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 MS9 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

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

Fascin knockdown in chronically HTLV-1-infected T-cells impairs virus release and infection of co-cultured T-cells.

<p><b>(A)</b> Scheme of co-culture experiments using HuT-102 cells and reporter Jurkat T-cells. <b>(A-C)</b> Chronically HTLV-1-infected HuT-102 cells with stable repression of Fascin (shFascin5) or control cells (untreated, shNonsense) were co-cultured with Jurkat T-cells that had been transfected 24h earlier with luciferase reporter vectors carrying the core promoter U3R of HTLV-1 (pGL3-U3R) or a control (pGL3-Basic). After 48h, relative light units (RLU) normalized on protein content and on background activity (pGL3-Basic) were determined. <b>(B)</b> Luciferase activity of co-cultures. The means of four independent experiments ± standard error (SE) are shown and compared to shNonsense using Student’s t-test (*: p<0.05). <b>(C)</b> Detection of Fascin and Tax-1 by western blot. β-actin (ACTB) served as control. <b>(D)</b> Scheme of infection experiments using MT-2 cells and Jurkat T-cells. <b>(D-F)</b> Chronically HTLV-1-infected MT-2 cells with stable repression of Fascin (shFascin5) or control cells (untreated, shNonsense) were co-cultured with Jurkat T-cells for 1h at 37°C. Thereafter, cells were stained for CD25 and gag p19 and analyzed by flow cytometry to detect the number of newly-infected Jurkat T-cells (CD25^- gagp19⁺). <b>(E)</b> Gag-positive Jurkat T-cells (%) co-cultured with the respective MT-2 cells. The means of four independent experiments ± standard error (SE) are shown and compared to shNonsense using Student’s t-test (*: p<0.05). <b>(F)</b> Detection of Fascin and Tax-Env by western blot. β-actin (ACTB) served as control. <b>(G-H)</b> Gag p19 ELISA using supernatants of <b>(G)</b> stable MT-2 cells (shNonsense, shFascin5) and <b>(H)</b> differently treated MT-2 cells. Equal numbers of cells (10⁵ cells/ml) were seeded and treated with cytochalasin D, nocodazole (each 5μM), or DMSO (control) for 48h. The means of at least four independent experiments ± standard error (SE) are shown and were compared to control cells (shNonsense or untreated) using Student’s t-test (*: p<0.05, **: p<0.01). <b>(I)</b> Detection of Fascin, Tax-Env and gag by western blot. Hsp90 α/β served as control.</p

Repression of endogenous Fascin impairs virus release and HTLV-1 reporter activity independent of the envelope type used.

<p><b>(A)</b> Scheme of experimental setup using single-cycle replication-dependent reporter vectors with 293T cells. <b>(A-D)</b> Stable 293T cells (sh293T) that carry one of two different shRNAs targeting Fascin (shFascin5, shFascin4) or the control (shNonsense) were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmids pCMVHT1M containing either HTLV-1 wildtype env (wt) or lacking env (Δenv). The latter were pseudotyped with VSV-G or supplemented with pcDNA3 (control). Cells were co-transfected with pEFTax or pEF (mock). Luciferase assays, ELISA and western blot were performed as shown in <b>A)</b>. Values were normalized on those obtained from shNonsense 293T cells transfected with inluc+wt, and the mean of four independent experiments ± standard error (SE) is shown. Values were compared to the respective mock (shNonsense+pEF or shNonsense+pEFTax) using Student’s t-test (*: p<0.05, **: p<0.01). <b>(B)</b> Luciferase activity (cell-to-cell transmission). Right panel: enlargement of dotted box. <b>(C)</b> Detection of gag p19 release by ELISA. <b>(D)</b> Detection of Fascin, Tax-1 and gag by western blot. Hsp90 α/β and β-actin (ACTB) served as control. Numbers indicate densitometric analysis of Fascin detection normalized on Hsp90 α/β.</p

Fascin and gag localize at cell-cell contacts and in long-distance connections between infected and uninfected T-cells.

<p><b>(A)</b> Confocal laser scanning microscopy of HTLV-1-infected MS-9 cells co-cultured with Jurkat T-cells. Jurkat T-cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Cells were co-cultured for 0 (k-p), 30 (a-j) or 60min (q-w) on poly-L-lysine-coated coverslips prior to drying (20min), fixation, and staining. <b>(A)</b> Stainings of Calcein (green), gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments showing clusters of Fascin (a-e) and gag (a-j), Fascin clutches (f-j) or long-distance connections (k-w) are depicted. Thin white arrows indicate gag of an infected cell clustering at the cell-cell contact towards an uninfected cell; framed white arrows indicate short-distance Fascin-containing membrane extensions; and thick white arrows indicate long-distance protrusions between uninfected and infected cells. Protrusions (k-o; q-u) were examined in more detail, and (p) the stains of gag and Fascin within the protrusion shown in (n) were enlarged; further, a region of interest (v-w) was analyzed showing the intensities of gag- (blue) and Fascin- specific (red) fluorescences shown in (t). <b>(B)</b> Detection of Fascin, Tax-1 and gag in HTLV-1-infected MS-9 cells and uninfected Jurkat T-cells by western blot. Hsp90 α/β served as control.</p

Model of Fascin’s role during HTLV-1 transmission.

<p>HTLV-1-infected T-cells express the transactivator Tax that upregulates Fascin expression via the NF-κB signaling pathway. Not only Tax-induced Fascin, but also endogenous (endog.) Fascin is required for virus release and cell-to-cell transmission. Beyond, adhesion of infected cells occurs Fascin-dependently, which may favor dissemination of infected cells <i>in vivo</i>. Functionally, Fascin makes room for gag clusters reminiscent of viral biofilms at the virological synapse (VS) and Fascin-containing short-distance membrane extensions clutch uninfected T-cells. Additionally, Fascin localizes with gag in long-distance connections between chronically infected and newly infected T-cells. A “mini VS” may be shaped at the tip of the long-distance connection towards the target cell. Overall, Fascin seems to be important for the transport of viral proteins to foster polarized budding, virus release and cell-to-cell transmission of HTLV-1.</p

Knockdown of Fascin does not affect cell aggregation but decreases cell adhesion of MT-2 cells in co-cultures with Jurkat T-cells.

<p><b>(A)</b> Scheme of immunofluorescence stains with MT-2 cells and Jurkat T-cells. <b>(A-D)</b> MT-2 cells with stable repression of Fascin (shFascin5) or control cells (shNonsense) were co-cultured with Calcein-stained Jurkat T-cells for 1h at 37°C either on poly-L-lysine- (a-e, k-o) or on fibronectin-coated (f-j, p-t) coverslips. <b>(B)</b> Immunofluorescence stainings of co-cultures. Jurkat cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Stainings of gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments are shown. <b>(C-D)</b> Results of automatic image analysis (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005916#ppat.1005916.s001" target="_blank">S1 Fig</a>). The means of three independent experiments ± standard error (SE) are shown and were compared to shNonsense using Student’s t-tests (*: p<0.05, **: p<0.01). <b>(C)</b> Percentage of MT-2 cells (shNonsense, shFascin5) with 0 to ≥5 cell-to-cell contacts to co-cultured Jurkat T-cells on the different coatings. <b>(D)</b> Percentage of adherent MT-2 cells (shNonsense, shFascin5) on the different coatings normalized on MT-2 shNonsense.</p

Repression of Tax-induced Fascin results in reduction of both virus release and cell-to-cell transmission.

<p><b>(A)</b> Scheme of experimental setup using single-cycle replication-dependent reporter vectors in Jurkat T-cells and Raji/CD4⁺ B-cells. <b>(A-D)</b> Jurkat T-cells were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmid pCMVHT1M encoding HTLV-1 with wildtype env (wt). Cells were co-transfected with pEFTax or pEF (mock) and one of two different shRNAs targeting Fascin (shFascin5, shFascin4) or the control (shNonsense). Luciferase assays, ELISA and western blot were performed as depicted in <b>A)</b>. <b>(B)</b> Luciferase activity (cell-to-cell transmission). The means of four independent experiments ± standard error (SE) are shown and were compared to the respective mock (shNonsense+pEF or shNonsense+pEFTax) using Student’s t-tests (*: p<0.05). <b>(C)</b> Detection of gag p19 release by ELISA. A representative experiment is shown. <b>(D)</b> Detection of Fascin, Tax-1 and gag by western blot. Hsp90 α/β served as control. Numbers indicate densitometric analysis of Fascin detection normalized on Hsp90 α/β. <b>(E-F)</b> Jurkat T-cells were co-transfected with the reporter vector pCRU5HT1M-inluc (inluc), the packaging plasmid pCMVHT1M (wt), pEFTax, and one of three different V5-tagged expression plasmids encoding a MOM-sequence and nanobodies FASNb2, FASNb5 or GFPNb (control). Luciferase assays and western blot were performed as depicted in <b>A)</b>. <b>(E)</b> Luciferase activity (cell-to-cell transmission). The means of six independent experiments ± standard error (SE) were normalized and compared to control samples (GFPNb) using Student’s t-tests (**: p<0.01). <b>(F)</b> Detection of Fascin, Tax-1, V5-tagged nanobodies and gag by western blot. β-actin (ACTB) served as control.</p

The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR–ABL- and JAK2V617F-Positive MPN

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression &gt;100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1–JNK-NF-κB–C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN