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

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns

Aligning the CMS Muon Chambers with the Muon Alignment System during an Extended Cosmic Ray Run

Peer reviewe

Molecular Mechanisms of HTLV-1 Cell-to-Cell Transmission

The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation

Quantitating the Transfer of the HTLV-1 p8 Protein Between T-Cells by Flow Cytometry

The Human T-cell leukemia virus type 1 (HTLV-1)-encoded accessory protein p8 is cleaved from the precursor protein p12 encoded by the HTLV-1 open reading frame I. Both p12 and p8 are thought to contribute to efficient viral persistence. Mechanistically, p8 induces T-cell conjugates and cellular conduits. The latter are considered to facilitate transfer of p8 to target cells and virus transmission. Transfer of p8 between p8-expressing T-cells and recipient cells has been analyzed by immunofluorescence and live imaging. However, automatic quantitation of p8-transfer between cells has not been studied yet. Here 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 a well-retained live cell dye. Upon quantitating the amount of p8 positive recipient cells with regard to the percentage of p8 expressing donor cells, time course experiments confirmed that p8 is rapidly transferred between Jurkat T-cells. 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. 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 the new 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 to co-cultured T-cells varies between different donor cell types since p8 transfer could hardly been detected in co-cultures of 293T donor cells with Jurkat acceptor cells. In summary, our novel assay allows automatic and rapid 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

A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-κB activity in T-cells

Background!#!Human T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4!##!Results!#!Here we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-β), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH!##!Conclusions!#!Together, these findings suggest that Tax's capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation

Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24–45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26–37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells

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 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

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