IL-2 withdrawal induces HTLV-1 expression through p38 activation in ATL cell lines.

Expression of human T-cell leukemia virus type-1 (HTLV-1) in adult T-cell leukemia (ATL) cells is known to be marginal in vivo and inducible in short-term culture. In this study, we demonstrated that withdrawal of interleukin (IL)-2 from IL-2-dependent ATL cell lines resulted in induction of HTLV-1 mRNA and protein expression, and that viral induction was associated with phosphorylation of the stress kinase p38 and its downstream CREB. Pharmacological inhibitors of the p38 pathway suppressed viral expression induced by IL-2 depletion. These results indicate that the stress-induced p38 pathway might up-regulate HTLV-1 gene expression through at least CREB activation.

Involvement of p38 MAPK signaling pathway in IFN-gamma and HTLV-I expression in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis.

We analyzed the relationship between the expression of interferon (IFN)-gamma and HTLV-I p19 antigen and activation of p38 mitogen-activated protein kinase (p38 MAPK) in two HTLV-I-infected T cell lines derived from two patients (HCT-1 and HCT-4) with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and three HTLV-I-infected T cell lines derived from three patients with adult T cell leukemia (ATL). Expression of phosphorylated (activated)-p38 MAPK was markedly increased concomitant with high levels of both IFN-gamma and HTLV-I p19 antigen expression in both HCT-1 and HCT-4 compared with cell lines derived from ATL patients. Treatment with SB203580, a specific inhibitor of p38 MAPK, suppressed IFN-gamma and HTLV-I p19 antigen expression levels in HCT-1, HCT-4 and peripheral blood CD4(+) T cells of HAM/TSP patients. These findings strongly suggest that activation of p38 MAPK signaling pathway is involved in the up-regulation of IFN-gamma expression with high HTLV-I proviral load in HAM/TSP patients.

Soluble recombinant HTLV-1 surface glycoprotein competitively inhibits syncytia formation and viral infection of cells.

Efficient entry into, and infection of, human cells by human T-cell leukaemia virus type-1 (HTLV-1) is mediated by the viral envelope glycoproteins, gp46 and gp21. The gp46 surface glycoprotein binds to an as yet unidentified cell surface receptor, thereby, allowing the gp21 transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral penetration and entry into the host cell cannot occur. The envelope glycoproteins are also a major target for neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and represent ideal constituents for a recombinant HTLV-1 vaccine. Given the importance of the envelope proteins in HTLV-1 pathogenesis there is increasing interest in obtaining sufficient quantities of these proteins for biochemical, biophysical and biological analyses. We have now developed a system for production of large amounts of a glycosylated and functional form of soluble recombinant gp46 (sRgp46), and have used this recombinant material for analysis of envelope function and receptor binding activity. We find that, the sRgp46 molecules expressed in our system are immunologically indistinguishable from the native virally expressed surface glycoproteins; that sRgp46 binds to T-cells in a dose dependent and saturable manner; and that cell surface binding by sRgp46 can be inhibited by neutralising antibodies. Importantly, we demonstrate that these sRgp46 molecules potently inhibit syncytia formation and viral infection of target cells, and that regions outwith the SU domain of envelope are not required for binding to target cells or for inhibiting membrane fusion. The sRgp46 produced in our study will provide new opportunities to investigate envelope-receptor interactions, and will be of utility in defining the conformationally sensitive antigenic determinants of the HTLV-1 surface glycoprotein.

Isolation and molecular characterization of a human T-cell lymphotropic virus type II (HTLV-II), subtype B, from a healthy Pygmy living in a remote area of Cameroon: an ancient origin for HTLV-II in Africa.

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-II-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-II and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-I gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-II-specific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus.

Calcium-mediated inhibition of phorbol ester and Tax trans-activation of the human T cell leukaemia virus type 1.

Human Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate-and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin-induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.

Spontaneous lymphocyte proliferation in human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infection: T-cell subset responses and their relationships to the presence of provirus and viral antigen production.

Spontaneous lymphocyte proliferation (SLP) during in vitro culture of mononuclear cells (MCs) characterizes over half of asymptomatic individuals infected with human T-cell lymphotropic virus type I (HTLV-I) or HTLV-II. Both CD4 and CD8 T-cell subsets within MC cultures are activated during SLP, as judged by high-density CD25 (CD25bright) expression; it is unclear, however, whether both cell subsets can directly undergo SLP. In the present investigation, the SLP capacities of purified CD8 and CD4 cells were examined in subjects infected with HTLV-I (n = 19) or HTLV-II (n = 54) in relation to the SLP status of MCs from each subject. No increase in SLP was observed for CD8 or CD4 cells from SLP-negative (SLP-) HTLV-infected subjects, whereas robust SLP characterized CD8 cells from all SLP-positive (SLP+) individuals, regardless of HTLV type. In contrast, SLP+ CD4 cells characterized only 23% (7 of 31) of HTLV-II+ SLP+ individuals, whereas SLP+ CD4 cells characterized 100% of HTLV-I+ SLP+ individuals. In cocultures of HTLV-II+ SLP+ CD8 cells and autologous SLP- CD4 cells, sizable proportions of both CD8 cells and CD4 cells coexpressed CD25bright, suggesting that SLP- CD4 cells were activated in the presence of SLP+ CD8 cells. PCR analysis for tax sequences detected provirus in most CD4- and CD8-cell preparations from HTLV-seropositive individuals, regardless of type and the SLP status of cell subsets. To determine whether SLP was associated with activation of viral genes, levels of HTLV-I and HTLV-II core antigen (Ag) in supernatants were measured. Viral Ag production and SLP responses were significantly correlated for both CD4 and CD8 cells in both HTLV-I and HTLV-II infections. However, inhibition of CD8- or CD4-cell SLP by cyclosporin A or anti-Tac (anti-CD25) did not reduce Ag production, indicating that Ag production is not coupled to SLP. These findings show that CD4 cells from SLP+ HTLV-I+ and SLP+ HTLV-II+ individuals differ in SLP capacity, that the absence of SLP does not indicate a lack of infection, and that production of viral Ag is associated with, but not dependent on, SLP.

Transient HTLV-I infection of a human glioma cell line following cell-free exposure.

The human T-lymphotropic virus type I (HTLV-I) has been recently associated with cases of tropical spastic paraparesis and human myelopathy. In order to study whether cells of neuroectodermic origin were susceptible to HTLV-I infection, a human glioma cell line T67 was exposed in vitro to HTLV-I by a cell-free method of virus transmission. The presence of HTLV-I proviral DNA was analyzed by polymerase chain reaction 3, 7, and 14 days after infection. The results showed the presence of LTR, pol, and tax sequences within glioma cell line 3 days after the infection. However after 7 and 14 days, detection of HTLV-I sequences remarkably decreased. P19 expression peaked 7 days after infection and decreased in the following week. These data provide evidence that cell-free transmission of HTLV-I results in transient infection of cells of glial origin.

In vivo infection of human T-cell leukemia virus type I in non-T cells.

Using three different procedures, immunomagnetic isolation, FACS and panning, each of the T4 cell, T8 cell, monocyte, and B cell population was fractionated from peripheral blood mononuclear cells (PBMC) obtained from 10 human T cell leukemia virus type I (HTLV-I)-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) cases, 3 adult T cell leukemia and lymphoma (ATLL) cases and 9 HTLV-I-infected healthy carriers. DNA from each fraction was then subjected to the quantitative polymerase chain reaction. Although T4 cells possessed the highest copy number of HTLV-I in general, we also detected a significant amount of HTLV-I provirus in T8 cells, monocytes, and B cells. To confirm virus infection of the non-T cell populations, especially the monocyte fraction, we performed further experiments and the following results were obtained: (1) HTLV-I tax RNA expression was confirmed simultaneously in the RNA of monocytes that were negative for T cell receptor beta chain (TCR beta) expression in vivo. (2) We cultured these monocytes for a short period of time and detected HTLV-I antigen expression in non-specific esterase-positive monocyte/macrophage cells. Our data indicate that HTLV-I has a broad host range in vivo and that monocytes or cells of monocyte lineage such as tissue macrophages might comprise a virus reservoir in vivo.

Identification of a putative cellular receptor for HTLV-I by a monoclonal antibody, Mab 34-23.

The ability of HTLV-I to infect cells is presumed to be dependent, in some part, on the attachment of the virus to a target cell via a specific cell surface receptor which is, as yet, unknown. Here we present evidence that a monoclonal antibody, Mab 34-23, inhibits the binding of HTLV-I to IL-2 and phytohemagglutinin-activated peripheral blood mononuclear cells and also inhibits virus entry into these cells. Analysis of a variety of target cells, including a human:mouse somatic hybrid which contains only human chromosome 17q, indicates that the binding of Mab 34-23 correlates with HTLV-I adsorption and entry. SDS-PAGE and Western blot analysis show that Mab 34-23 binds to four major proteins of MW 31, 45, 55, and 70 kDa and this binding can be inhibited by HTLV-I and not HIV proteins. HTLV-I virions bind to proteins of similar molecular weight and virus-binding to these proteins can be inhibited by preincubation with Mab 34-23. These data suggest that Mab 34-23 may identify a specific cell surface receptor(s) for HTLV-I.