lactate metabolism correlated with binding to the htlv receptor : lactate accumulation was not reduced in cells expressing the non - binding y114a rbd mutant and was minimally reduced in cells harboring the d106 rbd ( fig3 c ). similar results were obtained with h2 rbd harboring the same allelic mutations . these data favor a direct association between lactate - related metabolic alterations and htlv env receptor binding . extracellular lactate accumulates in cell cultures following its transport across cellular membranes by the mct1 monocarboxylate transporter [ garcia , 1994 ]. because htlv and mlv share a common organization of the extracellular envelope [ kim , 2000 ] and the receptors for mlv env are multispanning metabolite transporters [ overbaugh , 2001 ], we assessed whether the htlv rbd bound to mct1 . moreover , similar to our previous data concerning expression of the htlv receptor on t cells [ manel , 2003 ], expression of mct1 chaperone cd147 [ kirk , 2000 ] increases during t cell activation [ kasinrerk , 1992 ]. however , separate and combined overexpression of mct1 and cd 147 did not result in increased h1 rbd binding , arguing against a role for these molecules as receptors for htlv env . in addition to a decrease in extracellular lactate accumulation , expression of the htlv rbd also led to decreased intracellular lactate content , indicative of metabolic alterations upstream of lactate transport . in cell cultures , lactate accumulation results from the degradation of glucose during anaerobic glycolysis . therefore , we assessed whether the decreased accumulation of lactate observed upon expression of htlv rbd was linked to glucose metabolism . we measured glucose consumption as normalized to cellular protein content . glucose consumption of cells expressing an htlv rbd within the context of the h 183 fenv entire envelope or the h1 red was significantly decreased as compared to control cells ( fig1 b ) and this defect was detectable as early as 8 hours post transfection . to determine if this decrease in glucose consumption corresponded to a decrease in glucose transport across cellular membrane , we measured 2 - deoxyglucose and fructose uptake in control cells and cells expressing htlv rbd ( fig1 c ). we observed that expression of either htlv - 1 or htlv - 2 rbd induced an approximately 4 - fold decrease in 2 - deoxyglucose uptake , while a - mlv rbd had only a minor effect . inhibitors of glucose uptake , cytochalasin b and phloterin , also inhibited glucose uptake . theses results were also true for 3 - o - methylglucose transport . fructose uptake in the same cells was not altered by the presence of htlv - 1 nor htlv - 2 rbd however a - mlv rbd induced a slight decreased . we next evaluated the effect of glucose deprivation on the availability of the htlv receptor in both adherent human 293 t cells and suspension jurkat t cells . after overnight culture of cells in the absence of glucose , binding of h1 rbd was consistently increased by 2 - fold in both cell types ( fig1 d ). this effect of glucose deprivation was specific to htlv as amphotropic mlv rbd ( a rbd ) binding was only marginally affected ( fig1 d ). this phenomenon is reminiscent of a general metabolite transport feedback loop , whereby transporter availability at the cell surface increases upon substrate starvation [ martineau , 1972 ]. a simple model whereby the htlv envelope inhibits glucose consumption via direct binding to a glucose transporter can explain the metabolic effects described above . upon evaluation of the different glucose transporter candidates , glut - 1 appears to be the only one encompassing all the known properties of the htlv receptor . indeed , glut - 1 expression is increased upon glucose deprivation and is transports glucose in all vertebrate cells [ mueckler , 1985 ], while fructose is transported by glut - 5 . furthermore , glut - 1 is not expressed on resting primary t cells and its expression is induced upon t cell activation [ rathmell , 2000 ; chakrabarti , 1994 ] with kinetics that are strikingly similar to what we have reported for the htlv receptor [ manel , 2003 ]. since human but not murine erythrocytes have been described to be the cells exhibiting the highest concentration of glut - 1 [ mueckler , 1994 ], we evaluated htlv receptor availability on freshly isolated red blood cells . binding of h1 rbd on human erythrocytes was strikingly efficient , reaching levels higher than those observed on any other tested cell type , whereas a rbd binding to erythrocytes was minimal ( fig2 a ). on murine erythrocytes however , no significant h1 rbd binding could be detected , despite a similar a rbd binding on murine and human erythrocytes . furthermore , primary human hepatocytes do not express glut - 1 . accordingly , we were unable to detecte h1 red binding to human primary hepatocytes , while a rbd binding could be readily detected . in order to directly test the ability of htlv envelopes to bind glut - 1 , we derived a tagged glut - 1 expression vector and overexpressed this protein in hela cells . both h1 rbd and h2 rbd binding was dramatically increased upon glut - 1 overexpression ( fig4 b ). this interaction was specific as the htlv - 2 binding - defective mutant , d102a , as well as its htlv - 1 counterpart , d106a , did not bind glut - 1 ( fig4 a ). furthermore , h1 rbd and h2 rbd binding remained at background levels upon overexpression of the amphotropic mlv envelope receptor , the inorganic phosphate transporter pit2 [ miller , 1994 ]. conversely , binding of a rbd was not increased after glut - 1 overexpression but as expected , this interaction was increased upon transfection of pit2 ( fig4 a ). glut - 3 is the closest isoform to glut - 1 , and transports glucose with kinetics similar to that of glut - 1 . thus , we derived a tagged glut - 3 expression vector . albeit similar overexpression levels of glut - 1 and glut - 3 in 293t cells , glut - 3 did not induce any increase in h1 rbd binding ( fig4 b ), suggesting that increase h1 red binding in cells overexpressing glut - 1 is not an indirect consequence of increased glucose uptake . to determine if glut - 1 transfected cells were directly responsible for the observed increased in h1 rbd binding , we derived fluorescent tagged glut - 1 and glut - 3 to unequivocally identify glut - overexpressing cells in the course of our facs analysis . in this context , only cells overexpressing glut - 1 - dsred2 displayed an significant increase in hi red binding , while overexpressing glut - 3 - dsred2 had no effect on h1 rbd binding . consequently , we tested if htlv glycoproteins directly interacts with glut - 1 proteins . to this end , we evaluated the ability of h1 rbd to immunoprecipitate glut - 1 . as shown n fig is in fig4 c , glut - 1 could be readily detected upon immunoprecipitation with anti - rabbit - fc - beads when it was co - expressed with h1 rbd , but could not be detected when expressed alone or with the h1 rbd y114a mutant . moreover , a gfp - tagged htlv - 2 rbd colocalized with glut - 1 but not with pit2 as assessed by fluorescence microscopy . therefore , the glut - 1 glucose transporter is an essential component of the htlv envelope receptor . interaction of glut - 1 with its ligand cytochalasin b inhibits glucose transport [ kasahara , 1977 ]. since we showed that binding of htlv envelopes to glut - 1 inhibits glucose consumption and uptake , we tested whether cytochalasin b would abrogate htlv rbd binding . indeed , cytochalasin b treatment of jurkat t cells dramatically inhibited binding of h1 rbd , whereas binding of a rbd was not affected ( fig5 a ). thus , glut - 1 directed glucose transport as well as binding of htlv envelopes to glut - 1 are similarly inhibited by the cytochalasin b ligand . altogether , these data demonstrate that glut - 1 is a receptor for htlv envelopes . viral receptor permits entry and thus infection . no cellular system currently exists that lacks glut - 1 expression . thus , we developed a system in which htlv infection is specifically inhibited at the level of envelope - receptor interaction . in this system , overexpression of htlv - 2 rbd interferes with infecting incoming htlv particles and specifically decreases htlv titers by at least 2 logs , while no effect is detected on control amlv titers . to determine if glut - 1 is an entry receptor for htlv , we overexpressed glut - 1 , glut - 3 or pit2 in addition to the interfering h2 rbd . while pit2 and glut - 3 had no effect on htlv titers , glut - 1 completely alleviated the interference to infection induced by h2 rbd ( fig5 ). interestingly , both glut - 1 and glut - 3 , but not pit 2 , alleviated the alteration of glucose metabolism induced by the htlv rbd . thus , glut - 1 is an entry receptor for htlv . here we show that htlv - 1 and - 2 envelopes interact with glut - 1 through their receptor binding domains . this interaction strongly inhibits glucose consumption and glucose uptake , leading to decreased lactate production and a block in extracellular milieu acidification . mutations that specifically altered receptor binding of both htlv - 1 and 2 envelopes released the block in glucose consumption , indicative of a direct correlation between receptor binding determinants in the htlv envelopes and glucose transport . glucose starvation was rapidly followed by increased binding of htlv envelopes , highlighting a nutrient - sensing negative feedback loop between glucose availability and cell surface htlv receptor expression . further evidence converged to identify glut - 1 as the receptor , including increased binding of htlv rbd upon overexpression of glut - 1 but not glut - 3 , immunoprecipitation of glut - 1 by h1 rbd but not the receptor - binding mutant h1 rbd y114a , uppermost binding of htlv rbd on human erythrocytes , where glut - 1 is the major glucose transporter isoform , and no binding of htlv rbd on human primary hepatocytes and murine erythrocytes , where glut - 1 is minimally expressed . finally , glut - 1 could specifically alleviate interference to infection induced by htlv rbd . glut - 1 fits all other known properties of the htlv receptor . indeed , as previously demonstrated for the htlv receptor [ manel , 2003 ], glut - 1 , but not the glut 2 - 4 isoforms , is not expressed on resting t lymphocytes [ chakrabarti , 1994 ; korgun , 2002 ] and is induced upon immunological [ frauwirth , 2002 ; yu , 2003 ] or pharmacological [ chakrabarti , 1994 ] activation . moreover , glut - 1 orthologues are highly conserved among vertebrates , but are highly divergent between vertebrates and insects [ escher , 1999 ]. glut - 1 is thus a new member of the multimembrane spanning metabolite transporters that serve as receptors for retroviral envelopes . interestingly , until now , all envelopes that recognize these receptors have been encoded by retroviruses that have a so - called simple genetic organization , such as mlv , feline leukemia viruses , porcine endogenous retrovirus and the gibbon ape leukemia virus [ overbaugh , 2001 ], whereas htlv belongs to the so - called complex retroviruses which code for several additional regulatory proteins . however , we have shown that in contrast to the wide phylogenetic divergence of their genomic rna , the envelopes of htlv and mlv share a similar modular organization with some highly conserved amino acid motifs in their respective receptor binding domains [ kim , 2000 ]. cell - to - cell contact appears to be required for htlv transmission , and the cytoskeleton appears to play a major role in this process [ igakura , 2003 ]. indeed , we observed that the htlv receptor , despite pancellular expression , is specifically concentrated to mobile membrane regions and cell - to - cell contact areas . it should therefore be expected that the htlv envelope receptor is associated to the cytoskeleton . importantly , a cytoplasmic - binding partner of glut - 1 , glut1cbp , which encodes a pdz domain , has been reported to link glut - 1 to the cytoskeleton [ bunn , 1999 ]. it will therefore be interesting to evaluate the respective roles of the htlv envelope , its cytoskeleton - associated cellular partners , such as glut - 1 , glut1cbp and their immediate interacting cell components . because expression of the htlv receptor is induced upon glucose starvation , transmission of htlv may be more efficient in cells that are locally starved for glucose , such as lymphocytes in lymph nodes [ yu , 2003 ]. furthermore , the ability of circulating erythrocytes to dock htlv , as shown here , might provide a means to distribute htlv to such tissues . the identification of glut - 1 as a receptor for htlv envelopes provides additional clues as to the ubiquitous in vitro expression of the receptor on cell lines and the paradoxical restriction of htlv tropism to t lymphocytes in vivo . rapid and dramatic metabolic alterations associated with the blockade of glucose consumption are likely to take place upon expression of the htlv envelope in vivo , early after infection . therefore , we propose that in vivo , htlv infection initially spreads with a large tropism , however early after infection the vast majority of cells that are highly dependent on glut - 1 activity are rapidly eliminated . in contrast , resting t lymphocytes that have an extremely low metabolic rate and as such are much less dependent on glucose uptake , can tolerate this effect and are therefore maintained in vivo . furthermore , local imbalances in the access to glucose following htlv infection may lead to specific physiological alterations [ akaoka , 2001 ]. in this regard , it will be of interest to study the potential relationship between htlv - associated neuropathologies and the specific dependence of neurons on glut - 1 mediated glucose consumption [ siegel , 1998 ]. cell culture . 293t human embryonic kidney and hela cervical carcinoma cells were grown in dulbecco &# 39 ; s modified eagle medium ( dmem ) with high glucose ( 4 . 5 g / l ) and jurkat t - cells were grown in rpmi supplemented with 10 % fetal bovine serum ( fbs ) at 37 ° c . in a 5 % co 2 - 95 % air atmosphere . for glucose starvation experiments , cells were grown in either glucose - free dmem ( life technologies ) or glucose - free rpmi —( dutscher ) with 10 % dialyzed fbs ( life technologies ) and glucose ( 1 g / l ) was supplemented when indicated . expression vectors . full length envelope expression vectors for htlv - 1 ( pcel / 2 [ denesvre , 1995 ]) and friend ecotropic mlv ( pcel / f [ denesvre , 1995 ]), have been previously described . for the htlv - 2 envelope , a fragment from phte2 [ rosenberg , 1998 ] encompassing the tax , rex and env genes and the 3 ′ ltr was inserted in the pcsi [ battini , 1999 ] vector ( pcsix . h2 ). full length envelope expression vectors for amphotropic mlv ( pcsi . a ), or devoid of its r peptide ( pcsi . aδr ), and h 183 fenv that contains the n - terminal 183 amino acids of the htlv - 1 receptor - binding domain in the f - mlv envelope background , as well as truncated envelope expression vectors , derived from pcsi and encoding either of the first 215 residues of htlv - 1 su ( h1 rbd ), the first 178 residues of htlv2 - su ( h2 rbd ) or the first 397 residues of the amphotropic murine leukemia virus ( mlv ) su ( a rbd ), fused to a c - terminal rabbit igg fc tag ( rfc ) or to egfp ( h2 rbd - gfp ). all point mutations introduced in htlv - 1 and - 2 rbd constructs were generated using the quickchange site - directed mutagenesis method and mutations were verified by sequencing . human glut - 1 and glut - 3 cdna were amplified by pcr from the plib hela cdna library ( clontech ), and inserted into pchix , a modified version of the pcsi vector that contains a cassette comprising a factor xa cleavage site , two copies of the hemagglutinin ( ha ) tag , and a histidine tag . the resulting construct ( pchix . hglut1 ) encodes a glut - 1 protein with a ha - his tag at the c - terminal end . glut - 1 and glut - 3 were also inserted in a modified pcsi vector containing a dsred2 c - terminal tag . similarly , human cd147 was amplified from 293t total rna by rt - pcr and inserted into the pchix backbone in frame with the ha - his tag ( pchix . hcd147 ). envelope expression and metabolic measurements . 293t cells were transfected with the various envelope expression vectors using a modified version of the calcium phosphate method . after an overnight transfection , cells were washed in phosphate - buffered saline ( pbs ) and fresh medium was added . media were harvested at the indicated time points , filtered through a 0 . 45 - μm pore - size filter , and lactate and glucose were measured with enzymatic diagnostic kits ( sigma ). values were normalized to cellular protein content using the bradford assay ( sigma ) after solubilization of cells in lysis buffer ( 50 mm tris - hcl ph 8 . 0 , 150 mm nacl , 0 . 1 % sodium dodecyl sulfate , 1 . 0 % nonidet p - 40 , 0 . 5 % deoxycholate ) and clarification by centrifugation . assay of hexose uptake . 2 - deoxy - d [ 1 - − 3 h ] glucose , d [ u - 14 c ] fructose and 3 - o -[ 14 c ] methyl - d - glucose were obtained from amersham . hexose uptake assay were adapted from harrison et al ( ref harrison 1991 ). after transfection , approximatively 250 , 000 were seeded / well in 24 - well plates . the next day , cells were washed two times in pbs , incubated in serum - free dmem , washed one time in serum - free glucose - free dmem , and incubated for 20 ′ in 500 μl serum - free glucose - free dmem modulo inhibitors ( 20 μm cytochalasin b , 300 μm phloretin ; sigma ). uptake was initiated by adding labeled hexoses to a final concentration of 0 . 1 mm ( 2 μci / ml for 2 - 2 - deoxy - d [ 1 - 3 h ] glucose and 0 . 2 μci / ml for d [ u - 14 c ] fructose and 3 - o -[ 14 c ] methyl - d - glucose ) and cells were incubated for 5 ′ additional minutes . cells were then resuspended in 500 μl cold serum - free glucose - free dmem , wash one time in serum - free glucose - free dmem , and solubilized in 400 μl of 0 . 1 % sds . 3 μl was used for bradford normalization , while the rest was used for detection of either 3 h or 14 c by liquid scintillation in a beckman counter . western blots . culture media ( 10 μl ) from 293t cells expressing wild type or mutant htlv - 1 rbds , and / or glut - 1 or glut - 3 expression vecotor . were subjected to electrophoresis on sds - 15 % acrylamide gels , transferred onto nitrocellulose ( protran ; schleicher & amp ; schuell ), blocked in pbs containing 5 % powdered milk and 0 . 5 % tween 20 , probed with either a 1 : 5000 dilution of horseradish peroxidase - conjugated anti - rabbit immunoglobulin or 1 : 2000 dilution of anti - ha 12ca5 ( roche ) monoclonal antibody followed by a 1 : 5000 dilution of horseradish peroxidase - conjugated anti - mouse immunoglobulin , and visualized using an enhanced chemiluminescence kit ( amersham ). binding assays . binding assays were carried out as previously described [ manel , 2003 ]. briefly , 5 × 10 5 cells ( 293t , hela , jurkat or freshly isolated human erythrocytes ) were incubated with 500 μl of h 1 rbd , h2 rbd or a rbd supernatants for 30 min at 37 ° c ., washed with pba ( 1 % bsa , 0 . 1 % sodium azide in pbs ), and incubated with a sheep anti - rabbit igg antibody conjugated to fluorescein isothiocyanate ( sigma ). when indicated , cytochalasin b ( 20 μm ; sigma ) was added to cells for 1 hour prior to binding analyses . binding was analyzed on a facscalibur ( becton dickinson ) and data analysis was performed using cellquest ( becton dickinson ) and winmdi ( scripps ) softwares . infections . 293t cells were transfected in 6 - wells plate , and one day after transfection , medium was replaced by high glucose dmem supplemented with fructose ( 5 g / l ) and non - essential amino acids . the next day , infection was initiated by adding supernatants containing mlv particles pseuodtyped with either htlv - 2 or a - mlv envelopes . the following day , fresh medium was added , and 24 hours later cells were fixed and stained for alkaline phosphatase activity and dark focus of infection were counted . viral particles were obtained by transfecting 293t cells with plapsn , pgagpoule and either pcsix . h2 or pcsi . a , and harvesting the 0 . 45 μm - filtered supernatants 24 hours latter . fig1 expression of the htlv receptor - binding domain alters cellular metabolism . a , medium acidification and syncytia formation in 293t cells one day post - transfection with control dna or env expression vectors , including syncytial wild - type htlv - 1 env and htlv - 2 env , a non - syncytial chimeric h 183 fenv , and syncytial a - mlv δr env . b , extracellular lactate and glucose in the culture medium of 293t cells were measured two days following transfection with an irrelevant dna ( control ), f - mlv env , h 183 fenv , htlv - 1 rbd ( h1 rbd ) or amphotropic mlv rbd ( a rbd ) expression vectors . lactate and glucose concentrations were normalized to cellular protein content . c , 2 - deoxyglucose and fructose uptake following transfection of 293t with an irrelevant dna ( control ), h1 rbd , h2 rbd or a rbd expression vectors . control cells were also incubated with glucose transporter inhibitors cytochalasin and phloretin . data are the means of triplicate measures and are representative of two to three independent experiments . d , expression of the htlv and amphotropic - mlv receptors on 293t ( 1 ) and jurkat t ( 2 ) cells cultured overnight in the presence or absence of glucose was monitored by binding of h1 rbd and a rbd , respectively . fig2 htlv receptor properties correlates with glut1 properties . a , expression of the htlv and amphotropic - mlv receptors at the surface of human and murine erythrocytes , as well as human primary hepatocytes . b , h1 rbd and a rbd binding to jurkat cells in the absence or presence of the glut - 1 inhibitor cytochalasin b . fig3 htlv receptor - binding correlates with altered lactate metabolism . a , expression of h1 rbd and the derived mutants d106a and y114a was monitored by western blot analysis of the supernatants of 293t cells following transfection with the various expression plasmids . b , binding of h1 rbd and the d106a and y114a mutants to the htlv receptor on hela cells . c , extracellular lactate in the medium of 293t cells one day post transfection with an irrelevant dna ( control ), h1 rbd or the h1 rbd d106a and y114a mutants . data are representative of three independent experiments . fig4 glut - 1 is a receptor for htlv envelopes . a , binding of h1 rbd , h2 rbd , h2 rbd d102a mutant , and a rbd to control 293t cells or 293t cells overexpressing either glut - 1 or pit2 . b , binding of h2 rbd - egfp to cells overexpressing glut - 1 - ha or glut - 3 - ha , and corresponding immuoblots using an anti - ha antibody . c , immunprecipitation of glut - 1 - ha from 293t cells transfected with either an irrelevant construct , glut - 1 alone , h1 rbd alone , h1 rbd y114a alone , glut - 1 with h1 rbd or glut - 1 with h1 rbd y114a expression vectors . immunoprecipitation was performed using anti - rabbit - fc beads and probed with an anti - ha antibody . total cell extracts were blotted using an anti - rabbit fc or an anti - ha antibody . fig5 glut - 1 is an entry receptor for htlv . infections titer of mlv particles pseudotypes with htlv - 2 or a - mlv envelopes on 293t cells following transfection of an irrelevant or interfering h2 rbd expression vectors alone or in addition to glut - 1 , glut - 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