The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1).

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin 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 gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4+ B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed 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, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging 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, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission.

The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration.

BACKGROUND: The actin-bundling protein Fascin (FSCN1) is a tumor marker that is highly expressed in numerous types of cancer including lymphomas and is important for migration and metastasis of tumor cells. Fascin has also been detected in B lymphocytes that are freshly-infected with Epstein-Barr virus (EBV), however, both the inducers and the mechanisms of Fascin upregulation are still unclear. RESULTS: Here we show that the EBV-encoded oncoprotein latent membrane protein 1 (LMP1), a potent regulator of cellular signaling and transformation, is sufficient to induce both Fascin mRNA and protein in lymphocytes. Fascin expression is mainly regulated by LMP1 via the C-terminal activation region 2 (CTAR2). Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase ß (IKKß) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression. Furthermore, chemical inhibition of IKKß reduced both Fascin mRNA and protein levels in EBV-transformed lymphoblastoid cell lines, indicating that canonical NF-κB signaling is required for LMP1-mediated regulation of Fascin both in transfected and transformed lymphocytes. Beyond that, chemical inhibition of IKKß significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix. Transient transfection experiments revealed that Fascin contributed to LMP1-mediated enhancement of invasive migration through extracellular matrix. While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells. CONCLUSIONS: Thus, our data show that LMP1-mediated upregulation of Fascin depends on NF-κB and both NF-κB and Fascin contribute to invasive migration of LMP1-expressing lymphocytes.

The tumor marker Fascin is strongly induced by the Tax oncoprotein of HTLV-1 through NF-kappaB signals.

Oncogenic transformation of CD4(+) T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1-transformed and adult T-cell leukemia/lymphoma patient-derived CD4(+) T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.

Elevated cyclic AMP levels in T lymphocytes transformed by human T-cell lymphotropic virus type 1.

Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4(+) T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence.

The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo.

Treatment of human cytomegalovirus (HCMV) infections with any of the currently available antiviral agents is frequently associated with the occurrence of severe complications, seriously threatening the successful outcome of treatment. Therefore, the development of novel antiviral strategies is a challenging goal of current investigations. Previously, we reported that artesunate (ART) is an effective, non-cytotoxic inhibitor of HCMV in vitro. Here, we demonstrate that the efficacy of the antiviral effect of ART is augmented by co-treatment of HCMV-infected fibroblasts with ferrous iron, i.e. Ferrosanol, and/or the iron transfer-mediating molecule holo-transferrin. This could alleviate the HCMV-induced modulation of cell surface expression of adhesion molecule Thy-1, suggesting that ART might be able to prevent pro-inflammatory effects of infection. The iron-enhanced, antiviral effect of ART could also be demonstrated in cultured cells infected with rat cytomegalovirus. Experiments using the RCMV/rat model showed that both the viral DNA load and virus titers in the salivary glands from infected rats were significantly reduced upon treatment with ART. Furthermore, an additive antiviral effect for ART together with each one of conventional anti-HCMV drugs, i.e. ganciclovir, cidofovir or foscarnet, was detected in HCMV-infected fibroblasts. These findings might open new perspectives regarding the use of ART in clinical trials.

Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina.

Replication of human cytomegalovirus is limited at the level of nucleocytoplasmic transport of viral capsids, a process that requires the disassembly of the nuclear lamina. Deletion of the protein kinase gene UL97 from the viral genome showed that the activity of pUL97 plays an important role for viral capsid egress. Here, we report that p32, a novel cellular interactor of the viral kinase pUL97, promotes the accumulation of pUL97 at the nuclear membrane by recruiting the p32-pUL97 complex to the lamin B receptor. Transfection of active pUL97, but not a catalytically inactive mutant, induced a redistribution of lamina components as demonstrated for recombinant lamin B receptor-green fluorescent protein and endogenous lamins A and C. Consistent with this, p32 itself and lamins were phosphorylated by pUL97. Importantly, overexpression of p32 in human cytomegalovirus-infected cells resulted in increased efficiency of viral replication and release of viral particles. Thus, it is highly suggestive that the cellular protein p32 recruits pUL97 to induce a dissolution of the nuclear lamina thereby facilitating the nuclear export of viral capsids.