The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may represent a critical event in the lymphoproliferation induced by this human retrovirus, leading to leukemia.

Transcriptional activation of the vascular cell adhesion molecule-1 gene in T lymphocytes expressing human T-cell leukemia virus type 1 Tax protein.

Recruitment and extravasation of T cells through the blood-brain barrier are favored by adhesion molecule-mediated interactions of circulating T cells with endothelial cells. Since a common pathological finding in human T-cell leukemia virus type 1 (HTLV-1)-associated diseases is the infiltration of HTLV-1-infected T lymphocytes into various organs, we have looked for the profile of adhesion molecules expressed by HTLV-1-transformed T cells. Flow cytometry analysis indicated that these cells were expressing high levels of vascular cell adhesion molecule 1 (VCAM-1 [CD106]), a 110-kDa member of the immunoglobulin gene superfamily, first identified on endothelial cells stimulated with inflammatory cytokines. This adhesion molecule was also expressed by T cells obtained from one patient with HTLV-1-associated myelopathy/tropical spastic paraparesis but not by activated T cells isolated from one normal blood donor. The role of the viral trans-activator Tax protein in the induction of VCAM-1 was first indicated by the detection of this adhesion molecule on Jurkat T-cell clones stably expressing the tax gene. The effect of Tax on VCAM-1 gene transcription was next confirmed in JPX-9 cells, a subclone of Jurkat cells, carrying the tax sequences under the control of an inducible promoter. Furthermore, deletion and mutation analyses of the VCAM-1 promoter performed with chloramphenicol acetyltransferase constructs revealed that Tax was trans activating the VCAM-1 promoter via two NF-kappaB sites present at bp -72 and -57 in the VCAM-1 gene promoter, with both of them being required for the Tax-induced expression of this adhesion molecule. Finally, gel mobility shift assays demonstrated the nuclear translocation of proteins specifically bound to these two NF-kappaB motifs, confirming that VCAM-1 was induced on Tax-expressing cells in a kappaB-dependent manner. Collectively, these results therefore suggest that the exclusive Tax-induced expression of VCAM-1 on T cells may represent a pivotal event in the progression of HTLV-1-associated diseases.

Human T cell leukaemia virus type I env gene-transfected HeLa cells display a decrease in cell fusion ability.

The envelope (env) gene of human T cell leukaemia virus type I (HTLV-I) was inserted into an expression vector, referred to as phMTenv, under the transcriptional control of the human metallothionein IIa gene promoter (hMT-IIa). When this vector was transiently transfected in HeLa cells treated with hMT-IIa inducers, formation of multinucleated cells was observed, indicating the expression of functional surface and transmembrane glycoproteins. Of several HeLa cell clones transfected with phMTenv together with a plasmid carrying the neomycin resistance gene and isolated after selection in G418-containing medium, env mRNA was detected in only two, in the presence of hMT-IIa inducers. Viral glycoproteins were found to be weakly expressed as detected in immunoprecipitation assays of 125I-surface-labelled cells. These env-transfected HeLa cell clones, although unable to form syncytia when cocultivated with untransfected control HeLa cells, retained the capacity to fuse with HTLV-I-producing C91PL T cells. However, a significant decrease in their fusogenic ability was observed, after treatment with hMT-IIa inducers. Under identical experimental conditions, control HeLa cell clones stably transformed with the same plasmid, but lacking the env gene, were still able to fuse with C91PL cells. These observations suggest that a post-transcriptional step in HTLV-I env expression is impaired, probably leading to the establishment of superinfection interference.

Critical involvement of human T cell leukaemia virus type I virions in mediating the viral mitogenic effect.

Human T cell leukaemia virus type I (HTLV-I) is a direct activator of human resting T lymphocytes. The present study was undertaken to delineate further the role of viral particles and to define the involvement of envelope glycoproteins in the induction of T cell mitogenic stimulation. Virus-producing cells treated with paraformaldehyde (PFA) were found to be unable to induce the formation of syncytia, but still able to trigger the proliferation of resting T cells. Likewise, PFA-treated virus particles were still mitogenic. These results suggest that the mitogenic event is triggered before the fusion of the envelope with the cell membrane. Furthermore, HTLV-I envelope-expressing cells obtained after infection of C8166/45 cells (HTLV-I-transformed, but defective in virion production) with an HTLV-I envelope recombinant vaccinia virus were unable to activate normal T cells. Human immuno-deficiency virus type 1 particles produced by C8166/45 cells were also devoid of mitogenic ability. However, when HTLV-I viral preparations were purified by chromatography, only the virion-containing fractions were found to be mitogenic for human resting T lymphocytes. This mitogenic activity was partially abolished by preincubating the purified virus with a monoclonal antibody directed to the surface envelope glycoprotein. Finally, treatment of HTLV-I-transformed cells by tunicamycin, an inhibitor of N-linked glycosylation, led to the production of virus particles with a decreased mitogenic activity. Collectively, these observations suggest that the HTLV-I mitogenic activity is triggered by the contact of HTLV-I virions with T cells.