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)

Christine, Gross; Wiesmann, Veit; Sebastian, Millen; Martina, Katia; Wittenberg, Thomas; Gettemans, Jan; Thoma-Kress, Andrea K.

doi:10.1371/journal.ppat.1005916

Cited by 33 publications

(37 citation statements)

References 69 publications

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“…These viruses can even be transported directly from cell to cell through tunneling nanotubes; IRSp53 is also related to tunneling nanotubes, while fascin interacts directly with microtubules . Furthermore, it is already known that fascin is crucial for the release and cell–cell transmission of human T‐cell leukemia virus type1 (HTLV‐1) …”

Section: Discussionmentioning

confidence: 99%

“…[65] Furthermore, it is already known that fascin is crucial for the release and cell-cell transmission of human T-cell leukemia virus type1 (HTLV-1). [66] In addition to the relationship of the proteins discussed above (IRSp53, fascin, CDC42) to filopodia-like structure formation, other proteins that negatively regulate filopodia formation were also identified in the extracellular PRV virion preparation. These proteins include 14-3-3 proteins, destrin, and cofilin.…”

Section: Fascin Rac1 and Cdc42 In Prv Transmissionmentioning

confidence: 98%

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Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

Miao

Xia

et al. 2019

Proteomics

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Pseudorabies virus (PRV) has been widely used as a live trans‐synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co‐purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell–cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia‐like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.

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Section: Discussionmentioning

confidence: 99%

Section: Fascin Rac1 and Cdc42 In Prv Transmissionmentioning

confidence: 98%

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

Miao

Xia

et al. 2019

Proteomics

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“…It had been nicely shown by real-time live imaging that Gag-YFP expressed from an HTLV-1 construct and p8-mCherry are transported via protrusions to neighboring cells within minutes ( Van Prooyen et al, 2010b ). Combining the measurements of p8 transfer as described here and Gag transfer by flow cytometry ( Gross et al, 2016 ) opens up the possibility to quantitatively evaluate p8’s role in virus transmission.…”

Section: Discussionmentioning

confidence: 99%

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

2018

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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.

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“…A representative flow cytometry analysis of co-cultures after staining of HA shows that Jurkat T-cells ( Fig 9B, upper part, blue gate, acceptor cells) were discriminated from MT-2 cells (Fig 9B, upper part, red gate, donor cells) according to their FSC and SSC. In parallel, we also discriminated Jurkat T-cells from MT-2 cells by staining for the IL-2Rα chain CD25, which is present on MT-2 cells, but not on Jurkat T-cells [56] as described previously [52], which gave comparable results. A representative dot plot shows that ca.…”

Section: Vasp Is Important For P8-transfer In Chronically Infected T-mentioning

confidence: 92%

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

et al. 2020

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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.

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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)

Cited by 33 publications

References 69 publications

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

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

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

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