Human T cell leukemia virus type I (HTLV-I) is an RNA tumor virus identified as a causative agent of adult T cell leukemia and shown to be associated with several neurological and chronic disorders. An estimated 10 to 20 million people are infected by HTLV-I. Of these only a small percentage develop symptomatic disease, the majority of infections are silent. The mechanisms of the development and pathogenesis of HTLV-I associated diseases remain unknown. Studies of human viral isolates as well as evidence from animal models yield no data to correlate disease with genetic variation in the virus. Pulmonary angioinvasive T-cell lymphoma can be a feature of HTLV-1 infection and adult T-cell leukemia and lymphoma (ATLL). A similar T-cell angioinvasion also occurs in some non-Hodgkin?s lymphomas not associated with virus. To evaluate a model for studying malignant T-cell angioinvasive biology, rabbits were injected with either RH/K34 HTLV-1-transformed rabbit T-cells, or cultured primary lymphocytes, and evaluated for evidence of infection and disease. Transformed T-cells infiltrated pulmonary vascular walls and separated endothelium from the basement membrane zone. This change, including narrowed vessel lumens and perivascular cuffing by neoplastic T-cells, as well as multifocal consolidation of pulmonary parenchyma became more severe with time post inoculation. The severity of lesions correlated with increased HTLV provirus load. HTLV-1 was most concentrated in tissues with T-cell lymphoma-like infiltration including lung, liver and kidney. Controls inoculated with uninfected cells lacked these changes. Rabbits given RH/K34 develop infection and lesions closely mimicking some ATLL patients. The morphology of the lesions seen in these rabbits duplicates the invasive biology of malignant T-cells trafficking into blood vessels. We previously reported a flow cytometric assay that measures binding of human T-lymphotropic virus type 1 (HTLV-1) virions to target cells. This assay was used to investigate the binding process and to screen for compounds affecting viral binding. The cell-free virus preparations used here mediated infection of rabbits and in vitro infection of certain target cell lines. Optimum binding was observed with T-cell lines while monocytic lines showed weak but positive viral attachment. Results using human T-cell targets showed that adenosine receptor type 2 antagonists effectively inhibit viral binding at concentrations below 10 micromolar. No inhibition was seen when antagonist was used to pretreat cells or was added post binding, suggesting direct interference with virus attachment. Additional compounds affecting viral binding have been found this year. These inhibitors of viral binding also inhibit glucose uptake meditated by the glucose transporter, GLUT1. We have demonstrated that disruption of lipid rafts by cholesterol depletion compromises viral binding but does not affect glucose uptake. Comparison of GLUT1 expression in monocytic cell lines which bind virus weakly and T cell lines exhibiting the highest levels of viral binding revealed little difference in expression. These data suggests that viral binding is mediated by a multi-component receptor which may include GLUT1. Two real time PCR assays for quantification of HTLV-1 have been adapted for use in rabbits. These multiplex quantitative assays utilize rabbit beta globin as an internal control. The first assay for determining proviral load is a reliable and accurate tool measuring the distribution of proviral DNA in the PBMC and the organs of HTLV-1 infected rabbits. With this assay five copies of proviral HTLV-I DNA can be reliably detected and the assay has a six log range. The second assay is a quantitative assay to measure the level of HTLV-I virus in rabbit plasma. It is a single-tube real-time reverse transcription-PCR (RT-PCR) assay based on a fluorogenic probe and primers directed to highly conserved sequences in the gag region of the HTLV-I genome. As few as fifty HTLV-1 copies per ml of plasma can be detected using this assay.
