Evidence for the chronic in vivo production of human T cell leukemia virus type I Rof and Tof proteins from cytotoxic T lymphocytes directed against viral peptides.

Human T cell leukemia virus type I (HTLV-I) is a persistent virus that causes adult T cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Studies on rabbits have shown that viral proteins encoded by the open reading frames pX-I and pX-II are required for the establishment of the persistent infection. To examine the in vivo production of these proteins in humans, we have investigated whether cytotoxic T lymphocytes isolated from HTLV-I-infected individuals recognized pX-I and pX-II peptides. CD8(+) T lymphocytes to pX-I and pX-II peptides were detected in HTLV-I-infected individuals, whatever their clinical status, and even in the absence of any antigenic restimulation. These findings indicate that the HTLV-I pX-I and pX-II proteins are chronically synthesized in vivo, and are targets of the natural immune response to the virus.

Early assembly step of a retroviral envelope glycoprotein: analysis using a dominant negative assay.

As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.

New hematopoietic differentiation antigens detected by anti-K562 monoclonal antibodies.

Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immunoprecipitates a Mr 105,000 glycoprotein on K562 cells. (b) Two MoAbs, 14B6 and 12B1, react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-O-tetradecanoylphorbol-13-acetate treated K562 cells as a Mr 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-O-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (c) Two anti-K562 MoAbs, CF4 and HE10, recognize a myeloid differentiation antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an apparently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 recognizes the monocyte low affinity Fc receptor (Mr 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies a Mr 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of antibodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hematopoietic progenitors.

HLA class-I and class-II antigen expression by human leukemic K562 cells and by Burkitt-K562 hybrids: modulation by differentiation inducers and interferon.

K562 cells, which could be regarded as pluripotent hematopoietic progenitors, are usually considered as HLA class-I and class-II-negative cells. We show here that differentiation induction (with either sodium butyrate, 12-O-tetradecanoyl-phorbol-13-acetate, or teleocidin) or recombinant alpha- or gamma-interferon (IFN) treatment resulted in the augmentation of HLA class-I antigen expression. This augmentation of HLA class-I antigens was also observed in the Burkitt X K562 hybrid cells PUTKO and DUTKO (the latter coming from two presumably HLA-A, B-negative parents). HLA class-I genes are thus functional in K562 cells. In this system, alpha- and gamma-IFN had no clear differentiating capacity, since they were not able to modulate the expression of various hematopoietic markers, as chemical differentiation inducers did. On the other hand, neither differentiation induction nor interferon treatment could induce HLA class-II antigen expression on K562 cells. These molecules could be very faintly induced in PUTKO and DUTKO hybrids, in contrast with strong HLA class-II expression on the B parental lines. Whether these results are due to "lineage infidelity" in K562 cells or whether K562 cells represent the proliferation of HLA class-I-positive class-II-negative hematopoietic cells, with active suppression of HLA class-II antigen expression, is discussed.

HTLV-I p27rex regulates gag and env protein expression.

The experiments reported here show that the p27 product of the rex gene that is encoded by the 3' end of the human T cell leukemia virus type I (HTLV-I) is required for expression of the virion capsid and envelope proteins. The regulation of virion protein expression occurs via posttranscriptional regulation of viral mRNA. In some contexts, regulation of the virion proteins is achieved by p27rex-dependent accumulation of unspliced or singly spliced mRNAs that specify capsid or envelope proteins. In other contexts, the envelope protein production is dependent upon p27rex even in the presence of accumulation of envelope-specific mRNA. The consequences of p27 regulation for HTLV-I replication are discussed.

The potential for homeostatic regulation of the X region proteins of the human T cell leukemia virus type I.

The human T cell leukemia/lymphoma virus type I (HTLV-I) encodes two regulatory proteins, a 42 kDa trans-activator protein (p42tax) and a 27 kDa protein (p27rex), required for virus capsid protein synthesis. The experiments described here show that the p27rex protein negatively regulates the expression of the p42tax protein by suppressing the accumulation of the spliced messenger RNA from which the p42tax and p27rex proteins are made. It is proposed that such an interplay between the two regulatory genes results in a homeostatic system that may regulate the rate of viral replication as well as the growth of HTLV-I transformed cells.

Functional comparison between HTLV-I envelopes originating from TSP/HAM or ATL cell lines.

The human T-cell leukemia type I (HTLV-I) virus is associated with two different diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We have compared the viral envelopes originating from TSP/HAM and ATL patients, using the capacity of infected cells to form syncytia with receptor-expressing cells. We show that like the ATL cell lines, the TSP/HAM ones can form syncytia with a large panel of human target cells, including a variety of hematopoietic cell lines, as well as cell lines of neuroectodermal origin. None of the target cell lines tested was able to discriminate between TSP/HAM- and ATL-infected cell lines. When infected cells of TSP/HAM origin are cocultivated with cells of ATL origins, syncytia are never observed. This interference phenomenon suggests that the viruses expressed by the different cell lines utilize the same receptor.

Natural killer cell activity in human bone marrow recipients: early reappearance of peripheral natural killer activity in graft-versus-host disease.

Natural killer (NK) cell activity toward K562 target cells and antibody-dependent cell-mediated cytotoxicity (ADCC) toward L1210 cell sensitized with anti-L1210 antisera were sequentially tested in peripheral blood lymphocytes (PBLs) from 24 human bone marrow (BM) recipients. Although consistently decreased before the transplant, NK cell activity was restored in all of the patients tested that argues for a bone marrow origin of NK progenitors in humans. In patients without graft-versus-host disease (GVHD), peripheral NK cell activity remained low during the 1st month after the transplant, then rapidly increased and reached normal values usually between days 30 and 50. By contrast, peripheral ADCC appeared earlier restored (since day 13), suggesting that NK and ADCC are two distinct effector mechanisms. When restored, peripheral NK cell activity remained within normal range, except in seven cases with a drastic fall in NK cell values contemporary with a severe viral infection, mainly with cytomegalovirus (CMV). NK cells are thus suggested to play an important role in the control of viral infections in these deeply immunodepressed patients. In patients with acute GVHD, strikingly high NK values were observed early after the transplant, and during the 1st month a strong correlation did exist between high NK values and acute GVHD occurrence. These results suggest that cells involved in GVHD mechanism are able to exert NK cell activity at some stages of their maturation. The assessment of NK cell activity could be an attractive routine procedure for monitoring the prophylaxis of GVHD in human BM recipients.

HTLV-1 structural proteins.

HTLV-1 structural proteins do not appear to ensure virus transmission as efficiently as most other retrovirus structural proteins do, whereas all other retroviruses can be transmitted via either free virions or cell-to-cell contacts, infection by HTLV-1 by free virions is very inefficient, and effective infection requires the presence of HTLV-1 infected cells. This characteristic feature of HTLV-1 provides a unique tool which can be used to analyse retrovirus cellular transmission in the absence of simultaneous cell-free infection. Here we summarise what is known about HTLV-1 structural proteins and identify the questions about these proteins which remain to be answered.

The cytoplasmic domain of the human T-cell leukemia virus type I envelope can modulate envelope functions in a cell type-dependent manner.

C-terminal truncations of the human T-cell leukemia virus type I envelope affected the intracellular maturation and syncytium formation in a cell type-dependent manner. The intracytoplasmic domain appears dispensable for syncytium formation, but its truncation can modulate the envelope functionality in some cell types.

Multiple functions for the basic amino acids of the human T-cell leukemia virus type 1 matrix protein in viral transmission.

We studied the involvement of the human T-cell leukemia virus type 1 (HTLV-1) Gag matrix protein in the cell-to-cell transmission of the virus using missense mutations of the basic amino acids. These basic amino acids are clustered at the N terminus of the protein in other retroviruses and are responsible for targeting the Gag proteins to the plasma membrane. In the HTLV-bovine leukemia virus genus of retroviruses, the basic amino acids are distributed throughout the matrix protein sequence. The HTLV-1 matrix protein contains 11 such residues. A wild-type phenotype was obtained only for mutant viruses with mutations at one of two positions in the matrix protein. The phenotypes of the other nine mutant viruses showed that the basic amino acids are involved at various steps of the replication cycle, including some after membrane targeting. Most of these nine mutations allowed normal synthesis, transport, and cleavage of the Gag precursor, but particle release was greatly affected for seven of them. In addition, four mutated proteins with correct particle release and envelope glycoprotein incorporation did not however permit cell-to-cell transmission of HTLV-1. Thus, particle release, although required, is not sufficient for the cell-to-cell transmission of HTLV-1, and the basic residues of the matrix protein are involved in steps that occur after viral particle release.

Mutations introduced along the HTLV-I envelope gene result in a non-functional protein: a basis for envelope conservation?

The envelope protein of the human T-cell leukemia virus type I (HTLV-I) is highly conserved among the isolates sequenced so far, as opposed to what is observed for the human immunodeficiency virus (HIV) envelope. By linker insertion scanning, we have produced 33 random mutations along the HTLV-I envelope gene, cloned into a eukaryotic expression vector. The resulting envelope products were analysed by immunoprecipitation and syncytia formation after transfection into COS-1 cells. We show here that 25 out of 33 mutations result in a non-functional envelope product as assessed by the lack of ability to form syncytia. In the majority of these mutants, the processing of the envelope gp61 precursor into the mature gp45 and gp20 proteins was affected. We propose that conformational constraints for processing and fusion abilities tend to limit the variability of the HTLV-I envelope. In three mutants, processing was observed but no syncytia were formed. These mutations might affect regions important for HTLV-I envelope functions, such as the receptor binding region.

Human T-cell leukemia virus type I envelope protein maturation process: requirements for syncytium formation.

The human T-cell leukemia virus type I (HTLV-I) envelope protein is synthesized as a gp61 precursor product cleaved into two mature proteins, a gp45 exterior protein and a gp20 anchoring the envelope at the cell membrane. Using N-glycosylation inhibitors and site-directed mutagenesis of the potential glycosylation sites, we have studied the HTLV-I envelope intracellular maturation requirements for syncytium formation. We show here that experimental conditions resulting in the absence of precursor cleavage (tunicamycin, monensin treatments, and use of inhibitors of the reticulum steps of the N glycosylations) also result in no cell surface expression of envelope protein. The lack of syncytium formation observed in these cases is thus explained by incorrect intracellular transport. When the precursor is cleaved in the Golgi stack (no treatment or treatment with inhibitors of the Golgi steps of the N glycosylations), it is transported to the cell surface in all the cases examined. Syncytium formation is markedly reduced, however, when Golgi glycosylations are incorrect, which shows that the sugar moieties are involved in the envelope functions. Site-directed mutagenesis demonstrates that each of the five potential glycosylation sites is actually glycosylated. Glycosylation of sites 1 and 5 is required for normal maturation, whereas that of sites 2, 3, and 4 is dispensable. Glycosylation of each site, however, is required for normal syncytium formation. Altogether, the restraints exerted by the cell for the HTLV-I envelope to be transported and functional are very high, which might play a role in the observed conservation of the envelope amino acid sequence between various strains.

Target structure for natural killer cells: evidence against a unique role for transferrin receptor.

The transferrin receptor (TfR) was recently proposed as putative natural killer (NK) cell target structure. Here data are presented against this hypothesis and it is shown that low TfR expression and high NK sensitivity can occur concommitantly . K562 cells were studied at various stages of cell proliferation. No change in NK sensitivity could be observed between exponential growth and the plateau phase, whereas TfR expression completely disappeared during the latter. Protein synthesis inhibitors such as cycloheximide (1 microgram/ml, 48 h) and actinomycin D (50 micrograms/ml, 48 h), that abolished the TfR expression at the K562 cell surface, had no effect on NK sensitivity. Similarly, hemin induction (0.1 mM, 5 days) did not change NK susceptibility of K562 cells but considerably diminished TfR expression. Moreover, attempts to block NK sensitivity with anti-TfR monoclonal antibodies were unsuccessful, even when the 42.6 antibody, which is known to bind to the active site of TfR, was used. Finally, no blocking of NK sensitivity could be achieved when K562 cells were preincubated with saturating concentrations of transferrin or when transferrin was added during the NK assay. It therefore seems doubtful that TfR is the unique target structure for NK cells. It remains possible that TfR and NK target structures are often coexpressed on actively dividing cells.

Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level.

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.

Effect of a synthetic thymic factor (facteur thymique serique) on natural killer cell activity in humans.

The effect of the serum thymic factor, FTS, on human NK cells was studied. NK cell activity was measured in a 51Chromium-release assay in which effector cells were peripheral blood lymphocytes (PBL) or bone marrow (BM) lymphocytes from healthy individuals and patients, and targets were K562 cells. When added in vitro in this assay, FTS modulated NK cell activity of normal PBL. Low concentrations of FTS (10(-2) ng/ml) increased NK activity (P less than 0.001) whereas higher concentrations decreased it (P less than 0.01) for 10 and 10(2) ng/ml. FTS exhibited no effect on NK cell activity of BM lymphocytes. When administered in vivo to 4 cancer patients (10 micrograms/kg i.v. every 3 days), FTS progressively increased peripheral NK activity in two patients with low pre-treatment NK values, whereas it decreased NK activity in two patients with previously normal or high NK values. The mechanism by which FTS modulates NK cell activity is still unknown but such modulation suggests that NK cells belong in part to the T-lineage.

A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody.

An IgG2a monoclonal antibody, referred to as 12B1 and raised against the K562 cell line, reacted with adherent monocytes maintained in culture for several days but not with bone marrow or peripheral blood cells including freshly isolated monocytes. Among human leukemic cell lines, 12B1 reacted essentially with the promyelocytic HL60 cell line. 12-O-Tetradecanoylphorbol 13-acetate treatment, but no other differentiation inducer, strongly enhanced its reactivity on K562, HL60 and the histiocytic U937 cell line. Immunoperoxidase staining of sections of normal human tissues showed that 12B1 specifically recognized dendritic reticulum cells in germinal centers of lymph nodes, spleen and tonsils. The 12B1-detected antigen is a highly glycosylated polypeptide of an apparent molecular mass of 93-86 kDa. The 12B1 antigen appears to be a new glycoprotein marker shared by adherent monocytes and dendritic reticulum cells. The association of the 12B1 epitope with cells which present antigen and/or exert accessory function suggests that this molecule could play a role in these activities.

Identification of functional regions in the human T-cell leukemia virus type I SU glycoprotein.

Single conservative and nonconservative amino acid substitutions were introduced into the gp45 external envelope protein (SU) of human T-cell leukemia virus type I (HTLV-I). The mutated amino acids were those identified as being conserved in HTLV-I, HTLV-II, and simian T-cell leukemia virus type I (but not in bovine leukemia virus). The mutated envelopes were tested for intracellular maturation and for function. Mutants with three major phenotypes could be defined: (i) 9 mutants with a wild-type phenotype, which included most of the conservative amino acid changes (five of seven) distributed throughout the SU protein; (ii) 8 mutants with affected intracellular maturation, 6 of which define a region in the central part of the SU protein essential for correct folding of the protein; and (iii) 13 mutants with normal intracellular maturation but impaired syncytium formation. These mutations likely affect the receptor binding step or postbinding events required for fusion. Five of these mutations are located between amino acids 75 and 101 of the SU protein, in the amino-terminal third of the molecule. The other mutations involve positions 170, 181, 195, 197, 208, 233, and 286, suggesting that two other domains, one central and one carboxy terminal, are involved in HTLV-I envelope functions.

The HTLV-I envelope glycoproteins: structure and functions.

The human T-cell lymphotropic virus type I (HTLV-I) envelope has a structural organization shared by all retroviral envelopes, which contain two mature viral glycoproteins deriving from a common precursor: an external surface protein (SU), associated with a transmembrane protein (TM) responsible for anchoring the SU-TM complex at the cell surface or in the viral envelope. Our understanding of the tertiary structure of these proteins is extremely poor. The intracellular maturation follows the normal cellular secretory pathway, resulting in expression of the mature glycoproteins at the cell surface. The five potential N-glycosylation sites are glycosylated. Most mutations artificially introduced into the glycoproteins result in loss of function, mostly due to abnormal intracellular maturation. This probably indicates a very compact structure of these proteins, where the entire structure is involved in correct conformation. Studies using neutralizing antibodies or mutagenesis have defined functional domains in the SU protein, which is responsible for receptor binding. These domains occur throughout the SU glycoprotein. Sequence analysis of the HTLV-I TM predicts a structure, and probably functions, similar to other retrovirus TMs: involvement of this glycoprotein in the different oligomerization steps leading to a fusogenic SU-TM complex and in the fusion process itself. These features remain to be proven, and it is not yet understood why the free HTLV-I viral particle is not infectious.