HTLV-1 and T cell-polarity regulators: Interference of Tax with the subcellular localization of Disc Large 1.

MARZIALI FEDERICO, BUGNON VALDANO M, BRUNET AVALOS C, MORIENA L, CAVATORTA AL, GARDIOL D

Sienna

Workshop; EMBO Workshop Lymphocyte antigen receptor signaling; 2018

EMBO

Human T cell-leukemia virus type I (HLTV-1) is a retrovirus that infects 10-20 million people around the world. Although most of the infected people are asymptomatic, a 4% develop a neuro-inflammatory disease known as HTLV-1-associated myelopathy/ Tropical spastic paraparesis (HAM/TSP) and other associated autoimmune diseases. HAM/TSP is characterized by the infiltration of infected T cells into the central nervous system and severe inflammatory responses. Regulatory T cells (Tregs) were reported as the main reservoir of HTLV-1 in HAM/TSP patients and the infection was demonstrated to lead to Treg dysfunction. Evidence suggests a role of the viral protein Tax, which has been reported to interfere with cellular partners and participate in HTLV-1 replication. However, the complete scenario that describes Tax contribution to Treg dysfunction remains unknown. Treg signaling relies on complexes regulated by scaffolding proteins, like the polarity protein Disc Large 1 (DLG1). DLG1 is especially important, with a high amount of it being recruited to the immunological synapse of Tregs. Here, DLG1 activates NFAT and downregulates PI3K/Akt pathways, both necessary to maintain Treg phenotype. Interestingly, biochemical assays demonstrated that DLG1 can associate with Tax, however, the role of this association in viral pathogenesis has not been investigated.In this work, we aimed at studying Tax-DLG1 association in vivo to analyze possible contributions of polarity proteins targeting to Tregs disfunction. To this end, as a first approach we conducted transfection experiments, biochemical assays and confocal microscopy to analyze Tax influence on DLG1 expression in epithelial cell models. By doing so, we discovered that Tax clearly relocates DLG1 from cell borders to a defined perinuclear spot and redistributes DLG1 to the insoluble fraction of protein extracts. Furthermore, by FRET analysis we were able to demonstrate for the first time that Tax directly interacts with DLG1 in vivo. After that we analyzed co-localization of Tax-DLG1 complexes with organelle markers and detected a clear co-localization with the centrosome in close association with cis-Golgi. Having found this, we then extrapolated our analysis to lymphoid cell models. In this regard, we transfected lymphoid cells with Tax expressing plasmids and also analyzed HTLV-1 naturally infected cells. We obtained similar results, confirming therefore the targeting of DLG1 by Tax to such specific region of the cell. Our results demonstrate that Tax is able to directly interact with DLG1 and recruit it to the centrosome/cis-Golgi region. Considering a Treg model, this could impact negatively on DLG1- dependent signaling pathways and therefore explain in part the deficiency in Treg functions observed in HAM/TSP as well as the onset of autoimmune diseases. On the other hand, DLG1 sequestration to the centrosome might have a role in centrosome polarization, a process mediated by Tax and necessary for HTLV-1 replication and transmission. Although preliminary, our data provide new insight about possible mechanism underlaying pathological states of Tregs in HTLV-1 infected people.