12
Plenary and Slide l THE HTLV-I pl21 PROTEIN BINDS TO THE IMMATURE FORMS OF THE IL-2 RECEPTOR ß AND 7 CHAINS: POSSIBLE IMPLICATION IN IL-2R SIGNALING James C. Mullov1, E.Boeri1, R.C.Gallo1, W.J.Leonard2 and G.Franchini' Laboratory of Tumor Cell Biology, NCI, Bethesda, MD 20892 Laboratory of Cellular and Molecular Immunology, NIAID, Bethesda, MD 20892 The HTLV-I pl2' protein is a small hydrophobic protein that interacts with the 16 Kd component of the H* vacuolar ATPase and cooperates with the bovine papillomavirus (BPV)-l E5 oncoprotein in cell transformation. Since an important step in BPV-1 cell transformation appears to be the binding of the E5 protein to the platelet-derived growth factor (PDGF) receptor, we investigated whether the pl2' protein also interacts with cellular receptors, particularly those expressed in hematopoietic cells. Co- transfection experiments in HeLa/tat cells demonstrated that the pl2' protein binds to the ß and y chains of the IL-2R, but not to the IL-2R a or to the PDGF, EGF or EPO receptors. Mapping experiments using /1-chain deletion mutants and chimeric a-ß, a-y, and CD4-/Î receptors demonstrated that the pl2' protein binds mainly to the acidic domain of the IL-2R ß-chain, the same region that interacts with the T cell-associated protein tyrosine kinase, p56lck. Similarly, the cytoplasmic but not the extracellular portion of the 7 chain is involved in the interaction with pl2'. The ß and 7 chains associated with pl2' are endoglycosidase-H sensitive suggesting that the interactions among these proteins occur in the endoplasmic reticulum. Furthermore, in the presence of pl2' the endoglycosidase-H sensitive 7 chain appears to be stablized. It is possible that the pl2' protein intermolecular association with the IL-2R chains may be the underlying mechanism of ligand independence of HTLV-I transformed cells. 3 A PRIMATE T-LYMPHOTROPIC VIRUS, PTLV-L, DIFFERENT FROM HTLV-I AND HTLV-II IN A WILD CAUGHT BABOON P. Goubau. M. Van Brüssel, A-M. Vandamme, H-F. Liu, J. Desmyter. Rega Institute and University Hospitals, Katholieke Universiteit Leuven, Belgium. PBMC from a wild caught healthy baboon, with an HTLV-II-like antibody pattern in HTLV-I western blot, were cocultured with human cord lymphocytes. A human CD4+ cell line was obtained (PH969). With thin-section electron microscopy STLV-like virions are seen. The cells produce antigen which is recognised in indirect immunofluorescence by the baboon's own serum and by the serum of another baboon with similar antibody pattern, but the cell bound antigen reacts only weakly with HTLV-I and with African or American HTLV-II antibody positive sera. Antigen is detected in the supernatant fluid with a p24 HTLV-I/II antigen ELISA. The virus is transmissible by coculture of the mitomycin C-treated PH969 cells to fresh cocultured PBMC. Preliminary genomic sequence analysis indicates that the differences with the other known PTLVs are of the same order of magnitude as the differences between HTLV-I and HTLV-II and that this virus belongs to a new type, provisionally called PTLV-L. A second isolate of the same virus was obtained from a second independent bleed of the same baboon after coculture with PBMC from a seronegative baboon. The provirus was also detected in uncultured PBMC by PCR. The finding of a new PTLV type in an African baboon is further evidence of the wide variety of PTLV found on this continent. It remains to be seen whether PTLV-L resembles PTLV-I and PTLV-II in the extension of its host range to other primates, including man. Session Abstracts 2 HTLV-II INFECTION IN MONGOLIA W.W. Hall*, S.W. Zhu*, P. Horal", Y. Furuta", G. Zagaany", A. Vahlne0 *The Rockefeller University, New York, NY, USA "University of Goteburg, Goteburg, Sweden "Ministry of Health, Ulan-Bator, Mongolia Molecular analysis of mitochondrial DNA has suggested that the ancestors of present day native American Indians may have migrated in several waves from Asia tens of thousands of years ago. In this regard, it has been suggested that certain Mongolian populations and native Americans may have had a common ancestor. In view of the finding of endemic HTLV-II infection in native Americans, we have investigated whether HTLV-II infection also occur in Mongolia. Venous blood samples were collected from 96 patients admitted to two general hospitals; plasma samples were analyzed by Western Blot analysis and PBMC DNA by PCR amplification. Three samples were found to have patterns of reactivity suggestive of HTLV-II infection. PCR amplification confirmed this and nucleotide sequence analysis demonstrated that infection involved the HTLV-Ila subtype. The finding of HTLV-IIa infection in Mongolia does not support the view that HTLV-II be considered a New World Virus and may have important implications for our understanding of the origin of the virus. 4 ANALYSIS OF THE GENOMIC STRUCTURE OF A NEW TYPE OF PRIMATE T-LYMPHOTROPIC VIRUS : PTLV-L M. Van Brüssel. A-M. Vandamme, P. Goubau, J. Desmyter. Rega Institute and University Hospitals, Katholieke Universiteit Leuven, Belgium. A cell line (PH969) was obtained by cocultivation of human T-lymphocytes with blood from an African baboon whose serum had a PTLV-II type antibody pattern. We detected the presence of PTLV-like proviral DNA by nested PCR, with primers deduced from the very conserved taxlrex region of the PTLV genome. The PCR fragment was used as a probe to screen a cDNA library obtained from poly-A RNA of PH969 cells. Several positive clones were isolated and further analysed. One clone contained a 1802 bp cDNA fragment that extends from the env region, including the complete transmembrane protein, to part of the taxlrex gene. The overall nucleotide homology to HTLV-I and -IT (prototypes), was 62 % and 64 % respectively. Two homologous coding regions were identified. In the env region the homology was 65 % and 70 % to HTLV-I and -II respectively, and in the partial taxlrex region, 80 % to both HTLV-I and -II. In the 5' part of the pX region, a significant homology was only seen with HTLV-II (52 %). In this region, one additional open reading frame was identified. Phylogenetic analysis, based on the transmembrane protein coding region, indicates that this new virus represents a PTLV type with a long independent evolution from PTLV-I and -II. The high divergence of its nucleotide sequence and genomic structure, compared to PTLV-I and -II, justifies the distinction as a new PTLV type, provisionally designated here as PTLV-L. 443

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Page 1: Plenary and Slide Session Abstracts

Plenary and SlidelTHE HTLV-I pl21 PROTEIN BINDS TO THE IMMATURE FORMS OF THE IL-2RECEPTOR ß AND 7 CHAINS: POSSIBLE IMPLICATION IN IL-2R SIGNALING

James C. Mullov1, E.Boeri1, R.C.Gallo1, W.J.Leonard2 andG.Franchini'Laboratory of Tumor Cell Biology, NCI, Bethesda, MD 20892Laboratory of Cellular and Molecular Immunology, NIAID, Bethesda,

MD 20892

The HTLV-I pl2' protein is a small hydrophobic protein thatinteracts with the 16 Kd component of the H* vacuolar ATPase andcooperates with the bovine papillomavirus (BPV)-l E5 oncoproteinin cell transformation. Since an important step in BPV-1 celltransformation appears to be the binding of the E5 protein to theplatelet-derived growth factor (PDGF) receptor, we investigatedwhether the pl2' protein also interacts with cellular receptors,particularly those expressed in hematopoietic cells. Co-transfection experiments in HeLa/tat cells demonstrated that thepl2' protein binds to the ß and y chains of the IL-2R, but not tothe IL-2R a or to the PDGF, EGF or EPO receptors. Mappingexperiments using /1-chain deletion mutants and chimeric a-ß, a-y,and CD4-/Î receptors demonstrated that the pl2' protein binds mainlyto the acidic domain of the IL-2R ß-chain, the same region thatinteracts with the T cell-associated protein tyrosine kinase,p56lck. Similarly, the cytoplasmic but not the extracellular portionof the 7 chain is involved in the interaction with pl2'. The ß and7 chains associated with pl2' are endoglycosidase-H sensitivesuggesting that the interactions among these proteins occur in theendoplasmic reticulum. Furthermore, in the presence of pl2' theendoglycosidase-H sensitive 7 chain appears to be stablized. It ispossible that the pl2' protein intermolecular association with theIL-2R chains may be the underlying mechanism of ligand independenceof HTLV-I transformed cells.

3A PRIMATE T-LYMPHOTROPIC VIRUS, PTLV-L, DIFFERENT FROMHTLV-I AND HTLV-II IN A WILD CAUGHT BABOON

P. Goubau. M. Van Brüssel, A-M. Vandamme, H-F. Liu, J. Desmyter.Rega Institute and University Hospitals, Katholieke Universiteit Leuven,Belgium.PBMC from a wild caught healthy baboon, with an HTLV-II-like antibody patternin HTLV-I western blot, were cocultured with human cord lymphocytes. A humanCD4+ cell line was obtained (PH969). With thin-section electron microscopySTLV-like virions are seen. The cells produce antigen which is recognised inindirect immunofluorescence by the baboon's own serum and by the serum ofanother baboon with similar antibody pattern, but the cell bound antigen reactsonly weakly with HTLV-I and with African or American HTLV-II antibodypositive sera. Antigen is detected in the supernatant fluid with a p24 HTLV-I/IIantigen ELISA. The virus is transmissible by coculture of the mitomycin C-treatedPH969 cells to fresh cocultured PBMC. Preliminary genomic sequence analysisindicates that the differences with the other known PTLVs are of the same order ofmagnitude as the differences between HTLV-I and HTLV-II and that this virusbelongs to a new type, provisionally called PTLV-L. A second isolate of the samevirus was obtained from a second independent bleed of the same baboon aftercoculture with PBMC from a seronegative baboon. The provirus was also detectedin uncultured PBMC by PCR. The finding of a new PTLV type in an Africanbaboon is further evidence of the wide variety of PTLV found on this continent. Itremains to be seen whether PTLV-L resembles PTLV-I and PTLV-II in theextension of its host range to other primates, including man.

Session Abstracts2HTLV-II INFECTION IN MONGOLIA

W.W. Hall*, S.W. Zhu*, P. Horal", Y. Furuta", G.Zagaany", A. Vahlne0

*The Rockefeller University, New York, NY, USA"University of Goteburg, Goteburg, Sweden"Ministry of Health, Ulan-Bator, MongoliaMolecular analysis of mitochondrial DNA has suggestedthat the ancestors of present day native AmericanIndians may have migrated in several waves from Asiatens of thousands of years ago. In this regard, ithas been suggested that certain Mongolian populationsand native Americans may have had a common ancestor.In view of the finding of endemic HTLV-II infectionin native Americans, we have investigated whetherHTLV-II infection also occur in Mongolia. Venousblood samples were collected from 96 patientsadmitted to two general hospitals; plasma sampleswere analyzed by Western Blot analysis and PBMC DNAby PCR amplification. Three samples were found tohave patterns of reactivity suggestive of HTLV-IIinfection. PCR amplification confirmed this andnucleotide sequence analysis demonstrated thatinfection involved the HTLV-Ila subtype.

The finding of HTLV-IIa infection in Mongoliadoes not support the view that HTLV-II be considereda New World Virus and may have important implicationsfor our understanding of the origin of the virus.

4ANALYSIS OF THE GENOMIC STRUCTURE OF A NEW TYPE OFPRIMATE T-LYMPHOTROPIC VIRUS : PTLV-L

M. Van Brüssel. A-M. Vandamme, P. Goubau, J. Desmyter.Rega Institute and University Hospitals, Katholieke Universiteit Leuven,Belgium.A cell line (PH969) was obtained by cocultivation of human T-lymphocytes withblood from an African baboon whose serum had a PTLV-II type antibody pattern.We detected the presence of PTLV-like proviral DNA by nested PCR, withprimers deduced from the very conserved taxlrex region of the PTLV genome.The PCR fragment was used as a probe to screen a cDNA library obtained frompoly-A RNA of PH969 cells. Several positive clones were isolated and furtheranalysed. One clone contained a 1802 bp cDNA fragment that extends from theenv region, including the complete transmembrane protein, to part of the taxlrexgene. The overall nucleotide homology to HTLV-I and -IT (prototypes), was 62 %and 64 % respectively. Two homologous coding regions were identified. In theenv region the homology was 65 % and 70 % to HTLV-I and -II respectively, andin the partial taxlrex region, 80 % to both HTLV-I and -II. In the 5' part of the pXregion, a significant homology was only seen with HTLV-II (52 %). In thisregion, one additional open reading frame was identified. Phylogenetic analysis,based on the transmembrane protein coding region, indicates that this new virusrepresents a PTLV type with a long independent evolution from PTLV-I and -II.The high divergence of its nucleotide sequence and genomic structure, comparedto PTLV-I and -II, justifies the distinction as a new PTLV type, provisionallydesignated here as PTLV-L.

443

Page 2: Plenary and Slide Session Abstracts

5 6IMPACT OF HTLV-II INFECTION UPON CELLULAR IMMUNOLOGICRESPONSE TO HIV EXPOSURE IN HIV SERONEGATIVE DRUG USERS

S.H. Weiss. M. Clerici, R.K. Mayur, T.N. Denny, J.E.Quirinale, J.A. Berzofsky, G.M. Shearer.UMDNJ-New Jersey Medical School, Newark NJ; NationalCancer Institute, Bethesda MD. U.S.A.

Cell mediated immune (CMI) responses to HIV in long-termHIV seronegative (HIV-) injection drug users (IDU) froma 9-yr, high risk cohort study were assessed by IL-2production to influenza virus, alio, PHA, the non-immunogenic HIV peptide P2 3, and 5 HIV env peptides (Tl,T2, T4, P18IIIB< p18hn) • Spontaneous lymphocyteproliferation occurred in half of the HTLV-II positiveIDU. 65 non-IDU HIV- healthy controls (HC) and 47 HIV-IDU responded to influenza, alio and PHA. A positiveCMI response to HIV env constituted > 2 responses to the5 HIV env peptides. 2/65 (3.1%) HC vs. 26/47 (55.3%)IDU responded to HIV env (p<.001). The response ratewas significantly higher among HTLV-II infected IDU(7/7, 100%) vs. HTLV-II negative IDU (19/40, 47.5%),p=.01. These long-term HIV- subjects with CMI to HIVmay represent "immunologie successes" among IDU who wereexposed to HIV, and stand in contrast to those IDU whodevelop HIV antibody (with the known risk of progressiveimmunodeficiency). HTLV-II infection may modify aperson's immunologie response to HIV exposure(s) andperhaps to other infectious agents as well. HTLV-IIinfected IDU should be selectively included in HIVvaccine efficacy trials. The effect of HTLV-II on humanimmunity merits further study.

7LIMITED T CELL RECEPTOR Va AND Vß CHAIN USAGE IN HTLV-ISPECIFIC CYTOTOXIC T LYMPHOCYTES IN HAM/TSP PATIENTS

S. Jacobson. U. Utz, S. Smith, I. ElovaaraNeuroimmunology Branch, National Institutes of Health, USA

In HAM/TSP, evidence has accumulated to support the hypothesis that within theCNS, proliferating HTLV-I specific T cells may be associated with thepathogenesis of this disorder. This includes demonstration of HTLV-I specificCD8+ CTL directly from lymphocytes obtained from the CSF, a high pCTLfrequencies of these cells in the CSF and peripheral blood, inflammatory CD8+cells in HAM/TSP spinal cord lesions, and HTLV-I genomic sequences as well asmRNA expression in CNS material. Recently, we have demonstrated that thepredominant CD8+ CTL response from these patients was directed toimmunodominant tax peptides of HTLV-I and restricted to particular HLA class Ialíeles. If the inflammatory CD8+ cells that predominate in HAM/TSP lesions arereflective of the CD8+ HTLV-I specific CTL that have been demonstrated in PBLand CSF of HAM/TSP patients, then analysis of the T cell repertoire of thesevirus-specific functional T cells would be important. Here we describe PCR-based analysis of T cell receptor Va and Vß chain gene expression of CD8+cloned lymphocytes from the peripheral blood of patients with HAM/TSP. RNAwas isolated from either HLA A2 or HLA-B14 restricted CTL specific for thenanopeptides LLFGYPVY or VPYKRIEEL, respectively. cDNA was analyzedby PCR amplification using Va and Vß chain family specific oligonucleotideprimers. The results indicate that CD8+ cytotoxic T cell lines from HLA-A2HAM/TSP patients express a limited repertoire of T cell receptor chains.Importantly, there were clear differences in the T cell receptor repertoire betweenCTL lines obtained from patients with different severity and duration of disease.These observations suggest that restricted functional HTLV-I specific T cell linesmay be clinically significant in the development of HAM/TSP and offersstrategies for immunotherapeutic intervention.

ANALYSIS OF THE INFILTRATING CELLS OF HTLV-1 UVEITIS

T. Watanabe1, A. Ono12, M. Mochizuki2, K. Yamaguchi3, S. Araki2, N.Miyata". 1, Inst Med Sei, Univ Tokyo, 2, Kurume Univ Med Sch, 3,Kumamoto Univ Med Sch, 4, Miyata Eye Hospital, Japan

The infiltrating cells in the anterior chamber of HTLV-1 uveitis werecharacterized morphologically, immunocytochemically, immunologicallyand virologically. They were predominantly lymphocytes (91.8-95.0%)and no neutrophils or monocytes were found in all the 5 patientsexamined. T cells constituted the majority (60.8-92.3%). CD4-positivecells were mostly less than half of total cells. In 36 of 38 HTLV-1 uveitispatients (94.7%), HTLV-1 provirus were demonstrated by PCR in theinfiltrating cells. Population of the infected cells was compared bydetecting HTLV-1 provirus in samples simultaneously collected fromperpheral blood and aqueous humor. In 3 of 4 patients studied, virusgenome was detected only in the infiltrating cells, suggestingaccumulation of the virus infected cells in the affected eye. Expression ofviral genes (env and/or pX) in the infiltrating cells was shown by RT-PCRin all the 7 patients studied. On the other hand, it was detected in 4 of 11fresh PBMC samples. As for cytokine gene expression, IL-6 was detectedby RT-PCR in 7 out of 12 patients (58.3%). However, other cytokines suchas IL-1aand IFNy were not detected except for the latter in one case.These results suggest that HTLV-1 infected T cells may play a major rolein the pathogenesis of uveitis.

8Regional specificity of HTLV-I proviral integration inthe human genome S. Zoubak*,b, J.H. Richardson0, Äj_Rvnditch*'b. P. Höllsbergd, D.A. Hafler0^, E. Boeri",A.M.L. Lever0 and G. Bernardi

a. Instituí Jacques Monod, Paris, France, b. Institute of Molecular Biology and Genetics, Kiev, Ukraine,University of Cambridge, U.K. and Harvard Medical School, Boston, MA, USA.The location of HTLV-I (human T-cell leukemia virustype 1) proviral sequences in the genome of infectedhuman cells was explored by hybridization of a viralprobe with compositional fractions of host-cell DNAs.In the twelve cases examined, HTLV-I secjuences wereabsent from the GC-poorest 40% of the host genome(namely, from isochores that are below 39% GC).Transcriptionally inactive proviral secjuences werelocalized in GC-poor isochores (comprised between 39%and 42-44% GC) of the human genome, which arecharacterized by a constant and low geneconcentration. In contrast, transcriptionally activeproviral sequences were found in the GC-rich and veryGC-rich isochores, which are gene rich,transcriptionally and recombinationally active, andendowed with an open chromatin structure. Since GC-rich isochores are present in R'-bands and very GC-rich isochores form T-bands, these results alsoprovide information on the location of HtLV-1 proviralsequences in human chromosomes. The results obtainedwith HTLV-1 are in agreement with the non-random,compartmentalized integration of animal retroviralsequences that had been previously observed in otherviral-host systems. They provide, however, much moredetailed information on the regional location ofproviral sequences in the host genome and on thecorrelation between their transcription and theirlocation.

<M4

Page 3: Plenary and Slide Session Abstracts

9RELATIONSHIP OF HLA TO HTLV-I SEROCONVERSION

A. Manns. D. Mann, R. Wilks, B. Hanchard, W. BlattnerNational Cancer Institute, Bethesda, MD and the University ofthe West Indies, Kingston, Jamaica

To evaluate whether genetic factors contribute to susceptibilityto HTLV-I infection following virus exposure, serological typingof human leukocyte antigen (HLA) was conducted for a Jamaicanpopulation of transfusion recipients (TR). All available samplesfrom transfusion recipients enrolled in a transfusion transmissionstudy (N=109) consisting of HTLV-I+ seroconverters (SC), HTLV-I-exposed nonconverters (NC) and unexposed, HTLV- controls (CO) wereevaluated. Class I HLA typing was available for SC (96%), NC (95%),and CO (88%) from the original cohort. HLA frequencies werecalculated and comparisons were made with Chi2 Yates continuitycorrection or two-tailed, Fisher's exact test (*P<0.05). Among TR,important HLA associations were as follows:HLA SC (23) HTLV- TR (86) NC (40) COÍ46)B8% 9* 0* 0 0B51% 22* 8 13 4*Cw3% 0* 17* 23* 13Cw4% 44* 26 18* 33HTLV-I seroconversion was associated with increased HLA Cw4 andabsence of HLA Cw3, resulting in enhanced susceptibility toinfection. These data suggest that risk for HTLV-I infection is insome instances dependent on host immunogenetic factors. Furtherevaluation of this cohort with Class II and PCR typing iswarranted.

11INDUCTION OF ANERGY IN TAX-REACTIVE CD8 T CELL CLONES.P HollshergU. J.H. Richardson». W. Weber'.3. F. Dangond'-3. andDAHafler13.'Brigham and Women's Hospital and 2Dana Färber Cancer Institute, 3HarvardMedical School, Boston, MA 02115.

It has been shown that presentation of peptides by MHC class I has thepotential of inducing T cell tolerance. However, the consequence of T cellpresentation of viral peptides are unknown since T cells are normally not the hostof human viruses. To study whether anergy can be induced by presentation ofHTLV-I tax peptides by immune cells to cytotoxic CD8 T cells, we have clonedCD8 T cells specific for the tax peptide 11-19 from an HLA-A2+ subject withHTLV-I associated myelopathy. Autologous EBV transformed B cells pulsedwith taxi 1-19 as well as tax transfected HLA-A2+ CD4 T cells were able toinduce proliferation in CD8 T cells specific for the taxi 1-19 peptide. However,when taxi 1-19 stimulated CD8 T cells were restimulated 6 days later with thesame antigen, ffiey were unresponsive (anergic). In contrast, CD8 T cells thatwere mock treated in the primary stimulation were responsive to secondarypresentation of taxi 1-19. The anergic taxi 1-19 stimulated CD8 T cells wereequally responsive to rIL-2 as secondary stimuli as unstrimulated CD8 T cells.Lack of antigen responsiveness in the CD8 T cells was not due to down-regulation of the T cell receptor. Thus, presentation ofHTLV-I encoded peptidesto viral specific CD8 T cells may result in proliferative anergy to subsequentrestimulation.

10GENETIC IMMUNIZATION AGAINST HTLV-I

]'2K.E. Ugen ,1»3 M.G. Agadjanyan, 1>2B. Wang, 1-2M. Mcrva,]*2W.V. Williamsand ^D. B. Weiner.1 University of Pennsylvania, ^Institute of Biotechnology and AdvancedMolecular Medicine, Phila, PA, and •institute of Viral Preparation, Moscow ,

Russia

Recently, we have demonstrated cellular and humoral immune responses againstthe gpl60 envelope precursor protein of HIV-1 after intramuscular inoculation(genetic immunization) of mice with a plasmid containing DNA for this protein.

Investigations have been initiated on the intramuscular inoculation of aplasmid containing DNA encoding the HTLV-I envelope glycoprotein. HTLV-I,a human retrovirus associated with adult T-cell leukemia (ATL), is endemic inseveral areas of the world. Since rabbits and rats can be productively infectedwith HTLV-I, this system can test the ability of genetic inoculation to produce invivo protective immune responses against a human retrovirus by prevention ofinfection after virus challenge. Rabbits were injected intramuscularly with 0.5$bupivicaine-HCL 24 hours before intramuscular inoculation with a DNAconstruct encoding the envelope glycoprotein of HTLV-1 (100 |ug / animal).Subsequent expression of the construct was analyzed. Rabbits demonstrated,after genetic inoculation, immune responses against HTLV-I recombinantproteins / peptides representing immunogenic regions of the envelopeglycoproteins. The immunity induced after DNA construct inoculaion appearedto be functional since two thirds of rabbit sera samples demonstrated anii-syncytial activity (at a 1:30-1:40 dilution). In addition, several of the geneticallyinoculated rabbits demonstrated T cell proliferative activity. We have recentlyexpanded our studies to include inbred rat strains which will allow assays ofpotential cytotoxic T cell activity elicited by this novel immunization technique.These studies provide a useful model for testing the utility of genetic inoculationfor the production of protective immune responses and provides a potentiallynovel vaccination strategy against HTLV-I infection.

12DETECTION OF HTLV-I TAX IN THE CNS FROM HAM/TSPPATIENTS BY IN SITU HYBRIDIZATION

T.J. Lehky. C. H. Fox, S. Koenig, S. Izumo, E. Sato,C. S. Raine, M. Osame, S. Jacobson

Neuroimmunology Branch, NIH, Bethesda, MD USA

The predominant pathological finding in HAM/TSP is atrophy of thethoracic spinal cord with axonal degeneration and demyelination andperivascular infiltrates throughout the CNS. Despite these findings andthe observed presence of HTLV-I in the CNS by PCR, it has beendifficult to visualize the virus in neural cells of HAM/TSP specimens.Autopsy specimens from three patients with HAM/TSP were examinedfor the presence of HTLV-I by in situ hybridization. HTLV-I taxmRNA transcripts were observed in isolated cells within the whitematter of the spinal cord and cerebellum of these HAM/TSP pathologicalspecimens. In the spinal cord, these cells were observed in the anteriorand lateral funiculus. In the cerebellum, they were observed within thedeep white matter. Some of the positive cells were observed near theperivascular infiltrates, but not specifically within the perivascularinfiltrates or inflammed meninges. On average, only two to threepositive cells were seen per tissue section signifying the rareness ofthese events. These observations provide evidence for the in situpresence of HTLV-I expression in neural cells of HAM/TSP patients.

Page 4: Plenary and Slide Session Abstracts

13INTERACTION OF HTLV-I TAX, WITH CELLULAR PROTEINSR. Reid, A. Mireskandari and J. N. Bradv

Laboratory of Molecular Virology, National Cancer Institute, NationalInstitutes of Health, Bethesda, MD 20892

The HTLV-I Tax, protein plays a critical role in viral transformationand transcriptional regulation. We have been interested in identifyingtranscription factors and cellular regulatory proteins with which Tax,specifically interacts. Jurkat T lymphocyte cDNA libraries have beenprobed with purified HTLV-I Tax, protein. We have isolated a

partial length cDNA clone for a cellular protein, TRX, that interactswith Tax,. The TRX cDNA insert is 808 base pairs in length andencodes an open reading frame of 167 amino acids. Western blotanalysis of MT4, C81 and Jurkat cell extracts with TRX anti-peptidesera showed specific reaction with a 28 kD protein. The expressionof TRX mRNA was analyzed using a multiple tissue northern blot.The predominant transcript migrated at 3.5 kB in all tissues tested.Minor RNA species of 9.5, 2.3 and 1.6 kB were also detected.Northern blot analysis further demonstrated that TRX mRNA was

expressed at similar levels in HTLV-I transformed cell lines as well as

uninfected Jurkat T lymphocytes. DNA and amino acid sequenceanalysis in GenBank demonstrated that TRX was a novel eukaryoticgene product. The possible functions of TRX in HTLV-I Tax,transcription regulation and transformation will be discussed. In vitroexperiments which analyze the molecular mechanism of Tax,transactivation through interaction with transcription factors will alsobe discussed.

15MUTATION IN TAX OF HTLV-I IS ASSOCIATED WITH TSP/HAM

B. Reniifo and M. EssexDepartment of Cancer Biology, Harvard School of Public Health.Boston. USA

Human infection by the HTLV-I retrovirus is associated with at leasttwo types of diseases, Adult T-cell Leukemia/lymphoma (ATL) andthe neurological disease Tropical Spastic Paraparesis/HTLV-IAssociated Myelopathy (TSP/HAM).Initial attempts to describe genomic variants of HTLV-I associatedwith these diseases have been unsuccessful. We studied HTLV-Iisolates from HTLV-I asymptomatic carriers and TSP patients fromTumaco (Colombia), where the rate of TSP cases is one of thehighest in the world. Also included in this study were ATL and TSPpatients, as well as HTLV-I asymptomatic carriers from Japan.Peripheral blood lymphocyte genomic DNA was amplified usingprimers for U3, env, rex and tax genes of HTLV-I. A comparison of theobtained sequences with published sequences revealed thatmutations in U3, env, rex and tax tended to segregate according togeographical origin. However a mutation in the tax gene wasstatistically associated with TSP despite the geographical origin ofthe sample. Sequences and statistical analysis of this study will bepresented.

14

ABNORMAL EXPRESSION OF L-SELECTIN IN ATL CELLSAND TRANSACTIVATION OF ITS GENE EXPRESSION BYHTLV-1 TAX.

M. Tatewaki1, K. Yamaguchi2, S. Mori', T. Watanabe'.1, The Institute of Medical Science, The University of Tokyo, Tokyo,Japan, 2, Kumamoto University Medical School, Kumamoto, Japan.Massive infiltration of mononuclear cells in the organs is one of thecommon clinico-pathological features of HTLV-1 associated diseasesand is speculated to be relevant to the pathogenesis of TSP/HAM andHU. L-selectin is an adhesion molecule expressed on leukocytes andmediates their rolling on vascular endothelium, which is important in theinitial step of extravasation. In normal lymphocytes the expression isdown-regulated upon stimulation, while ATL cells express it along withtypical activation antigens. Therefore we studied its expression andregulation in ATL cells. Here we report that L-selectin is constitutivelyover-expressed by ATL cells and its expression is aberrantly up-regulated by stimulation with PHA and PMA. We also found that thepromotor of L-selectin gene, which we first identified by "Oligo-capmethod", was transactivated by HTLV-1 Tax.These results suggest thattransactivation by Tax may lead to its over-expression in ATL cells andthat this may be one of the basis for tissue infiltration of lymphocytescommonly observed in HTLV-1 associated diseases.

16ANTIBODIES TO HTLV-I IN GINGIVAL CREVICULAR FLUID OF TSP ANDSEROPOSITIVE INDIVIDUALS FROM TUMACO, COLOMBIA.L.E. Soto-Ramirez.'') F. Garcia,'") A. Vergara,<2> I. Borrero,«2) R. Marlink,<»and M.Essex."'"»Department of Cancer Biology, Harvard School of Public Health, Boston MA.U.S.A. <2>MolecularBiology Laboratory, Universidad del Valle, Cali, Colombia.In order to asses the value of saliva samples for the diagnosis of HTLV-I infectionand to evaluate the oral antibody response against this virus, the presence ofHTLV-I specific antibodies (Abs), IgG, IgA and secretory IgA(sIgA), in gingivalcrevicular fluid(GCF) of patients with Tropical Spastic Paraparesis (TSP) andhealthy seropositive (HS) individuals from Tumaco, Colombia, an area of highratio of TSP cases per HTLV-I infected individuals(l:30, Trujillo et. al. 1992), wasevaluated with ELISA. Using a commercial device (OraSure, Epitope, Inc.) thatcollects mainly GCF(a serum transudate)but also saliva, we obtained GCF from 79individuals, 33 with TSP, 22 HS and 24 seronegatives. In 62 of those individuals(30 TSP, 11 HS, 20 seronegatives) a serum sample was also obtained, and was

evaluated with ELISA for IgG and IgA. The results for GCF were as follows:IgG(%) IgA(%) sIgA(%)

TSP(33) 33(100) 27(82) 15(45)HS(22) 19(86) 4(18) 4(18)Seronegatives(24) 0(0) 0(0) 0(0)pfTSPvsHS) 0.5 0.000003 0.03The presence of slgA correlated in 15 TSP and 4 HS with IgA detection. In 11TSP, only IgA was detected. When IgG and IgA results were compared with thosefrom serum samples, the correlation was 100% for IgG in both groups. In 3 cases,two with TSP and one HS, IgA was present in saliva but not in serum; all 3 hadhigh levels of slgA. In two HS individuals with no serum sample, IgG in GCF was

negative. In spite of the small numbers, ELISA detection of IgG in GCF from TSPpatients can be used as a diagnostic tool. The fact that IgA and slgA are detectedmore frequently when TSP is already developed, may suggest high viralreplication; in that case slgA may protect against viral transmission. The infectivityof saliva and the neutralizing capacity of these Abs should be determined.

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17 18HTLV-I ASSOCIATED TSP IN A COMMUNITY IN JAMAICA

A. Pan eau, S. Richards, P. Rodgers-Johnson, B. Cranston, O.Morgan, G. RománNeuroepidemiology Branch, NINDS/NIH, Bethesda, Maryland and TheUniversity of the West Indies, Mona Kingston, Jamaica

A field survey, conducted primarily in remote mountainous areasof St. Catherine Parish from July-October 1993, identified 385persons with difficulty walking. Subsequent neurological exam-inations were performed in their homes. Criteria for a fielddiagnosis of TSP were a slowly progressive myelopathy withobjective weakness and spasticity of one or both lower ex-tremities, increased reflexes in the legs, at least minimal signsof involvement of the posterior columns, with or without bladderdysfunction, constipation, cerebellar impairment or impotence.Possible TSP: at least one motor component often accompanied bydiminished vibratory sensation, lower extremity paresthesias,bowel, bladder or sexual dysfunction. Not TSP: clear signs,symptoms or history of other causes including other myelopathiesor trauma observed or documented.Of 350 serum samples tested by ELISA, 52 were HTLV-I+ (14.9%).Thirteen of 32 (40.6%) persons with a field diagnosis of TSP and20/80 (25%) diagnosed Possible TSP were HTLV-I+. In the Not TSPgroup, 14/201 (7.0%) were seropositive.Our study found a clear association of seropositivity withneurological symptoms. Some of the HTLV-I+/Possible TSP groupmay represent a forme fruste or early stages of the disorder.Preliminary results of this field survey indicate a higherprevalence than previously estimated by passive surveillancetechniques. Because of public health issues, field surveillanceinvolving a realistic estimate of prevalence is desirable. Oneimplication of this is it would be reasonable to monitor theblood supply for HTLV-I in Jamaica and in countries receivingimmigrants from the Caribbean and other tropical areas.

19EXCESS MORTALITY AMONG PERSONS INFECTED WITH HUMANT-CELL LYMPHOTROPIC VIRUS TYPE II (HTLV-II)S.H. Weiss. R.K. Mayur, J.E. Quirinale, J. French,R. Goode, W.A. BlattnerUMDNJ-New Jersey Medical School, Newark NJ; NJDepartment of Health, Trenton NJ; National CancerInstitute, Bethesda MD. U.S.A.

The causes of mortality were assessed in a cohort of 573HIV seronegative injecting drug users (IDU) originallyenrolled in 1984 from drug treatment programs. KnownHIV seroconverters were excluded. The total mortalitywas significantly associated in logistic regressionanalysis with HTLV-II seropositivity (OR=2.62, p=0.03),older age (OR=1.07, p=0.02), intermittent injection druguse (OR=2.60, p=0.03), denial of heroin use in the prior5 years (OR=2.20, p=0.04) and cough (OR=3.03, p=0.02).The mortality attributable to medical causes wasassociated only with HTLV-II seropositivity (OR=3.5,p=0.02) and older age (0R=1.11, p=0.002), and thesefindings were confirmed by lifetable and Coxproportional hazards analyses. All deaths due to asthmaor adenocarcinoma of the lung occurred in HTLV-IIseropositive subjects. Respiratory causes of deathcompared to other medical causes of death were morefrequent in the HTLV-II positive IDU (0R=5.5, p=.15).In summary, infection with the orphan virus HTLV-II isassociated with increased medical mortality. ImprovedHTLV-II screening procedures for the blood supply may bewarranted. Guidelines for HTLV-II counselling should bereassessed periodically. The possible etiologic role ofHTLV-II in select respiratory diseases and the effect ofHTLV-II on the immune system merit further study.

INFECTIVE DERMATITIS IN TRINIDAD AND TOBAGO

M. Suite, N. Jack. K. Basdeo-Maharaj, J. Edwards, F. White, W. Blattner,C. Bartholomew.UWI, CAREC, General Hospital, Port-of-Spain, Trinidad and DCE/NCI/NIH,USA.

In 1990, La Grenade et al demonstrated a strong association between infectivedermatitis and HTLV-1 infection in 11 Jamaican children. We now report 15cases of infective dermatitis identified at the dermatology clinics in Trinidad andTobago between January 1991 and July 1993. All subjects were of African descentbetween ages 2 and 36 (mean=11.7), and all were HTLV-1 antibody positive.Chronic relapsing eczema was associated with positive cultures for B-haemolyticstrept. and/or staph. aurais infection. The repeated use of oral antibiotics was

required for resolution of the skin condition in 12 of the cases. Consistently absentwas the chronic nasal discharge described by La Grenade, and unlike the Jamaicanpatients, 33.3% of our patients had eczema of the antecubital flexures. 80.0% hadlymphocytosis and ATL cells were seen on blood films in 46.7% of cases. Inaddition, we report for the first time, the occurrence of familial infective dermatitisamong three siblings of one family, and two cases with facial nerve palsy at ages2 and 5. La Grenade only studied 2 mothers of the 11 cases; both were HTLV-1antibody positive. Twelve of 15 (80.0%) mothers in our study were HTLV-1antibody positive. Of the three cases having HTLV-1 seronegative mothers, onewas age 13 (currently 24) at disease onset and denies sexual activity before 17years. The second was 5 years (currently 11) at onset of disease, with a brotherwho was also HTLV-1 positive. The third case acquired infective dermatitis as anadult (35 years). She received 4 blood transfusions in the previous 10 years. Thissuggests that infective dermatitis can result from infection other than by mother tochild transmission.

20A Search for Diseases Associated with HTLV-II Among GuaymiPatients, Panama.R.M.Giusti*,F.Gracia**,K.Stephens***,K.Fukuda****,C.Vitek****,P.Hartge*,A.Levin***,W.Blattner*,P.Levine*J.Kaplan****--*Viral Epidemiology Branch, NCI,**Gorgas Memorial Laboratory,Panama,***Research Triangle Institute,****Retrovirus DiseasesBranch, CDC

Objective: To investigate i 1Iness associated with HTLV-IIinfection in a population with high HTLV-II seroprevalence focusingon hématologie malignancies, neurologic, skin and joint diseases.Methods: Sera were obtained from 1,134 Guaymi Indians seen at theChanguinola Hospital internal medicine, dermatology, psychiatry,and TB clinics, and inpatlent medicine and pediatrics services,Dec.1990

-

Sept.1993 and were screened for antibodies to HTLV-I/IIby Dupont ELISA and Cambridge Bioscience Envelope Assay. Reactivesamples were confirmed by p2IE-enhanced Western Blot (WB).Demographic data, history of surgery and transfusion, and primaryand secondary diagnoses were obtained through patient interview andmedical record review. Age-sex-specific rates derived from a

population seroservey of 3, 629 Changuinola Guaymi were used tocalculate expected seroprevalence in the hospital groups.Results: Overall, 9.3% (106/1,134) of patients were seropositive.This did not differ from the expected rate of 9.7%. Of 16 pat lentswith asthma, more were seropositive than expected (0/E=6/2.3,p=.03). Of 31 patients with acute glomerulonephrit is (AGM) morewere seropositive than expected (0/8*4/1*5) but this was notsignificant (p=.07). None of 4 patients with hématologiemalignancies were seropositive. No patients with TSP/HAM wereseen. Patients with skin and musculoskeletal diseases were no more1 ike1y than expected to be seropositive. More pédiatrie inpatients(N=186) were seropositive than expected (O/E=10/5.0, p=.03),however, record review of pédiatrie seropositives revealed no

specific disease correlates.Conclusion: This is the f irst report of asthma associated withHTLV-II. No significant association was found between HTLV-II andmusculoskeletal or skin diseases. Additional surveillance would bereguired to rule out HTLV-II association with rare disease such asATL or TSP/HAM. Clinical and laboratory studies are needed toassess the potential role of HTLV-II in asthma and AGN.

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21 22RISK FACTORS FOR HTLV-II SEROCONVERSION AMONGINJECTING DRUG USERS IN BALTIMORED. Vlahov. R. Khabbaz, S. Cohn, N. Galai, E. Taylor, J. Kaplan. The JohnsHopkins Univ., Baltimore, MD; Centers for Disease Control, Atlanta, GA.OBJECTIVE; To estimate incidence and risk factors for HTLV-IIseroconversion (SC) among injecting drug users (IDUs).METHODS: IDUs recruited into the ALIVE Study in 1988/1989 were

assayed at baseline for antibody to HTLV using ELISA and Western blot(Cambridge Bioscience). Participants were followed semiannually withvenipuncture and interviews. In 1992, the most recent sera of HTLV-negative participants were tested for HTLV using ELISA (Cambridge Bio-science) and confirmed and typed by Western blot (DBL 2.3). Assays werethen performed for all intervening visits to determine interval of SC.Incidence rates were estimated using person-time techniques. Risk factoranalysis used a nested case-control design with up to 5 controls per casematched by time of study entry and duration of follow-up.RESULTS: At baseline, we identified 256 HTLV positive, 17 indeterminantand 2574 HTLV seronegative IDUs. Follow-up of the seronegatives identi-fied 38 SC (all HTLV-II) over 11,341.9 person semesters for a rate of.68/100 person years. Median lag time for SC was 6.8 mos. Factorsassociated with HTLV-II SC included backloading within the previous sixmonths (OR=6.52; 95% CI= 1.94-21.95), and a history of receiving moneyfor sex at baseline (OR=3.36; 95% CI=1.32-8.56). Of those with > 1 sex

partner in the past 6 months, women were more likely than men toseroconvert (OR=5.77; 95% CI=I.33-25.1).CONCLUSIONS: HTLV-II SC's continue to occur among IDUs and areassociated with sharing injection equipment and possibly sexual transmission.

23HTLV-I/n-INFECnON RESULTS IN ALTERATIONS OF CELLULAR RECEPTORSINCLUDING THE UP-MODULATION OF T CELL LIGANDS: CD40, CXJ54, AND CD80 (B7)C.S. Dezzutti. D.L. Rudolph, and R.B. Lai Retrovims Diseases Branch,

CDC, Atlanta, GA 30333.

Twelve HTLV-I-infected, 11 HTLV-II-infected and 6 virus negative T celllines were examined by flow cytometric analysis for the expression of variouslineage markers and receptor/counter-receptor pairs responsible for cellularactivation. Of the progenitor/myeloid markers examined (CD9, CD13, CD33,CD34, and CD63), only the expression of CD9 was significantly (p < 0.05)increased on HTLV-I and II-infected cell lines (85.3% and 96.5%,respectively) as compared with virus negative cell lines (28.3%). The 3groups of cell lines expressed similar amounts of the leukocyte markersexamined (CD43, CD44, and CD53). Of the ßl integrin molecules examined(CD29, CD49d, and CD49e), CD49e was significantly (p < 0.001) decreasedon HTLV-infected cell lines. While no difference was detected for theexpression of the 132 integrin molecules (CD18 and CDlla) on the infectedcell lines, their cell surface density was increased. The CDllc molecule wasexclusively expressed on the HTLV-I and II-infected cell lines. Severalreceptor/counter-receptor pairs (CD2/CD58, CD45RO/CD22, CD5/CD72,CDlla/CD54, gp39/CD40, and CD28/CD80) were examined next. The threegroups of cell lines expressed comparable amounts of the T cell markers(CD2, CD45RO, CD5, and CD28) and of some of the counter-receptors (CD58,CD22, and CD72). Interestingly, the HTLV-I and II-infected cell linesdemonstrated an increase in expression of CD54 (88.3% and 72.7%,respectively), CD40 (73.1% and 38.4%, respectively), and CD80 (98.3% and95.8%, respectively) as compared with the vims negative cell lines (17.6% forCD54, 0% for CD40, and 5.5% for CD80). These results indicate that long-term infection of T cells by HTLV-I or -II can lead to the up-modulation ofT cell counter-receptors, which are normally expressed on B cells/antigenpresenting cells. The significance of the T cell counter-receptor expression onHTLV-I and II-infected T cells is currently under investigation.

DETECTION OF HTLV-1 PROVIRUS IN THE SPINAL CORDS FROMHAM/TSP PATIENTS BY IN SITU POLYMERASE CHAN REACTION

K.Hashimoto. N.Kashio, S.Izumo, F.Umehara, E.Sato, M.Yoshida, M.C.YoshidaM.OsameKagoshima University, Faculty of Medicine, Kagoshima, Japan

We demonstrate HTLV-1 proviral DNA in the autopsied spinal cord lesions fromHAM/TSP patients by in situ polymerase chain reaction (IS-PCR). Sections fromsix Japanese patients with HAM/TSP were studied. Using the technique of IS-PCR with multiple primer pairs, pX regions of HTLV-1 proviral DNA were

amplified and hybridized to digoxigenin-labeled oligonucleotide probes. InHAM/TSP patients, specific signals of HTLV-1 proviral DNA were obtained on

the nuclei of infiltrated mononuclear cells around the vessels. The localization ofthe signals of HTLV-1 proviral DNA correlated with the distribution of OPD4 (asurface marker for helper/inducer T cell) positive cells. The signals of HTLV-1were more frequently detected in the sections of patients with shorter durations ofillness. To detect the tropism of HTLV-1 proviral DNA, we establish the doublestaining techniques of IS-PCR and immunocytechemical staining, these resultswill be shown.

24A cDNA ENCODING THE PUTATIVE CELL SURFACE RECEPTOR FOR HTLV-I

J. Johnson. N. Fan, R. Brandon, C.A. Batt, L. Papsidero, H.J. Lane,B.J. Poiesz, J. GavalchinSUNY Health Science Center, Syracuse, NY, USA

Retrovirus entry into susceptible cells is mediated by specificbinding of the virus envelope glycoprotein to a cell membranereceptor. Our laboratory has derived a monoclonal antibody, Hab34-23, that inhibits both HTLV-I binding and infection ofsusceptible cells and thus may recognize a putative HTLV-Ireceptor. Using this Mab and recombinant HTLV-I env protein, aPHA-activated PBH 'Agtll library was screened to identify cDNAswhich may encode this receptor. Eight clones were identified thatbound either Mab 34-23 or recombinant HTLV-I env protein. Theirspecific reactivity was confirmed by Western Blot analysis of ß-galfusion proteins. An additional clone, TE3-1B, bound both. TE3-1Bis a partial cDNA approximately 400bp in length that does not sharesequence homology with any known cell surface antigen, but is 90%homologous with an mRNA of unknown function transcribed in the CEMcell line. Since TE3-1B binds both Mab 34-23 and HTLV-I env, itmost likely encodes the neutralizing epitope of the HTLV-Ireceptor.

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25 26NEUTRALISATION CHARACTERISTICS OF ANTI HTLV POSITIVESERA FROM DIVERSE ETHNIC GROUPS.

JHC Tosswill1, I Bastian2, JN Weber3.

2Central Public Health Laboratory and 3St Mary'sHospital, London, UK, 2Menzies School of HealthResearch, Darwin, Australia.

To investigate the neutralising capacity of sera fromdiverse ethnic groups, syncytium inhibition (SI)assays were performed using a prototype HTLV-I virus(c91pl) and indicator cells (C8166). The sera testedwere: anti HTLV-I positive sera from bothasymptomatics and from patients with HTLV associateddiseases who originated from Africa, West Indies andMelanesia; anti HTLV-II positive sera; and sera givingindeterminate Western blot (WB) results from UK blooddonors and Papua New Guineans.

Neither the anti HTLV-II positive nor the WBindeterminate sera inhibited syncytium formation.Among the HTLV-I positive sera, 16/17 (94%) from AfroCaribbeans (ACs) and 3/11 (27%) from AustralianAborigines (AAs) inhibited syncytia. Only one AAserum inhibited syncytia at a dilution of > 1/10: thispatient had ATLL.

The differences in neutralising capacity between ACsand AAs probably reflect known differences betweenprototype and Austro/Melanesian strains of HTLV-I.

27HTLV-I INFECTION AND IL-2 RECEPTOR EXPRESSION

M. Nakamura. K. Ohbo, N. Takasawa, K. SugamuraTohoku University School of Medicine, Sendai, Japan

Adult T-cell leukemia (ATL) cells infected with human HTLV-I constitutivelyexpress the high-affinity IL-2 receptor. lL-2-induced proliferation of T cellsinfected with HTLV-I is thought to be one of the early processes inleukemogenesis by HTLV-I. Thus, the constitutive expression of the high affinityreceptor is thought to be involved in leukimogenesis through HTLV-I trans-actingtranscriptional activator Tax-dependent a chain subunit induction. The high-affinity receptor contains, in addition to the previously identified a and ß chains,the third component, y chain, which we have cloned. In this study we askedwhether expression of the y chain is affected by HTLV-I Tax. The regulatoryregion of the y chain was cloned and linked to a luciferase reporter plasmid. Theluciferase activity was significantly enhanced, when HTLV-I Tax was expressed,demonstrating that HTLV-I Tax trans-activates the IL-2 receptor y chain.Interestingly, we and others recently demonstrated that the y chain is alsocommonly used by receptors for IL-4 and IL-7, which are involved in T cellgrowth and differentiation. Therefore, it may be possible that HTLV-I Tax-induced modulation of the y chain expression may contribute to theleukemogenesis through function of the y chain as subunits for the receptors forIL-4 and IL-7.

THE PHYLOGENY OF THE PRIMATE T-CELL LYMPHOMA/LEUKEMIA VIRUSES

B. Poiesz. S. Dube, M. Sherman, N. Saksena, D. Dube, J. Love andPTLV Working Group.SUNY Health Science Center, Syracuse, New York, USA.

Over 4 million human and 1,000 non-human primates from around theworld were screened for antibodies to HTLV-I/11 or STLV-I usingELISA and Western blot assays. Representative positives fromvarious racial, ethnic, geographic, risk behavior, diseased groupsor species were selected for extensive PCR or RT-PCR analyses usingprimer pairs that span the PTLV genome. Subsequent cloning andsequence analyses indicate that the 140 bp of the p_ol regionflanked by primers SKI10/SK111 accurately reflect the sequencerelationships among the entire genomes of the PTLV and otherretroviruses including BLV, RSV and MMTV. Phylogenetic analysesof this region utilizing the PAUP and bootstrap programs and 2 BLVisolates as the outgroup identified several discrete subgroups ofthe PTLV. The HTLV-II isolates can be divided into subgroups A andB with the former being the dominant strain in North America andthe latter being endemic to Paleo-Amerindians of Florida andCentral and South America. STLV-I isolates are divided intoAsian/Indonesian and African substrains, while HTLV-I isolates canbe divided into Austronesian and African substrains. Mostcosmopolitan HTLV-I strains are highly homologous to West AfricanHTLV-I. The data indicate that interspecies transmission of HTLV-Iand/or STLV-I has probably occurred multiple times over manymillenia. Relative rate tests comparing the divergence of each ofthe major subgroups to their common outgroup indicate that the PTLVhave mutated at a constant rate. Given certain assumptions basedon anthropologie data and PTLV sequences, we would speculate that1% of divergence in the PTLV genome is equivalent to 500-1000 yr.of separation. Regions of sequence conservation and variabilityand their biological implications will be discussed.

28AGREGATION OF THE CYTOPLASMIC TAIL OF THE BLVTRANSMEMBRANE GLYCOPROTEIN TRIGGERS TYROSINEPHOSPHORYLATION AND IL-2 SECRETION.

P. Beaufils1. R. Z. Mamoun2. R. Daculsi2, N. Rebeyrottc2 and B. Malissen1.1 Centre d'Immunologie de MarseûTe-Luminy, 13288 Marseille cedex 9.2 URA 1456 CNRS, Université de Bordeaux II, 33076 Bordeaux.

The mechanisms that govern the leukemogenic process induced by theBLV/HTLV retroviral subgroup are poorly understood. Recently,similarities to a consensus sequence of membrane cellular receptorsresponsible for signal transduction were found in the intracytoplasmicdomain of the BLV TM envelope protein. To demonstrate that the TMcytoplasmic tail shares both structural and functional properties withtransducing subunits associated with receptors, we chose to express the tailin lymphoid cells. In BLV-infected cells, the expression of the envelope iscontrolled by the Rex regulatory protein. To circumvent this requirement,we analysed the signalling capacity of the TM cytoplasmic tailindependently of other viral components by constructing a chimericprotein in which the cytoplasmic tail of the mouse CD8a polypeptide isexchanged with that of BLV. We obtained stable expression of theCD8/TM chimera in T and B mouse transfected cells. In the same manner

we expressed chimeric proteins mutagenized in the consensus sequence ofthe putative transducing subunit. The transfected cells were next tested fortheir ability to trigger lymphokine production and protein phosphorylationin response to cross-linking with anti-CD8 antibodies. The results ofthese experiments and their implications for BLV oncogenicity will bepresented.

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29 30ISOLATION OF A NOVEL SIMIAN T-CELL LYMPHOTROPIC VIRUS FROM PANPANISCUS, STLV„n , RELATED TO THE HUMAN T-CELL LYMPHOTROPIC VIRUSTYPES I AND II

G.Franchini.1 P.Markham,2 A.Giri,' L.Digilio,1 G.Hurteau,2 and R.C.GäTTö1'Laboratory of Tumor Cell Biology, NCI, Bethesda, Maryland2Advanced Bioscience Laboratories, Inc., Kensington, MarylandAn unusual serological profile against HTLV-I/II proteins wasreported in several human pigmy tribes in Zaire and Cameroon withserum antibodies reactive with gp21 and p24. Here we describe asimilar pattern of serum antibodies in a colony of captive pigmychimpanzees and the isolation of a novel retrovirus (STLV ) fromthe peripheral blood mononuclear cells (PBMC) from several of theseseropositive animals. Cocultures of PBMC from three seropositivepigmy chimpanzees with human cord blood mononuclear cells led tothe expression of an HTLV-I/II related virus initially demonstratedby electron microscopy. Furthermore, several of these coculturesbecame immortalized T-cell line expressing the CD4*, CD8* and DRpositive phenotype of mature activated T-cells. Southern blot andPCR analysis of DNA from these cell cultures suggested a distanthomology of these viruses to HTLV-I and II, and distinct from theknown STLV isolates. We designated this virus as STLVpan

.

Agenealogical analysis of the captive pigmy chimpanzee colony,originated from wild caught animals, revealed a prevalence ofseropositive offspring from infected mothers, as also observed forHTLVs. The presence in this old African great ape species of avirus, which is genetically quite distinct from HTLV-I and II,could provide new insights in the phylogenesis of STLVs and HTLVsand be instrumental in the discovery of related human viruses.

31DOES AN ENDEMIC HTLV-II EXIST IN AFRICA? SEROEPIDEMIOLOGICAL

AND MOLECULAR EVIDENCE

A GESSAIN. Unité d'épidémiologie des virus oncogènes Institut PasteurParis. France.

The origin and routes of dissemination of HTLV-I, HTLV-II and STLV-Iaround the world, remain a matter of debate. Based on seroepidemiolo-gical and phylogenetic analysis, HTLV-I and STLV-I appear as Old Worldviruses, whereas HTLV-II, appears as a New World virus. We haverecently shown the presence of HTLV-II specific antibodies (HTLV 2-3WB positive with p24 > p19, reactivity to K55 peptide, and higher titers onC19 cells than on MT2 cells) In sera of 2 out of 1783 blood donors fromConakry-Guinea (1), and in 3 out of 150 prostitutes from Douala-Cameroun (2). These data taken together with the recent serologicalevidence of other sporadic HTLV-II infection in West and central Africaraised the posibility that HTLV-II has also been present in Africa over along time period. However, except for the Pygmy data for which nosequences are available, imported infections, especially in sex workerscould not be excluded. Nevertheless, we suggest that HTLV-II hasexisted in Africa over a long period of time in micro-endemic clustersbecause of 1) the presence of clear cut serological HTLV-II results inisolated Pygmies groups 2) our findings of an HTLV-II intra-familialtransmission in gaboneese individuals without any evidence of knowncontact or risk factors for New World connection (3). 3) our sequencedata showing that this gabonese HTLV-II Is a variant of the subtype B.Isolation and molecular characterization of HTLV-II West and CentralAfrican, including Pygmies, are ongoing in order to get new insights onthe origin and molecular relationship of this human retroviruses whosegeographical distribution appears not to be restricted to the New World.1) Gessain et al JAIDS, 1993, 6, 324-325. 2) Mauclère et al AIDS, 1993,10,1394-1395.3)Tuppin et al submitted. 4) Gessain et al submitted.

INCREASED RISK FOR A SECOND RETROVIRAL INFECTION (SIV ORSTLV) FOR WILD AFRICAN GREEN MONKEYS AND PATASMONKEYS ALREADY INFECTED BY ONE RETROVIRUS

P. Tuooin1. J. P. Durand2, P. Maison1, G. Galat3, A. Galat-LuoniJ3, D.Jeannel', G. de Thé1

.Epidemiologie des virus oncogènes,Institut Pasteur, Paris, FranceIMTSSA, Marseille, France3 ORSTOM, BP 1386, Dakar, SenegalThis study was conducted among 221 patas monkeys (PM) and 171african green monkeys (AGM) in two areas of Senegal. In Kedougou,between 1981 and 1982, 215 monkeys were killed during a yellowfever control, and in Sine Saloum 177 were trapped between 1989and 1991. Sera were screened for SIV by ELISA assay (HIV 1 andHIV 2) and confirmed by WB using a SIVagm as antigen. For STLV,ELISA, IF assays and WB were used. Stepwise logistic regressionsanalysis were performed to select determinants of seropositivity toSIV or STLV. SIV seroprevalence rates were 3,2% for PM and38,6% for AGM, and for STLV, 22,2% and 35,6% respectively. InAGM an increase risk of seropositivity to SIV was independentlyassociated with being adult (RR = 4.9, p<0.0001) and being STLVpositive (RR = 3.2, p = 0.001), and in PM in Kedougou an increaserisk was also found with being seropositive to STLV (OR = 15,6p = 0.01). In AGM an increased risk of seropositivity for STLV wasassociated with two indépendant factors: SIV seropositivity(RR = 4.4, p<0.0001) and being located in Kedougou (RR = 2.4,p = 0.03), and in PM, in Kedougou, with three indépendant factorsbeing SIV seropositive (RR = 15.7,p = 0.03), being adult (RR = 5.7,p = 0.01) and being male (RR = 4.6, p = 0.0001). These data suggesta dépendance of infection between these two endemic simianretroviruses in two species with different social and sexualbehaviors, and could represent a model of coinfections in humans.

32INTRAFAMILIAL TRANSMISSION OF A HTLV-II SUBTYPE BMOLECULAR VARIANT IN GABON, CENTRAL AFRICA.

P. Tuppin1. A. Gessain1, M. Kazanji1 , J. Y. Cosnefroy2, M. C. Georges-Courbot2, A. Georges2, G. de Thé1.1 Epidemiologie des Virus Oncogènes, Institut Pasteur, Paris, France. 2Centre International de Recherches Médicales, Franceville, Gabon.

Recent serological evidence of sporadic infection of HTLV-II raised thepossibility of a presence for HTLV-II in Africa since a long time. Except forpygmies, for which no sequence data are availaible, imported infectionscould not be excluded. During a survey performed in 1993 in the area ofFranceville (South West Gabon), we found a 60 years old woman withserological evidence of antibodies to HTLV-II. A family study wasconducted among 45 family members, 7 were found to be HTLV-IIseropositive as demonstrated by higher titer on C19 (HTLV-II producingcells) than on MT2 (HTLV-I producing cells) and WB (HTLV 2.3 DiagnosticBiotechnology) pattern showing rgp21, p24, low p19 and a K55 peptide.This HTLV-II came from the family of the second husband (58 years) ofthe index case, who also infected infected his third wife. A sister (56years) of this man was also infected suggesting the presence of HTLV-II inthis family since more than 58 years. One child of the sister and one chidof the index subject were also infected. No blood transfusion were

reported for these subjects, who did not have close contacts withforeigners. PCR performed on DNA extracted from uncultured PBMC ofthese HTLV-II seropositive individuals were found positive for HTLV-IIusing specific primers of the gag, pol, and tax regions, but negative forHTLV-I. Sequence data (172 nucleotides from the gp21 env gene) from 5of these 7 DNAs revealed a new identical molecular variant of the subtypeB HTLV-II (NRA) prototype (7 bases substitution : 4,1%). Theseepidemiological and biological data suggest that HTLV-II is not only a NewWorld virus but might have been present in Africa over a long period oftime.

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33 34EVIDENCE FOR SEXUAL AND MOTHER-TO-CHILD TRANSMISSION OF HTLV-II AMONG GUAYMI INDIANS, PANAMA

Charles R. Vitek", F. Gracia*", K. Fukuda", D.Green", R.Giusti"", R. Khabbaz", P.Levine"*,J. Kaplan*, W. Blattner™

"Retrovirus Diseases Branch, CDC, Atlanta, GA;"Viral Epidemiology Section, NCI, Bethesda, MD;"™ Gorgas Memorial Laboratory, Panama City, Panama

Objective: To evaluate the modes of transmission of HTLV-IIamong Guaymi Indians, a non-IDU population in which HTLV-IIis endemic.Methods: All consenting Guaymi in ten fincas in Changuinola,Panama were enrolled. Demographic information and familialrelationships were ascertained; serum samples were obtainedfrom subjects >1 year of age. HTLV testing was performed byenzyme-linked immunoBorbent assay and Western blot.Results: We obtained serum specimens from 3686 (82.8%) of4451 eligible Guaymi participants. Of these 3686, 352 (9.5%)were seropositive for HTLV-II. Seroprevalence increased inboth sexes with age, beginning in adolescence. Of 592married couples, 71 were concordantly seropositive {expected,21) and 439 were concordantly seronegative (expected, 389;p<0.001). Seroprevalence of HTLV-II was higher amongchildren <10 years of age whose mothers were seropositive(36/219, 16.4%) than among those whose mothers were

seronegative (3/997, 0.3%); p<0.0001).Conclusion: These data suggest that HTLV-II is transmittedbidirectionally between sexual partners and from mother tochild in the Guaymi.

35DEVELOPMENT OF A NEW COMPETITIVE PCR SYSTEM FOR THEQUANTITATIVE ANALYSIS OF HTLV-I AND HTLV-II PROVIRUSES

A. Cimarelli, O Angelin Duelos, U. Bertazzoni.Istituto di Genética Biochimica ed Evoluzionistica del C.N.R. Pavia, Italy.The identification of proviral sequences of HTLV-I and HTLV-II by PCRprovides an invaluable tool for the distinction between the two viruses.The recent development of quantitative PCR has paved the way to thedetermination of the number of copies of the provirus present in PBMCs,to better correlate the burden of viral infection with other prognosticmarkers. The quantitative PCR technique is based on the concomitantamplification of the wild type DNA target and of a competitor sequence,differing in size but using the same primers.We have developed a new method of competitve PCR for HTLV-I andHTLV-II by amplifying a conserved region of the tax gene with a singleprimer pair common to both viruses (D. Zella et al., Lancet 336: 575-576,1990). The target sequence of 112 bp was cloned, cleaved with Clal andelongated by inserting a fragment of 24 bp obtained from pBR322digested with Mspl. The new plasmid, named pICCS, contained themodified sequence of 136 bp which was used for the competitive PCR,followed by the separation of the PCR products on 10% acrylamide geland by the densitometric scanning of the two bands. This competitvePCR system allowed the quantitative determination of HTLV-II proviralsequences in isolates obtained from drug addicts and in PBMCs ofinfected individuals.

HTLV-II SCREENING ISSUES: FALSE NEGATIVE HTLV ELISARESULTS WITHIN A HIGH RISK IDU COHORT.

S.H. Weiss, T.N. Denny. J. Quirinale, R.K. Mayur,P.E. Palumbo, A. Heredia, E.P. Cowan, I. Hewlett,B.J. McCreedyUMDNJ

-

New Jersey Medical School Newark, NJ, USA; Foodand Drug Administration, Rockville, MD; Roche BiomédicalLaboratories, Research Triangle Park, NC.

From 1989 to 1993, 282 persons from our injection druguser (IDU) cohort were screened for HTLV infection withthe Abbott HTLV ELISA at least once. 57 (20.2%) IDUwere reactive on ELISA and confirmed positive. Of 48persistently ELISA reactive subjects typed by PCR, 47(97.9%) were HTLV-II and 1 (2.1%) was PCR negative. Inaddition, there were 6 subjects on whom the Abbott ELISAvaried over time; PCR was positive for HTLV-II on 4,positive for HTLV (type undetermined) on one, andnegative on one. In light of the high prevalence ofHTLV-II infection, we explored the sensitivity for HTLV-II of the Abbott ELISA by further testing of ELISAseronegative IDU. We further tested 100 subjects whowere seronegative on the Abbott HTLV ELISA by PCR. Six(6%) subjects were typed and confirmed as HTLV-II by DNAPCR. Applying this rate to all 218 seronegative IDU, weestimate that 13 HTLV-II IDU may have been missedoverall, representing about 1 in 6 PCR-confirmed HTLV-IIinfections. The HTLV screening methodology mostcommonly used in U.S. blood banks has limitedsensitivity for HTLV-II.

36TRANSMISSION OF HTLV-II BY HTLV-I SCREENED BLOOD PRODUCTS

M Rios. R. F. Khabbaz, J.E. Kaplan, D. Kessler, W. Hall and C. Bianco. NewYork Blood Center, New York, NY., Centers for Disease Control, Atlanta, GA.

Concerns about transmission ofHTLV-I by transfusion led to the licensure ofblood donor screening tests in December, 1988. Screening for HTLV-II was anindirect benefit of the screening for HTLV-I because ofextensive cross-reactivitybetween antibodies to the two viruses. However, recent reports suggest thatHTLV-I based assays may not detect some individuals who are infected withHTLV-II. We reviewed cases of Iookback for HTLV-II at our institution andidentified 6 cases ofHTLV-II transmission by 5 donors. The HTLV subtype of 4donor-recipient pairs was determined by PCR amplification of the env region ofHTLV-II and was the same in each pair (3 were HTLV-IIa and 1 was HTLV-IIb).Two of the implicated donors had donated 1 to 6 times after HTLV-I screeningwas introduced and before the index donation, with negative screening test results.These units infected three recipients. In conclusion, HTLV-I tests may haveinadequate sensitivity for HTLV-II and HTLV-II contaminated units missed byscreening may transmit infection. The development of specific combined tests forHTLV-I and for HTLV-II may constitute a more effective approach to theprevention ofHTLV transmission by transfusion.

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37 38GENETIC BACKGROUND OF ATL AND HAM/TSP AMONG JAPANESEAND OTHER ETHNIC GROUPS

S.Sonoda, S.Yashiki, T.Fujiyoshi, M.Osame, H.Tanaka,V.Zaninovic, M.Blank, A.Blank, M.Hayami and K. TajimaKagoshima University, Kagoshima, Japan. ValleUniversity, Cali, Colombia. Institute for VirusResearch, Kyoto University, Kyoto, Japan. Aichi CancerCenter Research Institute, Nagoya, Japan.

Adult T-cell leukemia/lymphoma(ATL) and HTLV-I-associated myelopathy(HAM/TSP) are clinically differententities of disease which are segregated familially andethnically. In order to elucidate the genetic factorsinvoled in the disease segregation between ATL andHAM/TSP, we investigated HLA DRB1*-DQB1* haplotypes andHTLV-I immune responsiveness using peripheral bloodlymphocytes (PBL) of ATL and HAM/TSP patients. Of 34cases of Japanese ATL, 27 patients (79.4%) had aunique category of HLA haplotypes which was not foundamong HAM/TSP patients. Meanwhile, 87.3% (48/55 cases)of Japanese HAM/TSP patients had another category ofHLA haplotypes which were not found among ATL patients.The HLA haplotypes of Japanese ATL and HAM/TSP werefound in one of two Colombian black ATL patients and in2 of 5 Colombian black HAM/TSP patients, respectively.In vitro assay for T-cell response against HTLV-Iantigens revealed that HLA haplotypes of ATL patientswere associated with the low responder to HTLV-I whilethose of HAM/TSP patients were the high responders.

These results suggest that HLA DRB1*-DQB1*haplotypes may determine the immunogeneticsusceptibility to ATL and HAM/TSP of Japanese andColombian patients.

39INFECTION OF HUMAN THYMOCYTES WITH HTLV-I: MODULATIONOF THE CD2 AND CD3 ANTIGENS.

V.Maguer, L. Gazzolo.M Due Dodon.Immuno-Virologie Moléculaire et Cellulaire UMR30 CNRS-Université Lyon IFaculté de Médecine Alexis Carrel, 69372 Lyon Cedex 08, France.

The Adult T-cell Leukemia (ATL) appears after a long incubation period andthe risk of developing ATL is preferentially linked to neonatal HTLV-Iinfection. These observations indicate that infection of precursor T cells early inlife may be directly correlated with the development of ATL. Two mainthymocyte subsets could be distinguished: the less frequent immatureCD2+CD3- thymocytes present in the thymic cortex, and the matureCD2+CD3+ thymocytes in the medulla. We have previously shown that HTLV-I was able to trigger the proliferation of these two thymocyte subsets(J.Virol.1993, 67, 5529-5537). The present study was undertaken to furtherinvestigate the implication of HTLV-I infection of these thymocytes in thelymphoproliferative process induced by this virus. Mature or immaturethymocytes were infected by cocultivation with mitomycin C-treated HTLV-I-producing cells. Unseparated thymocytes which were productively infected,displayed an immature CD2+CD3- phenotype. Most of them were expressingthe CD4 together with the CD25 and B7 antigens. The CD2+CD3- immaturesubset which was also productively infected by HTLV-I, retained its originalimmature phenotype, and also expressed the CD4, the CD25 and B7 antigens.Both types of infected thymocytes were found to produce interleukin 6, acytokine normally synthesized by thymic epithelial cells. These cells whencultivated in the absence of IL2 stopped to proliferate and most of the survivingcells were displaying a CD2-CD3- phenotype. Upon addition of 112, thesedouble negative cells were proliferating and matured, as indicated by themembrane re-expression of the CD2 antigen.

After thymus infection by HTLV-I, the infected CD2-CD3- thymocytesmight constitute a reservoir of quiescent cells, among which clones of leukemiccells could be recruited during the life of an HTLV-I infected individual.

HLA-CLASS II EXPRESSION ON HTLV-I-INFECTED NEUROBLASTOMAS

E.P. Cowan'. T.J. Lehky2, T. Tsuchida2, W.E. Biddison2, and S. Jacobson2'Division of Transfusion Transmitted Diseases, CBER, FDA and2Neuroimmunology Branch, NINDS, NTH, Bethesda, MD

To examine the influence of HTLV-I infection on MHC expression in neuronalcells as a possible factor in the pathogenesis observed in HAM/TSP,neuroblastoma cell lines were infected with HTLV-I by coculture with HUT-102cells. The neuroblastomas examined after complete removal of HUT-102 cells atthe end of the coculture were infected with HTLV-I based on results of flowcytometry and in situ hybridization experiments. Furthermore, the infected cellsexpressed both HLA-class I and class II by detection at the cell surface and bydetection of transcripts by Northern blotting. The neuroblastomas also retainedexpression of neurofilaments when infected. Whereas class I expression on theneuroblastomas could be induced by cytokines, class II expression was onlyobserved in HTLV-I-infected cells. A time course experiment indicated thatdetection of viral antigens preceded the appearance of class II expression. HTLV-I-infected neuroblastomas expressing class II molecules could also be specificallyrecognized by superantigen-specific, class Il-dependent CTL lines when pulsedwith antigen, demonstrating that class II is functionally expressed on the targetcells. Class II expression on the neuroblastomas correlated with increased DNA-binding activity to the X2 region of the HLA-DRA promoter, suggesting a

molecular mechanism for class II induction on these cells. Neuroblastomasexpressing the HTLV-I Tax gene are being examined to determine the influence ofTax on class II expression. Collectively, these data indicate that HTLV-I infectionof neuronal cells can result in HLA-class II expression.

40DETECTION AND QUANTITATION OF ALTERNATIVELY -SPLICED pXmRNAs IN ADULT T-CELL LEUKEMIA (ATL) AND TROPICAL SPASTICPARAPARESIS (TSP/HAM)A. Cereseto. Z. Bememan, I. Koralnlk, G. Franchini, R.C. Gallo, M.E.KlotmanLaboratory of Tumor Cell Biology, NIH, Bethesda, Md., 20892Human T-cell leukemia virus type I (HTLV-I) contains the pX sequencewhich codes for the virus regulatory proteins. Several lines of evidencesuggest that expression of proteins encoded by the pX region isassociated with leukemogenesis by HTLV-I. The pX gene products (Tax,Rex and p21rex) are translated from a major doubly-spliced mRNA.Recently, seven alternatively spliced mRNA from the pX region with uniqueprotein coding capacities were identified (monocistronic p21rex, orf I, orf II,rex-orfl, tax-orfl, tax-orfll, pX A17, env A17). In this study, we used theRNase protection assay to a) demonstrate that these new transcriptsisolated by RT-PCR are not artifacts, b) determine the relative quantity ofall pX messages in different cell lines and c) compare the expression ofalternatively spliced pX mRNAs among cell lines established from patientswith ATL and TSP/HAM and uncultured PBMCs from patients with ATL

All alternatively spliced pX messages identified and cloned by RT-PCR were detected in RNA from both ATL and TSP/HAM cell lines. Inthree primary samples that were analyzed, all mRNAs were detected aswell although protected fragments were of unexpected size suggestingsequence variation. Analyzing the relative quantity of all transcripts in anATL cell line (C10/MJ), we observed that the main transcripts are tax-orf II(60%) that have a coding capacity for a unique 241 amino acid protein,p30. A TSP/HAM cell line showed a predominant expression of the p21rexRNA (40%). The expression of alternatively spliced HTLV-I pX mRNAs,found in both cell lines and in PBMCs from patients with ATL, supports arole for the encoded proteins in both ATL and TSP/HAM pathogenesis.

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41 42MATERNAL ANTI-HTLV-1 E/WANTIBODY TITER AS A MARKER OFMOTHER-TO-CHILD TRANSMISSION: DIFFERENTIAL RISK INBOTTLE-FED AND BREAST-FED CHILDREN

1S. Hino, 2S. Katamine, 2T. Miyamoto, 2H. Doi, 2Y. Tsuji, 2T. Yamabe,3J. E. Kaplan, 3D. L Rudolph, 3R. B. Lai

'Faculty of Medicine, Tottori University, Yonago 683, Japan; 2NagasakiUniversity School of Medicine, Nagasaki 852, Japan; 3Centers forDisease Control, Atlanta GA 30333

Endemicity of human T-lymphotropic virus type 1 (HTLV-1) primarilydepends on breast-feeding; substitution of breast-feeding with bottle-feeding reduced the transmission from 20% to 3%. To determine thecorrelate of transmission, we analyzed antibody titers of carrier mothersusing synthetic and recombinant antigens covering a wide area of theHTLV-1 genome. Average antibody titers of 33 mothers whotransmitted HTLV-1 to breast-fed children in all 3 env assays (Env1/5,MTA-1 and RE3), 244, 1403 and 718, were significantly higher thanthose of 56 non-transmitters, 106, 279 and 320, respectively (p<0.006).In contrast, average antibody titers of 22 transmitters who bottle-fedchildren in Env1/5 and RE3 assays, 80 and 113, were significantlylower than those of 28 non-transmitters, 269 and 418 (p<0.002). Theanti-gag (Gag1 a and r24) and anti-fax (Tax8/22-24) titers could notdiscriminate transmitters from non-transmitters. The results suggestthat the high env antibody titer presumably associated with high viralload is a marker for milk-borne transmission of HTLV-1, but that thehigher maternal antibody level may protect bottle-fed babies.

43ANALYSIS OF COMPLEMENTATION OF HIV REV FUNCTION BYHTLV REX.

M.-L. Hammarskjöld.D, Rekosh, M. Orsini, N. Lewis, M. Bray, R.Ernst and K. DamrothMyles H. Thaler Center for AIDS and Human Retrovirus Research,University ofVirginia, Charlottesville, Virginia, USAWe have analyzed HTLV rex function in a system in which the rexprotein was used to complement HIV rev. This system utilizes vectorscontaining the gag/pol or env genes of HIV-1 and the Rev ResponseElement (RRE) from either HIV-1 or HIV-2. Our results show that theHTLV rex protein functions much more efficiently in conjunction withthe HIV-2 RRE compared to the HIV-1 RRE. Transdominant revproteins are able to inhibit rex action on the RREs of HIV-1 as well asHIV-2. Using Ul snRNA suppressor mutants we have also shown thatrex complementation requires a 5' splice site and complex formationwith Ul snRNA. These results suggest that rex interacts with splicingfactors to promote transport of intron containing RNA from thenucleus to the cytoplasm. We have recently identified a cis-actingelement in the genome of the type D retrovirus Mason-Pfizer MonkeyVirus (MPMV) that promotes the nuclear export of intron-containingMPMV RNA. This element substitutes efficiently for rev and the RREin HIV expression. This indicates the existence of a cellular pathwayfor transport of incompletely spliced RNAs. We are presentlyinvestigatingwhether the MPMV element is capable ofpromotingexpression of HTLV structural proteins and nuclear export of HTLVmRNA in lieu of rex and the RxRE. The results of these experimentswill be presented.

LEUKEMOGENIC ACTIVITY OF HTLV-1

L. Ratner. J.T. Kimata, W. Grossman, F.H. WongWashington University School of Medicine, St Louis, Missouri, USA

Three model systems were used to explore the lymphoproliferativemechanisms employed by HTLV-1. First, with in vitro studies oflymphocyte proliferation induced by HTLV-1, we found that antibodies toLFA-3, CD2, and CD3 were inhibitory, whereas antibodies to Env, Gag,Tax, MHCII, and TCR did not affect this activity. No evidence for TCRVbeta-specific effects were noted arguing against the involvement of a

superantigen. Second, in transgenic mice with Tax expressed under thecontrol of a lymphocyte-specific promoter, tumors developed which are ofnatural killer cell origin. Similarities were noted to large granular cellleukemias in humans, a disorder which may be etiologically related toHTLV-1. Third, to decipher the viral determinants of leukemogenesis, wehave demonstrated that full length proviral clones of HTLV-1 can be stablypropagated in plasmid form, and are capable of producing viral RNA,proteins, and particles as demonstrated by sucrose gradient and electronmicroscopic analyses. Studies of virus infectivity and immortalizing capacityare underway.

44BIOLOGICAL CHARACTERIZATION OF A CLONED HTLV-I PROVIRUS

D. Derse , J. Mikovits , M. Polianova , and F.Ruscetti^Laboratory of Viral Carcinogenesis, 'Laboratory of LeukocyteBiology, National Cancer Institute and 3BCDP, PRI-DynCorp, NCI-FCRDC, Frederick MarylandSeveral molecular clones of HTLV-I have been reported and shown toexpress mRNAs and proteins in transiently transfected cells buttheir infectivity has not yet been demonstrated. While defects inthe cloned proviruses have been suggested, it is likely that theinability to show infectivity results from a combination of i)highly restricted expression which limits both detection and spreadof the virus through the culture; ii) low infectivity per particle,perhaps due to virion instability; and iii) cytopathic effects ofthe virus on productively infected cells. We have attempted tooptimize conditions to examine the infectivity of virions releasedfrom cells transfected with the proviral clone, pCS-HTLV. This clonewas previously shown to direct the expression of viral mRNAs andproteins in transfected HeLa cells. A human cell line was obtainedwhich is both very efficiently transfected and permissive for HTLV-1expression. Transiently transfected cells released virus particlesat high levels, as shown by sucrose gradient centrifugation.Significant cell death accompanied viral gene expression. Continuouspassage of the transfected cells gave rise to a cell line thatreleases HTLV-I particles at low levels. Growth medium fromtransiently transfected cells was used for cell-free infections ofseveral lymphoid cell lines and primary lymphocytes. In addition,infections of established T-cell lines by cocultivation with eitherthe transiently or stably transfected cells were performed. Theinfectivity of this molecular clone will be discussed.

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45 46RECOGNITION OF HTLV-1 ENV RECOMBINANT FRAGMENTS BYCD4+ HUMAN T HELPER LINES FROM SERONEGATIVEINDIVIDUALS

F. Manca. G. Li Pira, D. Fenoglio, M.T. Valle, A.Kunkl, A. Ferraris, L. Mortara, R. Balderas, A.Theofilopoulos. Departments of Immunology, Universityof Genoa, 16132 Italy and Scripps Research Institute,La Jolla, CA 90027, USA.

Since T helper (Th) cells are critical for immuneresponses in retroviral infections, Th cell linesspecific for HTLV-1) env have been generated fromseronegative donors to study the repertoire of nonimmune individuals. Four recombinant fragments (REÍ,aa 26-200; RE3, aa 166-307; RE5, aa 308-401; RE6, aa

166-401) were used. Th lines specific for one or more

fragments were obtained from 6/7 individuals tested.REÍ was recognized by 4/6 individuals, and the otherfragments were recognized by a fraction of theindividuals. Since the RE fragments contain 97 to245 aa, they may carry numerous T epitopes anddifferent T clones may recognize the same fragment. Totest clonal heterogeneity, T lines specific for REÍand RE6 were analyzed for expression of different TCRVß families with an RNAse protection assay. The REÍline contained specific T cells expressing at least11 Vß gene families, the RE6 line at least 6 Vß genefamilies. Thus most of the individuals carry Thprecursors specific for HTLV-1 env and clonalheterogeneity of HTLV-1 env specific T cells can bedetected in the human Th repertoire.

47IN VIVO LYMPHOCYTE TROPISM OF HTLV-II IS NOT RESTRICTEDTO CD8 CELLS.R.B. Lai. S.M. Owen, D.L. Rudolph, C. Dawson, H. PrinceRetrovirus Disease Branch, CDC, Atlanta, and Cellular Immunology Branch,American Red Cross Blood Services, Los Angeles, CA

We have examined the in vivo and in vitro susceptibility of lymphocytesubpopulations to HTLV-II to determine the cellular tropism for this virus.MAbs to T-cell subsets were used to obtain purified CD4+ and CD8+ cellsfrom peripheral blood lymphocytes from 35 individuals previously shown to beinfected with HTLV-II. The highly purified subpopulations were analyzed forHTLV-II provirus (pol and tax) by PCR and for virus expression by p24E"santigen detection of cultured lymphocytes. Based on nested amplification inpoland tax gene region, both CD8+ subsets (91%) and CD4+ subsets (69% to80%) from most infected subjects demonstrated HTLV-II provirus. Spontaneouslymphocyte proliferation (SLP) was higher in CD8+ subset (17986+4675cpm), when compared to CD4+ subset (2333+826) and viral antigenproduction was also higher in CD8+ cells from SLP+ individuals (median 115pg/ml; range 14-1360 pg/ml), as compared to CD4+ cells (9 pg/ml; 2-250pg/ml), suggesting higher proviral load in CD8 cells. Indeed, limiting-cell-dilution PCR analysis indicated that CD8 + subsets carried a higher HTLV-IIproviral frequency than CD4+ subsets. Infection of purified CD4+ and CD8+lymphocytes with irradiated HTLV-II cell lines resulted in productive infectionof both subsets. Cell sorting analysis of lymphocytes from 4 HTLV-II-infectedsubjects followed by PCR demonstrated that in addition to CD4+ and CD8+subsets, both CD45RO+ and CD45RO- as well as non-T cells (CD14, CD16,and CD19) also carried HTLV-II provirus. These data suggest that HTLV-IIpossesses a broad tropism for mononuclear cells.

DIFFERENTIAL HTLV-I REPLICATION IN CD4+ VERSUS CD8 + T-CELLSG.Newbound. L. Ratner, M. LairmoreCentre for Retrovirus Research, Department of Veterinary Pathobiology, The OhioState University, Columbus, Ohio, U.S.A.

HTLV-I infects CD4+ and CD8+ T lymphocytes, B cells and monocytesin vivo, various cell types in vitro, and preferentially transforms CD4 +lymphocytes both in vivo and in vitro. In infected persons CD4+ T lymphocytescontain higher proviral copy numbers compared to other infected cells. Therefore,although both CD4+ and CD8+ T-cells may be infected with HTLV-I in vivo andin vitro, we hypothesize that CD4+ T-cells possess a unique intracellularenvironment that is permissive to viral replication, cellular proliferation andtransformation. We co-cultured a lethally irradiated HTLV-I producing cell line(HUT 102) with highly enriched (>98% pure) populations of CD4+ and CD8 +T lymphocytes. CD4+ T-cells proliferated for longer periods of time and togreater numbers than did CD8+ T-cells. Additionally, 7 wks post co-cultivation,CD4+ cells produced 15-fold more viral p24 antigen as CD8+ cells. Followingco-tranfection of CD4+ and CD8+ T-cells with an HTLV-LTR/CAT plasmid(pU3RI-CAT) and an HTLV-LTR/Tax plasmid (pK-Tax), reporter gene activity48 hrs post-transfection was 8.5 times greater in CD4+ cells compared to CD8 +cells. Additionally, 48 hrs after transfection of a full length genomic clone ofHTLV-I, CD4+ cells produced twice as much viral p24 than did CD8+ cells.Together these data indicate that CD4 + T-cells are more permissive than CD8 +T-cells for viral replication. These observations suggest that unique factors withinCD4+ T-cells may interact with Tax to enhance HTLV-I LTR activity in thistarget cell.

(Funded by NIH CA55195 and the Medical Research Council of Canada)

48

Molecular Epidemiology of HTLV-I

Guy de The, Unite d'Epidemiol ogle des Virus Oncogènes. Institut Pasteur, 75724Paris Cedex 15. France. Presently at the Fogarty International Center. NIH.Bethesda. Maryland.

The question whether there exist specific gene mutations directingthe HTLV-I tissue tropism and pathogenesis led us, since 1988, tocompare genetic sequences of various HTLV-I isolates around theworld. Performed mostly on LTR. pol and env genes theses studiesshowed a very low genetic variability of HTLV-I, but exhibitedrestricted geographical subtypes. The puzzling epidemiologicalHTLV-I distribution, of molecular clusters, and the recentdiscovery of distant HTLV-I variants in Central Africa and inMelanesia raised new questions on the origin and evolution of HTLV-I, whose low genetic drift can be used as a molecular tool inpaleo-anthropological studies. Based on RFLP and DNA sequenceanalysis. 5 major subtypes have been recognized: the Cosmopolitan,the Japanese, the West and Central Africans and the Melanesian.The Cosmopolitan subtypes is widely distributed in the world,including South Japanese islands, probably spread through recentmigrations of infected individuals. The African appears as anensemble of quasi species, with at least two aggregates. TheJapanese subtype has recently also been found in India. Assuming asimilar mutation rate for this virus around the world, the topologyof the phylogenetic trees may well reflect the evolution andmigration of virally infected populations in recent or distantpast.

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