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Ahmad, M. U. D. et al. Nano-differential scanning fluorimetry for screening in fragment-based lead discovery. Journal of Visualized Experiments: JoVE. (171), e62469 (2021). Barbieri, L., Luchinat, E. Monitoring protein-ligand interactions in human cells by real-time quantitative in-cell NMR using a high cell density bioreactor. Journal of Visualized Experiments: JoVE. (169), e62323 (2021). Berg, H. et al. NMR-based fragment screening in a minimum sample but maximum automation mode. Journal of Visualized Experiments: JoVE. (172), e62262 (2021). Beriashvili, D., Schellevis, R. D., Napoli, F., Weingarth, M., Baldus, M. High-resolution studies of proteins in natural membranes by solid-state NMR. Journal of Visualized Experiments: JoVE. (169), e62197 (2021). Cornaciu, I. et al. The automated crystallography pipelines at the EMBL HTX facility in grenoble. Journal of Visualized Experiments: JoVE. (172), e62200 (2021). Crawshaw, A. et al. A sample preparation pipeline for microcrystals at the VMXm beamline. Journal of Visualized Experiments: JoVE. (172), e62306 (2021). Douangamath, A. et al. Achieving efficient fragment screening at XChem facility at diamond light source. Journal of Visualized Experiments: JoVE. (171), e62414 (2021). Groen, J. et al. A 3D cartographic description of the cell by Cryo Soft X-ray tomography. Journal of Visualized Experiments: JoVE. (169), e62190 (2021). Horrell, S. et al. Fixed target serial data collection at diamond light source. Journal of Visualized Experiments: JoVE. (168), e62491 (2021). Jiménez-Moreno, A. et al. Cryo-EM and single-particle analysis with scipion. Journal of Visualized Experiments: JoVE. (171), e62261 (2021). Moravcová, J., Pinkas, M., Holbová, R., Nováček, J. Preparation and CryoFIB micromachining of Saccharomyces cerevisiae for CryoElectron tomography. Journal of Visualized Experiments: JoVE. e62351 (2021). Vyas, N. et al. Cryo-structured illumination microscopic data collection from cryogenically preserved cells. Journal of Visualized Experiments: JoVE. (171), e62274 (2021). Weis, F., Hagen, W. J. H., Schorb, M., Mattei, S. Strategies for optimization of cryogenic electron tomography data acquisition. Journal of Visualized Experiments: JoVE. (169), e62383 (2021). White, J. B. R. et al. Single particle cryo-electron microscopy: From sample to structure. Journal of Visualized Experiments: JoVE. (171), e62415 (2021). Wollenhaupt, J. et al. Workflow and tools for crystallographic fragment screening at the helmholtz-zentrum Berlin. Journal of Visualized Experiments: JoVE. (169), e62208 (2021).

The ESFRI landscape includes fourteen infrastructures and several e-infrastructures in the health and food area. These cover vastly different structures and capabilities, but all provide access of some type to the biomedical sciences communities in Europe and, increasingly, globally. A major objective for the ESFRI Roadmap is to identify synergies between their infrastructures as well as any related regional and national facilities that integrate their services. This raises a major challenge to identify mechanisms that enable integration for user access, for service provision, for implementation of standards, for common practises and regulatory processes. The e-infrastructures bring existing ICT services that are used by research infrastructures and related projects and their integration relies on the co-design and development of new horizontal solutions that enable cross-disciplinary sharing. The aim of WP5 is to develop a common access framework that facilitates user access to services and resources across the RIs in the biomedical fields.

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. © 2011 Elsevier Inc.

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI- cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. © 2011 Elsevier Inc.

Using oligonucleotide primers that hybridize to conserved sequences in the reverse transcriptase (RT) gene, we have amplified by the polymerase chain reaction three sequence variants of HTLV-I from the genomic DNA of five patients with tropical spastic paraparesis (TSP), and a fourth sequence variant from a healthy carrier of HTLV-I. These results unequivocally identify the retrovirus associated with TSP as HTLV-I and suggest that no sequence variant is uniquely responsible for the condition. The same primers served to amplify two novel single-copy endogenous retroviral RT sequences related to the exogenous mammalian leukaemia viruses: and three KpnI (LINE1) family DNA repeats. This strategy, combining the sensitivity of PCR with cross-reactive primers, may be useful in the search for known or novel retroviruses in other diseases of possible retroviral aetiology.

Infection with human T cell leukemia virus type 1 is detected by screening programs and contact follow-up procedures. Where chronic infection results in overt pathology, this is treated largely symptomatically and control of transmission relies on physical and educational constraints. The poor infectious transmission rate of HTLV-1 has long been described but to date has not been exploited in preventative measures to combat the spread of the virus. We undertook to investigate some of the molecular steps involved in HTLV-1 cell-cell fusion, the main mechanism of transmission. We showed that poor transmission may relate in part to an inefficiency in adopting and maintaining a fusion competent conformation of the HTLV-1 envelope TM protein. In cell-cell fusion, this deficiency can be complemented by accessory molecules on both infected and target cells that stabilize the envelope/receptor interaction. In virion-cell fusion, this is less likely, leading to an inefficient interaction and poor infectious transmission by cell-free virus. A discussion of the accessory molecules involved in HTLV-1 fusion is presented. This weak envelope-dependent interaction with target cells in the host can be potently disrupted by peptides that destabilize the TM protein structure and significantly inhibit HTLV-1 fusion. These observations may be useful in the design of therapeutic agents to prevent HTLV-1 transmission.

Human T-cell leukemia virus type 1 (HTLV-1) is notable among retroviruses for its poor ability to infect permissive cells, particularly as cell free virus. The virus is most efficiently transmitted between individuals by infected cells, where it is presumed that intracellular particles and viral RNA are transferred to target cells following fusion. Although the mandatory first step for HTLV-1 fusion is the binding of envelope SU (gp46) to the receptor, the events which follow this interaction and lead to fusion and infection have not been well characterized. To investigate these events, we studied two HTLV-1 chronically infected cell lines with different abilities to fuse with K562 target cells. Although not inherently fusion incompetent, the HTLV-1 envelope protein on MT2 cells was poorly able to undergo a change in membrane hydrophobicity required for fusion with the target cell membrane after binding to the receptor. High level expression of a fusion-competent HTLV-1 envelope protein on MT2 cells had little effect on improving this suggesting that the defect was encoded by the parent cell. Visible syncytia were seen after incubation of these cells with K562 target cells but complete fusion as measured by transfer of cellular contents into the recipient cell was not observed. In C91-PL cells, binding of SU to the receptor resulted in a sustained hydrophobic change of envelope accompanied by a cytopathic effect in mixed cell cultures and complete fusion. However, in C91-PL cells, overexpression of envelope protein blocked the transfer of cell contents after receptor engagement and initiation of cytopathic membrane changes, indicating that post binding fusion events were blocked. These data suggest that HTLV-1 fusion is a multistep process which is susceptible to inhibition at two seperate stages of the fusion pathway post receptor binding. This, and the inefficient infection by cell-free virions, may explain the poor infectivity of HTLV-1 in vivo and suggests strategies for preventative therapy.

Naturally occurring mutations in Human T-cell Leukemia Virus Type 1 (HTLV-1) Tax protein lead to loss of recognition by cytotoxic T-lymphocytes. Most of these mutations also abolish or severely impair the transactivation function of Tax. Ninety percent of therexgene, which encodes the viral regulator of mRNA splicing (Rex), overlaps with thetaxgene. In this paper, we report that four previously described point mutations intaxthat abolished CTL recognition and activity did not alter either the dimerisation function or the ability to export viral mRNA of the corresponding Rex proteins. Rex proteins containing two other amino acid changes were likewise functional. However, five Rex deletion mutants, predominantly but not exclusively found in HAM/TSP patients, had all lost these functions. We conclude that, although the Tax protein is subject to strong CTL-mediated selection, there are stronger functional constraints on amino acid variation in Rex. This may limit the variation in thetax/rexnucleotide sequence which results in immune evasion.

There is a powerful, chronically activated cytotoxic T-lymphocyte (CTL) response to the Tax protein of human T-cell leukaemia virus type I (HTLV-I) in most people infected with the virus. The CTL select variant sequences of Tax which escape immune recognition and interfere with recognition of the wild-type protein. This positive selection process is more efficient in healthy HTLV-I carriers than in patients with tropical spastic paraparesis, an inflammatory neurological disease associated with HTLV-I. The mean virus load is more than 10-fold greater in patients with this neurological disease than in healthy carriers of HTLV-I. We conclude that anti-Tax CTL play an important part in limiting the rate of replication of HTLV-I. We suggest that the outcome of infection with HTLV-I is primarily determined by the CTL response of the individual: low CTL responders to HTLV-I develop a high virus load, resulting in widespread chronic activation of T cells. The activated T cells then invade the tissues and cause bystander tissue damage, probably by releasing cytokines and other soluble substances. An efficient CTL response to HTLV-I limits the equilibrium virus load, and so reduces the chance of developing inflammatory disease.

tax gene expression in a family cluster of three HTLV-I-infected asymptomatic individuals was investigated. Two carriers had normal tax mRNA, Tax-specific humoral antibody, and cell-mediated immune (CMI) response. In one carrier who had only weak Tax-specific humoral and no Tax-specific CMI response, an abnormal Tax-related mRNA product was detected. This product was sequenced and found to consist of two exons derived from the LTR gag and pX regions. The abnormal mRNA has an ORF predicting a 17-kDa protein, the translation of which is initiated in the first exon. The presence of this protein, of antibody to it, and of its function remain to be elucidated.

Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N- and C-terminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel beta-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PR14. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.

It is about a decade since SPINE – Structural Proteomics IN Europe – was envisaged, set-up and submitted as an idea to the European Commission fifth framework programme. Our concepts were built on the paradigm of the USA-based structural genomics projects (http://www.nigms.nih.gov/psi), the Japanese initiatives (http://www.rsgi.riken.go.jp/rsgi_e), and some national efforts within Europe. At the same time, benefiting from the experience and technology development of the preceding projects, SPINE took an approach different from that of the mainstream of structural genomics: a major success of SPINE was to bring the cutting-edge high-throughput (HTP) technologies to biomedically relevant targets, not only facilitating the determination of no less than 308 novel structures, but also achieving a highly collaborative spirit and a culture of exchange of knowledge in technology. SPINE-2 Complexes (S2C) was both a continuation of SPINE and a whole new venture in its own right. Having largely achieved the integration of HTP technologies at partner sites, S2C aimed to facilitate the study of macromolecular complexes in key areas of biological research (ubiquitin (de-)conjugation, cell cycle and apoptosis, synaptogenesis and neuronal signaling, kinases and phosphatases, transcription receptors and regulation, innate and acquired immunity, and mechanisms in viral infection). S2C also worked to advance ‘wet-lab’ technologies relevant to the study of challenging targets (e.g., production of multi-subunit complexes with an emphasis on eukaryotic systems, biophysical methods to characterize complexes, and library tools for HTP screening), and ‘dry-lab’ technologies (e.g., microcrystal handling, docking software, as well as data management, analysis and dissemination tools). This ambitious agenda was backed-up by the long standing interests of the participant labs in the corresponding research themes, and was supported by a shared enthusiasm for consolidation of technologies and conceptual approaches, which together allowed us to showcase the structural study of complex systems. This special issue appears at a time at which Integrative Structural Biology is beginning to blossom. It seems, therefore, to be appropriate to take the opportunity to look back at what was achieved in S2C, and to present papers summarizing progress in key areas of biological interest, as well as on describing new technological achievements and benchmarking and extending existing methodologies. In the Foreword of a special issue of Acta Crystallographica devoted to SPINE (vol. D62, part 10), Ray Stevens forecast beyond SPINE-2 Complexes (which at the time had just been funded) to SPINE-3 and beyond, a future that was never formally realized. It is likely, however, to be superseded by an even brighter future, largely as an outcome of SPINE and S2C: Instruct (http://www.structuralbiology.eu), via a coalition of biotechnology core centers in Europe, will provide access for European structural cell biologists to advanced instrumentation and to valuable expertise, thereby democratizing the ability to undertake technologically challenging research and increasing the competitiveness of European science. We hope that the research presented in this special issue, focusing on the work done within the SPINE networks, has helped to pave a bright European future for Structural Biology.

Epstein–Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins.

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates the HTLV-1 long terminal repeat and key regulatory proteins involved in inflammation, activation, and proliferation and may induce cell transformation. Tax is also the immunodominant target antigen for cytotoxic T cells in HTLV-1 infection. We found that Tax bound to assembled nuclear proteasomes, but Tax could not be detected in the cytoplasm. Confocal microscopy revealed a partial colocalization of Tax with nuclear proteasomes. As Tax translocated into the nucleus very quickly after synthesis, this process probably takes place prior to and independent of proteasome association. Tax mutants revealed that both the Tax N and C termini play a role in proteasome binding. We also found that proteasomes from Tax-transfected cells had enhanced proteolytic activity on prototypic peptide substrates. This effect was not due to the induction of the LMP2 and LMP7 proteasome subunits. Furthermore, Tax appeared to be a long-lived protein, with a half-life of around 15 h. These data suggest that the association of Tax with the proteasome and the enhanced proteolytic activity do not target Tax for rapid degradation and may not determine its immunodominance.

A prospective clinical study of 20 initially asymptomatic HTLV-I-seropositive carriers was commenced in 1991 to determine the natural history of the infection in relation to HTLV-I proviral load, immune responses, and lymphocyte phenotype. Proviral load varied widely between carriers but was relatively constant within an individual over time. The lymphocyte phenotype and prevalence of activated lymphocytes were not predictive of disease and the magnitude of the cytotoxic T-lymphocyte response to HTLV-I was independent of proviral load. Incident conditions, some related to HTLV-I infection, including a case of HTLV-I-associated myelopathy (HAM), were documented in 9 carriers. Development of myelopathy and uveitis was associated with high peripheral blood HTLV-I proviral load that predated symptoms. Persistently high proviral load appears to predate the development of HTLV-I-associated inflammation in neuro-ophthalmic tissue.

Human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) is a pathogenic retrovirus responsible for a number of inflammatory pathologies and adult T-cell leukaemia. Although T-cell tropic in vivo, HTLV-1 can infect a wide variety of cell types in vitro. Cell-to-cell spread of HTLV-1 may require specific binding of envelope to its cellular receptor, with other cell-surface molecules facilitating fusion. Here it is shown that intercellular adhesion molecule-1 or -3 (ICAM-1, ICAM-3) or vascular cell adhesion molecule-1 (VCAM-1) are required for syncytium formation of K562 with HTLV-1-infected MT2 cells but not C91-PL cells. The effect of ICAMs and VCAM-1 on MT2-induced fusion can be blocked by antibodies that bind beta-integrins. These fusion co-factor molecules are effective only when present in combination with HTLV-1 receptor-bearing cells and are not sufficient to mediate syncytium formation alone. The results suggest that engagement of HTLV-1-infected cells with susceptible target cells requires the simultaneous binding of viral envelope glycoprotein to the cellular receptor and co-factor molecules to beta-integrins. The tissue-specific expression of adhesion molecules might therefore influence HTLV-1 virus tropism and pathogenic changes associated with syncytium formation.

There is a high degree of intraisolate sequence heterogeneity in the tax gene of human T-cell leukemia virus type I (HTLV-I), although the sequence variation between patients is small compared with that of human immunodeficiency virus type 1. In the present study, we investigated whether naturally occurring amino acid substitutions changed the properties of the Tax protein in two respects: first, recognition of the protein by cytotoxic T lymphocytes (CTL), and second, the ability of the Tax protein to transactivate various promoters. We found that (i) all of the observed amino acid substitutions that occur in known CTL epitopes abolished the recognition of the synthetic peptide representing the respective epitope; (ii) these substitutions occurred significantly more frequently in subjects carrying HLA-A2; and (iii) most of the amino acid substitutions severely reduced the ability of Tax protein to transactivate three promoters: the HTLV-I long terminal repeat, the c-fos promoter, and the interleukin-2 receptor alpha chain promoter.

In human T cell lymphoma/leukemia virus (HTLV-1)-infected people with tropical spastic paraparesis (TSP), there are activated HTLV-1-specific cytotoxic T lymphocytes (CTL) in the circulation and lymphocytic infiltrates in spinal cord lesions that are rich in CD8+ T cells. These observations suggest a role for virus-specific CTL in the pathogenesis of TSP. We have examined the anti-HTLV-1 cytotoxic activity of freshly isolated CD8+ T cells from peripheral blood lymphocytes of eight subjects seropositive for HTLV-1. Four of five subjects with TSP had circulating activated anti-Tax CTL. However, two of three seropositive subjects without TSP also had activated anti-Tax CTL. These observations show that such activated CTL are not confined to patients with TSP and raise some uncertainty about their significance in the pathogenesis of the disease. In cultures of CD8+ T cells from two TSP subjects, we detected CTL with other HTLV-1 specificities, without exogenous antigenic stimulation. A CTL epitope in the middle region of Tax and one in the C terminus of Pol have been mapped at the peptide level and the HLA Class 1 molecules restricting their recognition have been defined.

We have amplified and sequenced DNA in the envelope (env) and long terminal repeat (LTR) regions of human T-cell lymphotropic virus type I (HTLV-I) proviruses from the peripheral blood of 10 HTLV-I-seropositive patients with tropical spastic paraparesis (TSP) and two patients with adult T-cell leukemia. The aim was to examine variation in these regions and to test the hypothesis that the sequences of leukemogenic HTLV-I isolates differ from those causing the neurological disease TSP. In 5 of the 12 HTLV-I-seropositive patients, more than one HTLV-I sequence variant was identified in the same individual. No two individuals shared identical sequences in either env or LTR U3. Sequence variations were found at 73 positions in 1,416 bases amplified in env. Sequence variability was found throughout the LTR-U3 region, including the sequences of two transcriptional enhancers. Several nucleotide changes common to both Caribbean and Japanese HTLV-I isolates allowed us to identify a consensus sequence that differs from the HTLV-I prototype sequence (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida, Proc. Natl. Acad. Sci. USA 80:3618-3622, 1983). No sequence in the env or LTR U3 region was found to be characteristic of isolates from TSP patients. Although each isolate was distinct at the nucleotide level, the predicted protein sequence of HTLV-I env is less variable than that of human immunodeficiency virus env, suggesting that these lymphotropic retroviruses use different strategies to evade host immune responses.

Division of Structural Biology, University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, England, and York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, England The concept of structural genomics arose in the mid to late 1990s in the USA and Japan as a response to the success of high-throughput (HTP) sequencing methods applied to whole genomes (see http://www.isgo.org). It was imagined that similar HTP methods could be applied to obtain three-dimensional structures of all the proteins (the ‘proteome’) of an organism, which would in particular be an efficient way of filling in the gaps in observed ‘fold-space’. This vision led to the investment of substantial sums of money into large-scale structural genomics projects in the USA [e.g. nine projects funded by the NIH/NIGMS Protein Structure Initiative (PSI) from September 2000 to June 2005, http://www.nigms.nih.gov/psi/] and Japan (e.g. the massive RIKEN project, http:// www.rsgi.riken.go.jp/). These were characterized by the concentration of resources into a small number of large centres, the development of novel, automated technologies to permit a HTP pipeline approach to structure determination, and a focus on novel folds as the major target criteria. The US-based projects, in addition, required immediate public deposition of structural data whereas the Japanese RIKEN project also aimed to support Japanese industry, precluding deposition in advance of patent evaluation. Europe was slower in implementing HTP approaches to structural biology. The Protein Structure Factory in Berlin, Germany (http://www.proteinstrukturfabrik.de/) led the way, followed by the Oxford Protein Production Facility (OPPF) in Oxford, UK (http:// www.oppf.ox.ac.uk/) and the Genopoles in France (notably Gif, Marseille and Strasbourg, http://rng.cnrg.fr/). However, it was not until October 2002 that the first Europe-wide project began. This was a three-year project funded by the EU FP5 programme called SPINE: Structural Proteomics IN Europe (http://www.spineurope.org). SPINE, a ‘second generation’ structural genomics project (indeed purposefully called a Structural Proteomics project to draw a distinction), made some radical departures from the firstgeneration initiatives, while at the same time obviously benefiting from the experience and technology development of the preceding projects. The challenge set for SPINE was to push forward with cutting-edge technologies aimed at biomedically relevant targets at the same time as generating a pan-European integration on biomedically focused structural proteomics. The SPINE consortium comprised

The synthesis of [N-benzyl-N′-(Nα-benzyloxycarbonyl-L-asparaginyl)hydrazino]sulfonyl-L-prolyl-L-isoleucyl-L-valine methyl ester 4, a potential HIV-1 proteinase inhibitor, is described.

The cytotoxic T-lymphocyte (CTL) response to HTLV-1 is directed mainly against the Tax protein. Circulating, activated Tax-specific CTL can be found in a majority of healthy carriers and patients with the HTLV-1-associated disease tropical spastic paraparesis (HAM/TSP). In this study we present data on the Tax-specific CTL response of 26 HTLV-1 carriers, including 10 newly recruited subjects. Rex-specific CTL were not found in any subjects investigated. Activated and memory CTL responses were determined separately in 4 healthy carriers, 3 HAM/TSP patients, and 1 “seronegative HAM/TSP.” In all subjects, the mean frequency of peptide-specific memory cells per epitope (1/1307) was high. There was no significant difference in mean memory CTL frequency per epitope or in the proportion of subjects with activated CTL between healthy carriers and HAM/TSP patients. One individual with HAM/TSP had an unusually high frequency response to two peptides, suggesting immunodominance of epitope recognition in this individual. We conclude that the magnitude and components of the HTLV-1-specific CTL response do not differ between healthy carriers and HAM/TSP patients. These data do not support a specific CTL-mediated component in the pathogenesis of HAM/TSP.

Some established techniques for B-cell enrichment include azide in the media. Here, B-cells were enriched using a technique which transiently exposed the cells of 15 mM sodium azide. After 3-4 days in culture, the precursor frequency of autoantibody-secreting cells in this population was 0.113%. In contrast, when B-cells were enriched using a technique which eliminated their exposure to sodium azide, the number of autoantibody-secreting precursor cells detected in cultures was increased up to 20-fold. Therefore, it is concluded that transient exposure to azide affects functional activities of B-cells in vitro.

Human T-cell leukemia virus type I (HTLV-1) causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. No association between a particular HTLV-I sequence and disease manifestation has been found in previous studies. We studied here the sequence variability of the gene for the HTLV-I Tax protein, which is the dominant target antigen of the very strong cytotoxic T-lymphocyte response to the virus. In HTLV-I infection, the intraisolate nucleotide variability is much greater than the variability between isolates. The predicted protein sequence of Tax was significantly more variable in the healthy seropositive individuals' provirus than in those of the patients with TSP. Thus, tax sequence heterogeneity, rather than the presence of particular sequences, distinguishes healthy HTLV-I-seropositive individuals from patients with TSP.