HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1)

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP, but never both. To better understand the gene expression changes in HTLV-1-associated diseases, we examined the mRNA profiles of CD4+ T cells isolated from 7 ATL, 12 HAM/TSP, 11 AC and 8 non-infected controls. Using genomic approaches followed by bioinformatic analysis, we identified gene expression pattern characteristic of HTLV-1 infected individuals and particular disease states. Of particular interest, the suppressor of cytokine signaling 1--SOCS1--was upregulated in HAM/TSP and AC patients but not in ATL. Moreover, SOCS1 was positively correlated with the expression of HTLV-1 mRNA in HAM/TSP patient samples. In primary PBMCs transfected with a HTLV-1 proviral clone and in HTLV-1-transformed MT-2 cells, HTLV-1 replication correlated with induction of SOCS1 and inhibition of IFN-α/β and IFN-stimulated gene expression. Targeting SOCS1 with siRNA restored type I IFN production and reduced HTLV-1 replication in MT-2 cells. Conversely, exogenous expression of SOCS1 resulted in enhanced HTLV-1 mRNA synthesis. In addition to inhibiting signaling downstream of the IFN receptor, SOCS1 inhibited IFN-β production by targeting IRF3 for ubiquitination and proteasomal degradation. These observations identify a novel SOCS1 driven mechanism of evasion of the type I IFN antiviral response against HTLV-1.     

HSP90 Protects the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Oncoprotein from Proteasomal Degradation To Support NF-κB Activation and HTLV-1 Replication

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.     

Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-kappaB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.     

Induction of lactoferrin gene expression in myeloid or mammary gland cells by human T-cell leukemia virus type 1 (HTLV-1) tax: implications for milk-borne transmission of HTLV-1

Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia, is transmitted vertically via breastfeeding. We have previously demonstrated that lactoferrin, a major milk protein, enhances HTLV-1 replication, at least in part by upregulating the HTLV-1 long terminal repeat promoter. We now report that HTLV-1 infection can induce lactoferrin gene expression. Coculture with HTLV-1-infected MT-2 cells increased the levels of lactoferrin mRNA in myeloid-differentiated HL-60 cells, as well as MCF-7 cells, models of two probable sources (neutrophils and mammary epithelium) of lactoferrin in breast milk. MT-2 cell coculture could be replaced with cell-free culture supernatants of MT-2 cells to exert the same effect. Furthermore, extracellularly administered Tax protein also induced lactoferrin gene expression at physiologically relevant concentrations. In transient-expression assays, Tax transactivated the lactoferrin gene promoter in HL-60 or MCF-7 cells. Experiments with Tax mutants, as well as site-directed mutants of the lactoferrin promoter reporters, indicated that the NF-kappaB transactivation pathway is critical for Tax induction of the lactoferrin gene promoter activity in myeloid-differentiated HL-60 cells, but not in MCF-7 cells. These results suggest that HTLV-1 infection may be able to induce expression of lactoferrin in a paracrine manner in the lactic compartment. Our findings, in conjunction with our previous study, implicate that mutual interaction between HTLV-1 and lactoferrin would benefit milk-borne transmission of this virus.     

Quantitative Comparison of HTLV-1 and HIV-1 Cell-to-Cell Infection with New Replication Dependent Vectors

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.     

Long noncoding RNA UCA1 regulates HCV replication and antiviral response via miR-145-5p/SOCS7/IFN pathway

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, which leads to the dysregulation of number of relevant genes including long noncoding RNAs (LncRNAs). LncRNA urothelial carcinoma-associated 1 (UCA1) has been reported to be upregulated in HCV-infected individuals. In a bid to elucidate on the contribution of UCA1 on HCV replication, we infected Huh7.5 cells with cell culture-derived HCV and found that UCA1 expression was elevated in time- and dose-dependent manners. Functionally, UCA1 knockdown by siRNA upregulated interferon (IFN) responses, thereby increasing the expression of interferon-stimulating genes (ISGs), and subsequently suppressing HCV replication. Bioinformatics analysis and experimental results indicated that, functioning as competitive endogenous RNA, UCA1 could sponge microRNA (miR)-145-5p, which targeted suppressor of cytokine signaling 7 (SOCS7) mRNA and subsequently mediated SOCS7 silencing. Moreover, SOCS7 protein exerted an inhibitory effect on IFN responses, thereby facilitating HCV replication. Taken together, at first, our findings demonstrate that UCA1 can counteract the expression of miR-145-5p, thereby upregulating the level of SOCS7, and in turn leading to the suppression of antiviral response in Huh7.5 cells.     

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.     

New insight into the oncogenic mechanism of the retroviral oncoprotein Tax

Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma (ATL), infects over 20 million people worldwide. About 1 million of HTLV-1-infected patients develop ATL, a highly aggressive non-Hodgkin's lymphoma without an effective therapy. The pX region of the HTLV-1 viral genome encodes an oncogenic protein, Tax, which plays a central role in transforming CD4+ T lymphocytes by deregulating oncogenic signaling pathways and promoting cell cycle progression. Expression of Tax following viral entry is critical for promoting survival and proliferation of human T cells and is required for initiation of oncogenesis. Tax exhibits diverse functions in host cells, and this oncoprotein primarily targets IκB kinase complex in the cytoplasm, resulting in persistent activation of NF-κB and upregulation of its responsive gene expressions that are crucial for T cell survival and cell cycle progression. We here review recent advances for the pathological roles of Tax in modulating IκB kinase activity. We also discuss our recent observation that Tax connects the IκB kinase complex to autophagy pathways. Understanding Tax-mediated pathogenesis will provide insights into development of new therapeutics in controlling HTLV-1- associated diseases.