Studies of HTLV-1p12(I) in calcium mediated signaling and T cell activation

Human T lymphotropic virus type 1 (HTLV-1) causes a variety of lymphoproliferative diseases including adult T-cell leukemia/lymphoma (ATLL). The mechanisms of early viral infection and subsequent T cell activation remain unclear. The complex retrovirus pX ORF I encoded protein, p12I, is critical for viral infection in vivo and in quiescent lymphocytes. HTLV-1 p12 I associates with interleukin-2 (IL-2) receptor beta and gamma chain and enhances Stat5 activation. Expression of p12I in T cells synergistically activates n&barbelow;uclear f&barbelow;actor of a&barbelow;ctivated T&barbelow; cells (NFAT) with Ras/MAPK pathway activation.; To characterize the subcellular localization and identify potential binding partners of p12I, we fractionated lysates of p12I transfected 293T cells and demonstrated that the viral protein accumulated in the endoplasmic reticulum (ER) and cis-Golgi apparatus. Immunofluorescence and immunoelectron microscopic studies further confirmed p12I localization. Moreover, p12I was associated with the calcium-modulating proteins calreticulin and calnexin in ER, suggesting a role of the viral protein in cellular calcium regulation.; We further tested the potential role of p12I on calcium mediated signaling. Expression of p12I increased basal calcium, decreased calcium available for release from ER stores and elevated extracellular calcium entry. These data indicate that p12I regulates ER calcium homeostasis to activate the downstream transcription factor, NFAT. Using chemical inhibitors, we demonstrated that both inositol 1,4,5-triphosphate receptor (IP3R) in ER membrane and c&barbelow;alcium r&barbelow;elease-a&barbelow;ctivated c&barbelow;alcium (CRAC) channels in plasma membrane contributed to p12I mediated NFAT activation.; Lastly, we tested the role of p12I on T cell activation. Expression of p12I in Jurkat T cells via a lentiviral vector enhanced interleukin-2 production when cell surface receptors were activated. The increased IL-2 secretion mediated by p12I was calcium pathway dependent as revealed by the blocking effect of calcium chelator and calcineurin inhibitor. This enhanced IL-2 production was most likely mediated by hyperactivated NFAT induced by p12I expression. Moreover, ER retargeting of a p12I truncated mutant demonstrated the requirement of ER localization for p12I mediated NFAT activation.; In conclusion, HTLV-1 p12I modulates calcium-mediated signals and facilitates T cell activation, providing initial requirements for HTLV-1 to establish productive infection of lymphocytes.