A-104 Tracing the intracellular journey of HTLV-1 HBZ during infection: From asymptomatic carriers to HAM/TSP ending to ATL: A one-way ticket?
Authors: Baratella Marco , Forlani Greta , Gessain Antoine , Jacobson Steve , Accolla Roberto 
Published: April 2019

HTLV-1 is the etiological agent of Adult T cell Leukemia (ATL) and of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) neurologic disorder. Although substantial progress has been made in understanding some cellular and molecular mechanisms underlying the pathology of the neoplastic and neurological diseases, still the intimate reason why some HTLV-1 infected patients develop HAM/TSP and others progress toward the neoplastic state are largely elusive. Two viral proteins, Tax-1 and HBZ, play an important role in both diseases. For example, Tax-1, the crucial transactivator needed for viral replication, has been implicated in the initiation but not in the maintenance of neoplastic process since leukemic cells may not express it. On the other hand, HBZ seems to be expressed throughout the infection and, importantly, is always expressed in leukemic cells. Nevertheless, the precise mechanism through which HBZ participates to initiation and/or maintenance of leukemic state is still unclear, as it is unclear the association of HBZ expression with initiation and/or maintenance of the inflammatory syndrome leading to HAM/TSP. Studies of this aspect have been partially prevented by the lack of suitable reagents to detect endogenous HBZ protein during the various phases of HTLV-1 infection and disease development. The recent isolation of a specific monoclonal antibody against HBZ, the 4D4-F3 mAb, by our group has allowed to precisely quantify and determine the subcellular distribution of the viral protein. We have demonstrated that HBZ, previously considered an exclusive nuclear protein, is mainly localized in the cytoplasm of PBMCs from HAM/TSP patients. Here, the analysis of a larger panel of patients has added new relevant informations on this point.

PBMC from 13 HTLV-1 infected asymptomatic carriers (AC), 14 HAM/TSP and 4 ATL patients were analysed. Expression and subcellular distribution of endogenous HBZ and Tax-1 proteins were assessed by confocal microscopy with the 4D4-F3 and A51-2 mAbs, respectively. HBZ expression was confined to the cytoplasm of PBMC of HAM/TSP patients, appearing as discrete dots. The percentage of HBZ-positive cells ranged between 1% and 10% of total PBMC with the exception of one negative patient. Tax-1 was expressed in all HAM/TSP cases analyzed, ranging from 2% to 15% of total PBMC. Tax-1 was preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. Interestingly, only in 1 case co-expression of HBZ and Tax-1 was observed in the same cells. In this unique case, however, although 8% and 15% of the cells were positive for HBZ and Tax-1, respectively, only 1% of cells co-expressed the 2 viral proteins. Of particular interest was the finding that PBMC of 6 out of 13 AC expressed HBZ (1%–4% of the cells) and again with an exclusive cytoplasmic localization. Tax-1 was expressed in 10 out of 13 cases (1%–11% of the cells). Only in 2 AC, HBZ and Tax-1 were found to be co-expressed in a very limited proportion of cells. The study of 4 distinct cases of ATL demonstrated, instead, that HBZ protein was expressed in the vast majority of the leukemic cells and importantly with an exclusive nuclear localization. Interestingly, none of these ATL cases were found to express Tax-1 protein.

These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the 2 diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that in AC, HBZ is expressed only in the cytoplasm strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1infection. Future studies will clarify whether the distinct HBZ intracellular localization is simply a marker or a causative event of disease evolution.