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

Abstract

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.

Inhibition of Tunneling Nanotube (TNT) Formation and Human T-cell Leukemia Virus Type 1 (HTLV-1) Transmission by Cytarabine

“…First, p12 is cleaved between amino acids in position 9 and 10 and then between amino acids 29 and 30, where the nature of the amino acids surrounding these cleavage sites determines the efficiency of cleavage and polymorphism has been found in HTLV-1 infected individuals 33 , 36 . Interestingly, also the p8 protein itself is transferred through cellular conduits to neighboring cells 34 , 38 . Transmission of HTLV-1 through these types of cell-to-cell contacts could provide protection from recognition by the immune system 34 .…”

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

“…Analysis of the co-immunoprecipitations by western blot (Fig 3B, right part) and subsequent densitometry ( Fig 3C) revealed that co-precipitation of p8 could be blocked by all peptides overlapping with the aforementioned sequence stretch 24 LFLLFLPLFFSLPLLLSPSLPL 45 of p8, while the control peptide p8(1-12) did not block but rather enhanced the VASP:p8 interaction. Among the peptides tested, peptide p8 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) led to a significant reduction (Fig 3C; p<0.01; n = 4) of co-precipitated p8. These in vitro data suggest that the peptide p8 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) may recognize the EVH1 domain of VASP, and thus, may also interfere with the VASP:p8 interaction in vivo.…”

“…Especially, vasodilator-stimulated phosphoprotein (VASP) and some members of the Wiskott-Aldrich Syndrome Protein (WASP) family as representatives of the EVH1-domain-containing proteins seemed to be interesting candidates because it is thought that VASP promotes actin filament elongation by preventing elongating actin-filaments from capping and by recruiting profilin-actin complexes [38]. Therefore, a putative VASP:p8 interaction could offer an explanation for the p8-mediated actin-dependent cell-cell conjugation, for protrusion formation in HTLV-infected cells, and for the transport of p8 to other cells [15,16,29]. In order to investigate whether p8 has potential PRMs which could bind to the VASP EVH1 domain, we defined a binding pattern based on the results of an experimental peptide screen published earlier (see Materials and Methods) [43].…”

“…Since viral particles are detectable at the contact site between conduits and target cells, but no "surfing" of HTLV-1 virions on cellular conduits has been observed [15], it is assumed that HTLV-1 buds from the conduit towards the target cell at a "mini-VS" [28]. Interestingly, p8 is also transferred to neighboring cells within minutes, invades target cells [15,29], and is supposed to induce T-cell anergy by decreasing T-cell receptor signaling [23]. The latter had been experimentally shown with constructs expressing both p12 and p8 [23].…”

“…Here we show that VASP is a novel interaction partner of HTLV-1 p8 and important for p8 and HTLV-1 Gag transfer between cells. Mapping the VASP:p8 interaction revealed that at least three different domains in VASP and a short sequence stretch in p8 (aa [26][27][28][29][30][31][32][33][34][35][36][37] are important for the VASP:p8 interaction. Functionally, repression of VASP by gene editing strategies demonstrated that VASP is not only crucial for the transfer of p8 to target T-cells, but also of HTLV-1 Gag, suggesting that HTLV-1 cell-to-cell transmission depends on VASP.…”