Endoplasmic reticulum and cis-Golgi localization of human T-lymphotropic virus type 1 p12^I: Association with calreticulin and calnexin

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12^I, in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12^I may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12¹ in cell activation, we characterized the subcellular localization of p12^I in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12^I accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12^I was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12¹ with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12^I directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12^I in the establishment of HTLV-1 infection by activation of host cells.