Endoplasmic Reticulum and cis-Golgi Localization of Human T-Lymphotropic Virus Type 1 p12I: 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 p12(I) 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 p12(I) 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.     

Proliferation Response to Interleukin-2 and Jak/Stat Activation of T Cells Immortalized by Human T-Cell Lymphotropic Virus Type 1 Is Independent of Open Reading Frame I Expression

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12(I) has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (alpha, beta, or gamma(c)) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.     

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

The p12(I) 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-gamma 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, p12(I) 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-lymphotropic virus type 1 open reading frame I p12(I) is required for efficient viral infectivity in primary lymphocytes

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. Emerging evidence indicates an important role for the pX ORF I-encoded accessory protein p12(I) in viral replication, but its contribution to viral pathogenesis remains to be defined. p12(I) is a conserved, membrane-associated protein containing four SH3-binding motifs (PXXP). Its interaction with the interleukin-2 (IL-2) receptor beta- and gamma-chains implies an involvement of p12(I) in intracellular signaling pathways. In addition, we have demonstrated that expression of pX ORF I p12(I) is essential for persistent infection in rabbits. In contrast, standard in vitro systems have thus far failed to demonstrate a contribution of p12(I) to viral infectivity and ultimately cellular transformation. In this study we developed multiple in vitro coculture assays to evaluate the role of p12(I) in viral infectivity in quiescent peripheral blood mononuclear cells to more accurately reflect the virus-cell interactions as they occur in vivo. Using these assays, we demonstrate a dramatic reduction in viral infectivity in quiescent T lymphocytes for a p12 mutant viral clone (ACH.p12) in comparison to the wild-type clone ACH. Moreover, addition of IL-2 and phytohemagglutinin during the infection completely rescued the ability of ACH.p12 to infect primary lymphocytes. When newly infected primary lymphocytes are used to passage virus, ACH.p12 also exhibited a reduced ability to productively infect activated lymphocytes. Our data are the first to demonstrate a functional role for pX ORF I in the infection of primary lymphocytes and suggest a role for p12(I) in activation of host cells during early stages of infection.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Human T-Cell Leukemia Virus Type 1 pX-I and pX-II Open Reading Frames Are Dispensable for the Immortalization of Primary Lymphocytes

Human T-cell leukemia virus type 1 (HTLV-1) infects and transforms CD4⁺ T-lymphocytes both in vivo and in vitro. Although the Tax protein of HTLV-1 has been strongly implicated as a transforming agent, other virally encoded proteins may also play a role in the transformation process. In addition to the rex and tax genes, the pX region of the HTLV-1 genome contains two open reading frames (pX-I and pX-II) which encode the putative viral accessory proteins known as p12^I, p30^II, and p13^II. Mutations in the ACH molecular clone of HTLV-1 that are predicted to abrogate the expression of p12^I, p13^II and p30^II were constructed. These mutations had no effect on viral replication or the immortalization of primary lymphocytes. Although these proteins are dispensable for viral replication and immortalization in vitro, it remains possible that they alter infection in vivo.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13(II) is required for viral infectivity in vivo

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13(II). p13(II) localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13(II) in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13(II) in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13(II) (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13(II) has an essential biological role during the early phase of virus infection in vivo.     

Free major histocompatibility complex class I heavy chain is preferentially targeted for degradation by human T-cell leukemia/lymphotropic virus type 1 p12(I) protein

Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.