p8 expression in Jurkat T cells significantly augmented the percentage of cells with conduits (Fig. strategies that may limit the expansion of infected cells in HTLV-1 carriers and decrease HTLV-1associated morbidity. Keywords:human leukemia retrovirus,orf-I The human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1associated myelopathy (13). The 3 end of the viral genome encodes the p12, p8, p13, p30, and HBZ proteins (47), whose functions are not fully understood. Here we focused on the function of the p8 protein. Expression of the singly splicedorf-ImRNA yields the endoplasmic reticulum (ER) resident precursor p12 protein. Removal of an ER retention/retrieval signal located at the amino terminus of p12 yields the p8 protein, which traffics to the cell surface (8,9). The p12 and p8 proteins exert contrasting effects on T cells. The p12 protein induces T-cell activation by increasing ER calcium influx and/or NFAT activity (10). Furthermore, p12 induces T-cell proliferation by binding the IL-2 receptor and chains (11) and by increasing STAT-5 phosphorylation and IL-2 production (12). In contrast, upon T-cell receptor (TCR) ligation, p8 is recruited to the immunological synapse (IS), the contact site between the antigen-presenting cell and the T lymphocytes. Upon T-cell activation, p8 down-regulates proximal TCR signaling and causes T-cell anergy (8,9). Prior work has demonstrated that, although theorf-Iprotein products increase T-cell contact by lymphocyte function-associated antigen-1 (LFA-1) clustering (13), they also decrease interaction among T cells by downregulating intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and the MHC-I at the cell surface to avoid recognition by natural killer (NK) cells and cytotoxic T cells (CTL) (14,15). Here we present data that reconcile these seemingly opposite SL910102 effects of the p12 and p8 proteins on T cells. We found that p8, but not p12, increases clustering of LFA-1 on the cell surface. In addition, we found that p8 increases the number and length of cellular conduits that enhance communication among several cell types (1618). Through these conduits, p8 is rapidly transferred to neighboring uninfected T cells, where it augments T-cell contact and HTLV-1 transmission. Thus, HTLV-1orf-Iencodes proteins that increase the proliferation of infected T cells and favor SL910102 their escape from immune recognition by downregulating MHC-I, ICAM-1, and ICAM-2, and, to the contrary, enhance T-cell contact while anergizing T cells, and induce conduit formation to favor virus transmission. == Results == == p8, but Not p12, Protein Increases T-cell Conjugation and HTLV-1 Transmission in T Cells. == To dissect the roles of p12 and p8 on HTLV-1 infection of T cells, we constructed cDNA of a natural allele oforf-Ithat carries a substitution of glycine for serine at position 29. This amino acid change severely impairs cleavage and results in the predominant expression of p12. We also generated a cDNA that encodes p8 (8). We used the HTLV-1 WT molecular clone, pAB, or the p12KO molecular clone deficient inorf-Iexpression (19). The Jurkat T cells transfected with pAB or p12KO plasmids produced equivalent amounts of virus (19). However, cocultivation with the reporter cell line (BHK1E6) (20), which contains the -gal gene under the control of the HTLV-1 LTR promoter, revealed that the p12KO virus was significantly less infectious than the WT virus (21) (Fig. 1A, lanes 1 and 2). The p12KO infectivity was rescued by the coexpression of p8, but not p12 (Fig. 1, lanes 3 and 4). The ability of p8 to rescue virus transmission required viral integration and the envelope protein, as demonstrated by the lack of infectivity of the Gpr20 integrase or envelope-defective HTLV-1 molecular clones (22) in the absence or presence of p8 (Fig. 1A, compare lanes 57 and 810, respectively). == Fig. 1. == p8 but not p12 increases T-cell contact and virus transmission. (A) Jurkat T cells were transfected with the WT HTLV-1 pAB, p12KO, env(defective in theenvelopegene), or int(defective in theintegrasegene) molecular clones with and without p12 or p8. After 24 h from transfection, SL910102 the Jurkat T cells were cocultured with the BHK1E6 cells. The data are representative of three independent experiments. Western blots analysis of p12-HA and p8-HA expression and tubulin as control are shown (Bottom). (B) p8-HA, p12-HA, and control pME transfected Jurkat T SL910102 cells were mixed at a 1:1 ratio with untransfected Jurkat T cells prestained with CellTracker Orange (blue) for 20 min, fixed, and stained with anti-HA antibody and Alexa-488 (red). (Scale bar, 10 m.) (C) Effects of inhibitors of microtubule (1 M nocodazole) and actin (1 M cytochalasin D) on cell conjugation (>300 cells were analyzed and counted.