Human T-cell lymphotropic virus type 1 p12(I) expression increases cytoplasmic calcium to enhance the activation of nuclear factor of activated T cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) establishes persistent infection and is associated with lymphoproliferative or neurodegenerative diseases. As a complex retrovirus, HTLV-1 contains typical structural and enzymatic genes, as well as regulatory and accessory genes encoded in the pX region. The early events necessary for HTLV-1 to establish infection in lymphocytes, its primary target cells, remain unresolved. Recent studies have demonstrated the importance of regulatory and accessory gene products in determining this virus-host interaction. Among these, pX open reading frame I, which encodes two proteins, p12(I) and p27(I), is required for establishing persistent infection in vivo and for infection in quiescent primary lymphocytes. In addition, p12(I) localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus and associates with a calcium binding protein, calreticulin. We recently reported that p12(I) expression induces the calcium-responsive T-cell transcription factor, nuclear factor of activated T cells (NFAT), in the presence of phorbol ester activation. Based on these studies, we hypothesize that p12(I) may modulate calcium release from the ER. Here, we report that p12(I) expression increases basal cytoplasmic calcium and concurrently diminishes calcium available for release from the ER stores. Overexpression of calreticulin, a calcium buffer protein, blocked p12(I)-mediated NFAT activation independently of its ability to bind p12(I). Chemical inhibition studies using inhibitors of inositol 1,4,5-triphosphate receptor and calcium release-activated calcium channels suggest that inositol 1,4,5-triphosphate receptor in the ER membrane and calcium release-activated calcium channels in the plasma membrane contribute to p12(I)-mediated NFAT activation. Collectively, our results are the first to demonstrate the role of p12(I) in elevating cytoplasmic calcium, an antecedent to T-cell activation, and further support the important role of this accessory protein in the early events of HTLV-1 infection.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Kinetics study of human retrovirus antigens expression in T lymphocytic cell lines by indirect immunofluorescence assay.

Indirect immunofluorescence assay (IFA) is a well-accepted assay for the confirmation of human retrovirus infection. Fluctuations in HIV-1 antigen expression in infected E-B2 cells depending on several factors have been reported. Cells kept in log phase expressed the highest levels of viral antigen. Thus, we studied the time kinetics of IFA positivity in MT-2 (HTLV-I), MO-T (HTLV-II), CEM, and H9 (HIV-1) cell lines. Uninfected T cell line, HT, was used as nonspecific control. Reference HTLV-I/II and HIV-1 serum panels from the Centers for Disease Control and Prevention (CDC) were tested by conventional IFA procedure on slides of each cell line made on different days. On the second day after subculture, HTLV-I strongly positive sera reacted on MT-2 and MO-T cells with a bright pericytoplasmic fluorescence pattern. Weakly positive sera showed a faint staining from the fifth day on, when all the sera showed the highest degree of fluorescence. With HIV-1 cell lines, sera predominantly reacted with a diffuse cytoplasmic pattern, although some sera showed a granular and pericytoplasmic capping staining. The highest degree of fluorescence was found at 3-5 days after subculture. We demonstrated that the sensitivity of the IFA for the detection of antibodies against human retroviruses depended on the day when the slides were assayed and on the serum antibody titer. The fifth day was the most appropriate for HTLV-I/II and HIV-1/H9 systems, whereas for HIV-1/CEM, the fourth day was better. Furthermore, the intensity of the immunofluorescence pattern differed with the antibody titers and the level of antigens expressed on the four cell lines studied. The IFA, improved in our laboratory, proved to be very sensitive, specific, and rapid and could be used as a supplementary/confirmatory assay for retrovirus infection.

Induction of HLA class I and class II expression in human T-lymphotropic virus type I-infected neuroblastoma cells.

Human T-lymphotropic virus type I (HTLV-I) is associated with a neurologic disease, HTLV-I-associated myelopathy-tropical spastic paraparesis, in which both pathological and immunological changes are observed within the central nervous system. The pathogenesis of infection in HTLV-I-associated myopathy-tropical spastic paraparesis is not well understood with respect to the cell tropism of HTLV-I and its relationship to the destruction of neural elements. In this study, neuroblastoma cells were infected with HTLV-I by coculturing with HUT-102 cells to demonstrate that cells of neuronal origin are susceptible to this retroviral infection. HTLV-I infection of the neuroblastoma cells was confirmed by verifying the presence of HTLV-I gp46 surface antigens by flow cytometry and by verifying the presence of HTLV-I pX RNA by Northern (RNA) blotting and in situ hybridization techniques. To determine whether HTLV-I infection could potentially lead to changes in cell surface recognition by the immune system, the infected neuroblastoma cells were analyzed for altered HLA expression. The HTLV-I-infected, cocultured neuroblastoma cells were shown, through cell surface antigen expression and RNA transcripts, to express HLA classes I and II. In contrast, cocultured neuroblastoma cells that did not become infected with HTLV-I expressed only HLA class I. HLA class I expression was enhanced by the cytokines tumor necrosis factor alpha and gamma interferon and in the presence of HUT-102 supernatant. In this system, expression of HLA class I and II molecules appeared to be regulated by different mechanisms. HLA class I expression was probably induced by cytokines present in the HUT-102 supernatant and was not dependent on HTLV-I infection. HLA class II expression required HTLV-I infection of the cells. The observation of HTLV-I infection leading to HLA induction in these neuroblastoma cells provides a possible mechanism for immunologic recognition of infected neuronal cells.