Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs

Abstract

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

FIG. 1.

Interaction of Tip wt, as well as mSH3B and ΔCSKH mutants, with Lck in T cells. (A) Lysates of monkey T cells transformed by wild-type herpesvirus saimiri C488 (HVS C488 wt) or by the recombinant virus HVS/Tip mSH3B were immunoprecipitated (IP) with rabbit anti-Lck (Lck) or anti-Tip (Tip) antiserum as indicated, and an immune complex kinase assay was performed. Precipitation without specific antiserum (−) was used as a control. (B) Jurkat T cells were transiently transfected with plasmids encoding AU1-tagged Tip (Tip wt) or the mutants Tip ΔCSKH (ΔCSKH) and Tip mSH3B (mSH3B) as indicated. The lysates were divided, and an immune complex kinase assay was performed using anti-CD4 (CD4), anti-Lck (Lck), or anti-AU1 (Tip) antibodies. Immunoprecipitation without specific antibodies (−) and nontransfected Jurkat cells (control) served as negative controls. Expression of Tip proteins was verified by Tip-specific immunoblotting on whole-cell lysates (IB α-Tip). The positions of Lck and Tip proteins are given on the right; molecular mass markers in kDa are on the left.

FIG. 2.

In vitro binding of Tip wt, ΔCSKH, mSH3B, and ΔCSKH mSH3B to Lck. Lysates (500 μl/300 μg each) of untreated COS-7 cells (−) or COS-7 cells transfected with expression plasmids coding for Lck (Lck wt), SH3-deficient Lck (Lck ΔSH3), Tip (wt), Tip ΔCSKH (ΔCSKH), Tip mSH3B (mSH3B), or the double mutant Tip ΔCSKH mSH3B (Δ + m) were mixed as indicated. Lck was immunoprecipitated from the mixtures with specific rabbit antibodies (IP α-Lck). The precipitates were divided into two aliquots and subjected to an in vitro kinase assay (IVKA) and Tip-specific immunoblotting (IB α-Tip). The presence of Tip in the lysates (12 μg total cellular protein/lane) was verified by immunoblotting. The positions of Lck and Tip proteins are given on the right; molecular mass markers are on the left. hc, immunoglobulin heavy chain.

FIG. 3.

Construction of recombinant herpesvirus saimiri C488 ΔCSKH, mSH3B, and ΔCSKH mSH3B. Mutated Tip-coding DNA fragments were introduced into plasmid pSTBlueStpCY114HN. The altered Bst1107I fragments from pSTBlue were then reinserted into the Bst1107I-digested cosmid 331ΔBst1107I. The resulting cosmids were linearized and cotransfected, together with cosmids 261, 291, 112, and 79, into permissive OMK cells. Recombinant virus from the culture supernatant was amplified by infection of OMK cells.

FIG. 4.

Phenotype of CBL cultures (donor 1749) infected with herpesvirus saimiri C488 (C488 wt) and recombinant viruses carrying wild-type sequences (M11 wt) or mutations within the Tip reading frame (ΔCSKH, mSH3B, and ΔCSKH mSH3B). (A) Morphology documented by photography 5 weeks postinfection (no virus, C488 wt, and M11 wt as in reference 35). (B) Surface expression of CD3, CD4, and CD8 antigens 6 weeks after infection. The histograms show fluorescence intensity in logarithmic scale on the x axis and cell numbers in linear scale on the y axis. The open graphs represent negative isotype controls, and the solid graphs represent specific staining.

FIG. 5.

T-cell transformation assay with herpesvirus saimiri C488 wild type (C488 wt and M11 wt), ΔCSKH, mSH3B, and ΔCSKH mSH3B. Stimulated human CBL were infected and cultured in the presence of exogenous IL-2. Proliferation was monitored by automated cell counting, and total cell numbers were calculated as outlined in Materials and Methods. The median values and standard deviation (error bars) of each time point were determined from three independent donors. C488 wt, wild-type isolate; M11 wt, wild type reconstituted from cosmids.

FIG. 6.

Lck binding and tyrosine phosphorylation of Tip Y/F mutants in T cells. Jurkat T cells were transfected with plasmids coding for Tip (wt), individual point mutants of Tip (Y114F, Y127F, and Y155F), HA-tagged Tip (HA-wt), or HA-tagged Tip with Y/F mutations at positions 94, 114, and 155 (HA-FFYF). The cells were harvested and lysed 24 h after transfection. (A) Tip was immunoprecipitated (IP) with a specific rabbit antiserum. (B) A guinea pig antiserum was used to precipitate Lck. Tyrosine-phosphorylated proteins were detected by immunoblotting (IB) with the monoclonal antibody 4G10. The membranes were reprobed with the Tip-specific rabbit serum and with a monoclonal antibody directed against Lck. Immunoprecipitations without antibody (w/o ab) or without cell lysate (ab w/o lysate) served as controls. pY, phosphotyrosine; pTip, tyrosine-phosphorylated Tip.

FIG. 7.

T-cell transformation assay with reconstituted herpesvirus saimiri wild-type (wt) and the virus expressing Tip with a mutation at position 127 (Y127F). Stimulated human CBL from 10 different donors were infected, and proliferation in the presence or absence of IL-2 was monitored by automated cell counting. An experiment was considered successful when viable cells were still present 15 weeks after infection when total cell numbers were determined as outlined in Materials and Methods. Diamonds, cell numbers of individual samples; horizontal lines, geometrical mean values.