Human T-cell leukemia virus type 1 (HTLV-1) is the only human retrovirus causing cancer. The virus is transmitted via breast-feeding, sexual contacts or direct exposure to HTLV-1-infected blood cells. Upon infection of its target cells, primarily CD4+ T lymphocytes, 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.
After 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 a neurological 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.
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 and 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 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 do HTLV-1 and related tumor viruses reprogram the cell to enhance infiltration and invasion of the transformed tumor cells?
(3) 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. The actin-bundling protein Fascin 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 (see below). Since Fascin is important for the stability of actin-filaments, we asked whether it contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, 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 contacts 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. Taken together, our work showed for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission (Figure 1). Thus, Fascin is an interesting novel target to counteract infections with HTLV-1.
The p8 protein is a cleavage product of the accessory p12 protein, and p8 increases both the number and the length of cellular, actin-dependent protrusions among T-cells (Figure 2). These protrusions are membrane extensions that are supposed to be formed by directed outgrowth of a filopodium-like protrusion towards a neighboring cell. Thereby, p8 is transferred to neighboring cells and supposed to induce T-cell anergy by decreasing T-cell receptor signaling in the target cell. Interestingly, p8 enhances HTLV-1 transmission through these protrusive structures. Host factors interacting with p8 that mediate p8 transfer to target cells and thus, virus transmission, have not been identified yet. We searched for novel interaction partners of p8 using bioinformatics, mass spectrometry, co-immunoprecipitations, peptide competition assays, and immunofluorescence. Bio-informatics identified p8 as a non-globular protein with putative linear motifs, which allowed predictions of potential interaction partners in the host cell. We have identified several candidate host factors, which specifically interact with p8 in transfected cells. We mapped the region of interaction making use of truncation constructs of one of these host cell factors. To decipher relevant amino acids in p8 that are required for efficient interaction, our collaboration partners Eileen Socher and Heinrich Sticht designed peptides that mimic different motifs in p8. Using this strategy we discovered a short amino acid stretch in the intracellular part of p8 that is responsible for the interaction with the host cell factor. Imaging analysis as depicted in Figure 2 revealed that p8 (red) is transferred to other T-cells (green) via protrusive structures that contain one of the identified host cell factors (blue). Repression of the novel p8 interaction partner by small hairpin RNAs led to decrease of p8 transfer to target cells. Thus, we identified a novel interaction partner of p8, which is crucial for the transfer of p8 to target T-cells. Currently, we test whether the novel host cell factor contributes to HTLV-1 transmission.
Since the actin-bundling protein Fascin (FSCN1) is specifically and strongly expressed in HTLV-1/Tax-transformed lymphocytes and plays an important role in HTLV-1 cell-to-cell-transmission, we asked how expression of Fascin is regulated. We found that two viral oncoproteins are potent inducers of Fascin, Tax-1 of HTLV-1, and LMP1 encoded by Epstein-Barr virus. During the last years, we characterized the regulation of Fascin making use of these viral oncoproteins. To decipher the mechanism of Fascin transactivation by Tax, we used luciferase reporter gene assays, chemical inhibitors, and quantitative PCR. We identified a 1.6 kb fragment upstream of the transcription start site in the human Fascin promoter (phF1.6) that carries a Tax-responsive region (TRR) with a putative Tax-response element (TRE; Figure 3). Tax-mediated activation of phF1.6 was NF-κB-dependent as Tax mutants deficient in NF-κB signaling and inhibitors of NF-κB signaling blocked Tax-mediated activation of phF1.6. However, the TRR lacks putative NF-κB-binding sites, and pharmaceutical activation of NF-κB was not sufficient to activate phF1.6. Thus, we screened for additional signaling pathways contributing to Tax-mediated activation of Fascin.
We found that the Src family kinase inhibitor PP2 impairs Tax-mediated upregulation of Fascin transcripts, but not Tax-mediated promoter transactivation. PP2 also reduced Fascin expression in Tax-transformed T-cells, suggesting that deregulation of Fascin upon transformation is mediated by both NF-κB and by a step that is inhibited by PP2. Together, our findings indicate that Tax-induced Fascin expression is mediated by activation of the Fascin promoter and by a novel, promoter-independent mechanism.
HTLV-1 persists for decades in vivo. Viral gene expression is silenced, which favors immune evasion of the virus and its immunodominant Tax antigen. Thus, a better understanding of the transcriptional complex guiding viral gene expression is required to elucidate strategies to enhance the immunogenicity of HTLV-1. Using microarray analysis, we could previously identify novel Tax targets within the group of transcription elongation factors. Among all known positive transcription elongation factors, only ELL2 was significantly upregulated in HTLV-1 positive cells. Further investigation of different HTLV-1-transformed cell lines, including patient-derived and in vitro transformed T-cell lines, verified upregulation of ELL2 as a common feature of HTLV-1 infection. Experiments in Tax-expressing 293T cells and in HTLV-1 in vitro transformed T-lymphocytes clearly connected ELL2 expression to Tax. Tax was sufficient to induce ELL2 transcripts and Tax alone was sufficient to transactivate the ELL2 promoter. While Tax was important in initiating ELL2 upregulation, results from HTLV-1-positive cell lines and patient samples lacking Tax expression demonstrated that once ELL2 is stably overexpressed, sustained Tax expression is no longer needed to maintain ELL2 expression. Functionally, we identified a synergistic effect of Tax and ELL2 on transactivation of the HTLV-1 promoter. Mechanistically, we found that ELL2 and Tax accumulate in the nucleus and are part of a common complex. This may explain the synergistic effect of ELL2 and Tax on viral gene expression as measured by reporter gene assays. Thus, Tax:ELL2 complexes play an important role in the control of viral gene expression.