Expression, subcellular localization and quantification of endogenous HBZ protein in HTLV-1 infected and in ATLL tumor cells as assessed by a newly generated anti-HBZ monoclonal antibody

Although the major documented viral oncogenic product of HTLV-1 is the Tax-1 protein, it has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, is also involved in the pathogenesis of Adult T cell Leukemia/Lymphoma (ATLL). The full role played by HBZ in oncogenesis is still to be explored in detail mainly owing to the unavailability of tools to study this protein in naturally infected cells and in ATLL cells. By the use of the first reported monoclonal antibody against HBZ, generated in our laboratory, we have carefully analyzed endogenous HBZ expression and subcellular localization. Endogenous HBZ is expressed in discrete speckle-like structures in the nucleus. HBZ localization overlaps partially with CBP and p300, but only minimally, if any, with JunB, JunD and CREB2. By confocal microscopy important differences were observed between previously reported distributions with GFP-HBZ chimeric proteins and the distributions detected by the mAb. A careful comparative analysis performed by quantitative western blotting, using purified HBZ recombinant protein as control and cell extracts from HTLV-1 infected C5MJ and ATLL ATL-2s cells indicates an average of 0,384x10-3 pg/cell corresponding to 9400 molecules/cell, and 0,570x10-3 pg/cell corresponding to 6300 molecules/cell, respectively. These values are 1000 fold less than those found in transiently HBZ transfected cells used by most authors to define the pattern of HBZ expression and its interaction with cellular factors. These studies open the way to more precisely assess the role of HBZ in the HTLV-1-mediated oncogenic process