Infections account for approximately 15-20% of human cancers. Human T-cell leukemia virus type 1 (HTLV-1), the only retrovirus causing cancer in humans, infects at least 5-10 mio. people worldwide and is the trigger for incurable neoplastic or inflammatory diseases. The viral Tax-1 (Tax) oncoprotein, a key player in initiating malignant transformation of infected CD4+ T-cells, deregulates cellular signaling pathways. Upon infection of T-cells, integrated HTLV-1 persists as a provirus in vivo. After a latency period, which may last up to decades, HTLV-1 may cause an aggressive and highly infiltrative leukemia of CD4+ T-cells, adult T-cell leukemia/lymphoma (ATL). Additionally, HTLV-1 is also the etiologic agent of the neurologic disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 persists lifelong in the presence of an active immune system. The virus has little cytotoxic effect on T-cells, but changes their growth properties. In contrast to normal T-cells, HTLV-1-infected lymphocytes can proliferate permanently in the absence of antigen stimulation and are resistant to apoptosis-inducing signals.
HTLV-1 is transmitted via breast-feeding, sexual contacts or direct exposure to HTLV-1-infected blood cells. After infection of its target cells, primarily CD4+ T-cells, HTLV-1 is reversely transcribed and integrates into the host cell genome. Transmission of HTLV-1 to other cells is strictly dependent on cell-cell contacts, and cell-free infection is very inefficient. Viral particles are transferred to other cells after polarized budding at a tight, confined cell-cell contact, the so-called virological synapse. Further, viruses are transmitted via viral biofilms at virological synapses. The viral biofilms consist of an agglomeration of viruses that are packaged on top of the infected cells in a biofilm-like structure. Beyond the transmission at tight cell-cell-contacts, HTLV-1 is supposed to be transferred to other cells via long-distance connections.
Two viral proteins have been shown to play a major role in virus transmission, Tax and p8. The regulatory protein Tax is the viral transactivator and also the major oncoprotein of HTLV-1. Tax not only enhances viral gene expression, but it also potently activates several cellular signaling pathways, amongst them cAMP-response element binding (CREB), nuclear factor kappa B (NF-κB), and the serum response factor pathway. Thus, Tax is a potent regulator of cellular gene expression, which contributes to viral pathogenesis and oncogenesis. Beyond, Tax has typical properties of an oncoprotein, since (1) Tax immortalizes primary rodent fibroblasts, (2) Tax induces leukemia and neurofibromas in transgenic mice, and (3) Tax initiates immortalization of primary human T lymphocytes. In late stages of HTLV-1-pathogenesis, however, Tax protein is barely detectable or only expressed in single cells in bursts and supposed to be no longer required to maintain the transformed phenotype. Since Tax is evoking a strong cytotoxic T-cell (CTL) response in vivo, silencing of Tax is an efficient strategy of immune evasion.
Therapeutic approaches have tried to reactivate Tax expression to enhance the CTL response, and thus, to eliminate virus-infected cells. Next to its role in initiating cellular transformation, Tax plays an essential role in remodeling of the host cell cytoskeleton during viral transmission at the virological synapse. Contrary, the accessory protein p8 induces cellular protrusions and is transferred to other cells to foster HTLV-1 cell-to-cell transmission. Thus far, detailed molecular mechanisms of HTLV-1 cell-to-cell transmission and the transfer of p8 are largely unknown.
Our major research aims are addressed by the following questions:
(1) How do the viral proteins Tax and p8 modulate the host cell to allow efficient cell-to-cell transmission of HTLV-1?
(2) How does HTLV-1 promote its own gene expression, and how can viral gene expression be manipulated?
The viral protein Tax and polarization of the host cell cytoskeleton are crucial for formation of the virological synapse, however, only little is known about the link between Tax and remodeling of the cytoskeleton to foster viral spread. Substantial insights into the different routes of HTLV-1-transmission have mainly been obtained by imaging techniques or by flow cytometry. Recently, strategies to quantify infection events with HTLV-1 improved. We use different quantitative methods to measure virus transmission in our laboratory. The methods are based on measuring gene activity of luciferase with a cost-saving in-house luciferase assay. First, we established a reporter Jurkat T-cell line carrying a luciferase gene under the control of the HTLV-1 core promoter U3R. Upon co-culture with chronically HTLV-1-infected T-cell lines, reporter cells are infected, and upon expression of the viral transactivator Tax, the viral promoter is activated resulting in enhanced luciferase activity. However, this assay does not exclude cell fusion as the mechanism allowing intracellular Tax-dependent activation of luciferase gene expression. Therefore, we make use of a second method, the single-cycle replication-dependent reporter system developed by Mazurov et al. (PLoS Pathog 6:e1000788, 2010) that allows quantitation of HTLV-1 infection in co-cultured cells. Combined use of both methods facilitates quantitation of HTLV-1 transmission and already helped to unravel pathways required for cell-to-cell transmission on a quantitative basis. We could recently show that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission. Fascin is an actin-bundling protein, which has evolved as a therapeutic target in several types of cancer. We previously identified Fascin as a novel target gene of Tax and also characterized the transcriptional regulation of Fascin in more detail. Since Fascin is important for the stability of actin-filaments, we asked whether it contributes to HTLV-1 transmission. Using the above mentioned quantitative techniques to measure HTLV-1 transmission, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired both gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, expression of Tax led to induction of Fascin expression, and this resulted in enhanced virus release and cell-to-cell transmission to Raji/CD4+ B-cells, which was reduced upon repression of Fascin. Analysis of chronically HTLV-1-infected T-cells revealed that repression of Fascin diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis uncovered that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin. This suggests that Fascin contributes to dissemination of infected T-cells. Confocal imaging analysis of chronically infected MS-9 T-cells in co-culture with Jurkat T-cells revealed that Fascin’s localization at tight cell-cell con-tacts is accompanied by gag polarization, suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Thus, Fascin is an interesting novel target to counteract infections with HTLV-1.
The HTLV-1 p8 protein is a cleavage product of the accessory p12 protein, and both p12 and p8 are thought to contribute to efficient viral persistence. Mechanistically, p8 increases the number and the length of cellular, actin-dependent protrusions among T-cells. The latter are considered to facilitate transfer of p8 to target cells and virus transmission. In the target cell, p8 is supposed to induce T-cell anergy by decreasing T-cell receptor signaling. Transfer of p8 between p8-expressing T-cells and recipient cells has been analyzed by immunofluorescence and live imaging. Since automatic quantitation of p8-transfer between cells had not been studied, we developed a novel method allowing time saving quantitation of p8 transfer between cells by flow cytometry. After establishing a protocol for the detection of intracellular p8 by flow cytometry and validation of p8 protein expression by western blot and immunofluorescence, we set up a co-culture assay between p8-expressing donor Jurkat T-cells and recipient Jurkat T-cells that had been prestained with the well-retained live cell dye CMAC-Blue (Figure 1A). Upon quantitating the amount of p8 positive recipient cells with regard to the percentage of p8 expressing donor cells, we performed time course experiments, which confirmed that p8 is rapidly transferred between Jurkat T-cells (Figure 1B). We found that p8 enters approximately 5% of recipient T-cells immediately upon co-culture for 5 min. Prolonged co-culture for up to 24 h revealed an increase of relative p8 transfer to approximately 23% of the recipient cells (Figure 1B). Western blot analysis of p8 (Figure 1C), immunofluorescence analysis of co-culture experiments and manual quantitation of p8 expression in fluorescence images confirmed the validity of the flow cytometry based assay. Application of our novel assay revealed that manipulation of actin polymerization significantly decreased p8 transfer between Jurkat T-cells suggesting an important role of actin dynamics contributing to p8 transfer. Further, transfer of p8 was cell type dependent. Contrary to co-cultures of Jurkat T-cells, p8 transfer could hardly been detected in co-cultures of 293T donor cells with Jurkat acceptor cells. In summary, our novel assay allows rapid and automatic quantitation of p8 transfer to target cells and might thus contribute to a better understanding of cellular processes and dynamics regulating p8 transfer and HTLV-1 transmission.
It has been known for years that constitutive activation of the classical and alternative NF-κB signaling pathways by Tax is a hallmark of HTLV-1-driven cancer. NF-κB-deficient Tax transgenic mice lack the induction of ATL-associated aggressive skin diseases. Further, animal studies therapeutically targeting NF-κB slow down and reduce tumor growth in ATL-like diseases. Although there are different reports whether NF-κB is critical for initiating cellular transformation, there is a strong connection between Tax, NF-κB, tumor formation and maintenance. Having found, against expectation, that activation of NF-κB signaling specifically enhances the abundance of Tax protein (Figure 2A), but not of Tax transcripts, we hypothesize that Tax establishes a novel positive feedback loop between itself and NF-κB activity, which results in enhanced protein expression of Tax and might thus serve as a novel therapeutic target to interfere with Tax-driven transformation (Figure 2B). To study the impact of NF-κB activity on Tax expression in primary T-cells, we optimized in collaboration with Dr. Ciminale (Padova, Italy) a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Our results showed that the RNA transfection technique combines high transfection efficiencies with low toxicity in primary T-cells. Together, these findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.