ABSTRACT
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.
Subject(s)
Cell Adhesion Molecules , Focal Adhesions , Gene Products, gag , Human T-lymphotropic virus 1 , Microfilament Proteins , Phosphoproteins , T-Lymphocytes , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Focal Adhesions/chemistry , Focal Adhesions/genetics , Focal Adhesions/metabolism , Focal Adhesions/virology , Gene Products, gag/chemistry , Gene Products, gag/genetics , Gene Products, gag/metabolism , Human T-lymphotropic virus 1/chemistry , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/metabolism , Humans , Jurkat Cells , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Domains , T-Lymphocytes/chemistry , T-Lymphocytes/metabolism , T-Lymphocytes/virologyABSTRACT
Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63ß isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63ß is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and ß. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63ß although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63ß in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63ß is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63ß therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63ß in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
