Inhibition of T-Cell Receptor Signal Transduction and Viral Expression by the Linker for Activation of T Cells-Interacting p12 ^I Protein of Human T-Cell Leukemia/Lymphoma Virus Type 1

Abstract

The p12I protein of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a small oncoprotein that increases calcium release following protein kinase C activation by phorbol myristate acetate, and importantly, this effect is linker for activation of T cells (LAT) independent. Here, we demonstrate that p12 I inhibits the phosphorylation of LAT, Vav, and phospholipase C-␥1 and decreases NFAT (nuclear factor of activated T cells) activation upon engagement of the T-cell receptor (TCR) with anti-CD3 antibody. Furthermore, we demonstrate that p12 I localizes to membrane lipid rafts and, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse. A p12 I knockout molecular clone of HTLV-1 expresses more virus upon antigen stimulation than the isogenic wild type, suggesting that, by decreasing T-cell responsiveness, p12I curtails viral expression. Thus, p12 I has contrasting effects on TCR signaling: it down-regulates TCR in a LAT-dependent manner on one hand, and on the other, it increases calcium release in a LAT-independent manner. The negative regulation of T-cell activation by p12 I may have evolved to minimize immune recognition of infected CD4 ؉ T cells, to impair the function of infected cytotoxic CD8 ؉ T cells, and to favor viral persistence in the infected host.Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) causes a rare, aggressive hematopoietic malignancy of mature T cells, designated adult T-cell leukemia/lymphoma, as well as a progressive myelopathy defined as HTLV-1-associated myelopathy/tropical spastic paraparesis (18,20,25,42,55). HTLV-1 infects human CD4 ϩ and CD8 ϩ (22,33,35,39,43) T cells of memory phenotype and is believed to confer a proliferative and survival advantage on infected memory T-cell clones, with consequent accumulation of somatic mutations and neoplastic transformation (16). The provirus is thought to be mostly latent, mainly in resting T cells (21). However, once T cells are stimulated by antigen, the proviral DNA can be expressed, endangering the survival of the infected cells because of immune recognition.The HTLV-1 genome, in addition to structural (Gag and Env) and enzymatic (Pol) proteins, encodes positive regulators of viral expression from open reading frames (ORFs) III and IV, such as Tax and Rex, as well as two negative regulators, p30 II (ORF II) (40) and HBZ, which is encoded from the minus strand RNA (19). ORF I encodes a 12-kDa protein (p12 I ) that in cells is localized in the endoplasmic reticulum (ER)/Golgi (13,28,32). The p12 I protein has been shown to have multiple functions. p12 I interacts with the interleukin-2 receptor (IL-2R) ␤ and ␥ c chains, increases STAT-5 activation, and decreases the threshold of the IL-2 requirement for T-cell proliferation (38, 41) in primary human lymphocytes. In the ER/Golgi, p12 I binds both the IL-2 receptor chains and the major histocompatibility complex (MHC) class I heavy chain and interferes with their trafficking to the cell surface (28, ...

Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission

“…MT-2 cells and Jurkat T cells (5 × 10 6 ) were electroporated (X-05 and H-10, respectively; Amaxa) with 10 μg of DNA plasmid. The HTLV-1 pAB WT, p12KO plasmids, Δenv (defective in the envelope gene), and Δint (defective in the integrase gene) were previously described (9,28,29,19). In the actin and tubulin remodeling studies, Jurkat T cells were incubated for 1 h at 37°C with 1 μM nocodazole (Sigma) and 1 μM cytochalasin D (Sigma).…”

“…Expression of the singly spliced orf-I mRNA 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.…”

“…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 the orf-I protein 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).…”

Viral Modulation of T-Cell Receptor Signaling

“…I inhibits the phosphorylation of LAT, Vav, and PLC-␥1 after TcR ligation, leading to decreased TcR-induced activation of NFAT (43). Interestingly, although both the 8-and 12-kDa forms of p12 I can be palmitoylated, only the smaller form localizes to rafts and downregulates TcR signaling (G. Franchini, personal communication).…”

“…The opposing stimulatory and inhibitory effects of HTLV-1 relate at least in part to opposing functions mediated by the p12 I protein. p12 I localizes to the endoplasmic reticulum/Golgi apparatus, to membrane lipid rafts, and also to the immunologic synapse after TcR stimulation (29, 43,65). While p12 I increases intracellular calcium, this effect is PLC-␥, LAT, and PI3K independent, suggesting that this effect bypasses proximal effectors in the TcR pathway (3).…”