Cytoplasmic localization of HTLV-1 HBZ oncoprotein: a biomarker of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of T cell neoplasia called Adult T cell Leukaemia (ATL) and of a neurologic disorder designated HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HBZ oncoprotein encoded by the minus strand of the HTLV-1 is thought to play an important role in both diseases. The recent isolation in our laboratory of the first described monoclonal antibody against HBZ protein has now permitted to investigate in detail the cellular and biochemical features of endogenous HBZ. In this direction our laboratory has recently established that HBZ is a nuclear protein in cells of ATL patients. My thesis was predominantly focused in expanding the analysis particularly in HAM/TSP patients, to assess similarities and/or diversities of HBZ subcellular distribution with respect to ATL patients and asymptomatic HTLV-1 carriers. Expression and localization of HBZ in peripheral blood mononuclear cells (PBMC) of ATL, HAM/TSP patients and in HTLV-1 asymptomatic carriers (AC) was analyzed by immunohistochemistry and confocal microscopy using the anti-HBZ 4D4-F3 mAb. Analysis of patients with HAM/TSP unequivocally showed that HBZ-positive cells presented an exclusive, never reported, cytoplasmic localization of the viral oncogenic protein. Interestingly, experiment with leptomycin B indicated that HBZ could not shuttle between cytoplasm and nucleus. This strict HBZ cytoplasmic localization was at variance with the distribution of the other HTLV-1 oncogenic protein, Tax-1, that could localize both in the cytoplasm and the nucleus, and could be sequestered totally in the nucleus after leptomycin B treatment of the cells. Additional extensive analysis of cells from ATL patients and asymptomatic recipients confirmed, instead, a nuclear localization of HBZ. I further supported this finding by studying HBZ and Tax-1 in the CIB cell line, a CD4+ IL-2-dependent T cell line derived from an HAM/TSP patient. The vast majority of CIB cells express significant amounts of HBZ exclusively in the cytoplasm, mostly in a speckle-like fashion. Tax-1 instead was expressed in 30% of the cells, either as a diffuse reticulum or distributed in a speckled-like fashion mainly in the cytoplasm. Interestingly, in cells co-expressing cytoplasmic HBZ and Tax-1, the two proteins did not co-localize, suggesting that they do not interact in vivo. Our results establish for the first time a distinctive and diverse pattern of sub-cellular localization of endogenous HBZ protein. Furthermore, and of potential importance in the pathogenesis of HTLV-1-associated diseases, our data suggest that the endogenous localization of HBZ protein in different cellular compartments may correlate with the different forms of the HTLV-1-mediated diseases.