Substitution of ras for the herpesvirus saimiri STP oncogene in lymphocyte transformation

Abstract

STP-C488 (STP of herpesvirus saimiri [HVS] group C strain 488 [C488]) is the only virus-encoded protein found to associate with cellular ras and activate ras signal transduction pathways. To investigate an important role for ras signal transduction in STP-dependent growth transformation, we constructed recombinant strains of HVS C488 in which the STP-C488 oncogene was replaced with cellular normal ras (c-ras) or viral oncogenic ras (v-ras). Recombinant HVS deltaSTP/v-ras immortalized primary common marmoset T lymphocytes to interleukin-2-independent growth as efficiently as wild-type HVS C488 (wt HVS), while recombinant HVS deltaSTP/c-ras did so with low efficiency. Whereas wt HVS immortalized CD4- CD8+ single-positive T lymphocytes, HVS deltaSTP/c-ras- and HVS deltaSTP/v-ras-immortalized cells were principally CD4+ CD8+ double-positive T lymphocytes. In addition, HVS deltaSTP/v-ras-immortalized T cells showed a high level of ras expression and exhibited an adherent macrophage-like morphology. These phenotypes were likely caused by the drastic activation of AP-1 transcriptional factor activity. Finally, HVS deltaSTP/v-ras and HVS deltaSTP/c-ras each induced lymphoma in one of two common marmosets, although onset of disease was more rapid with the v-ras virus. These results demonstrate that ras can substitute for the STP oncogene of HVS C488 to allow immortalized growth of primary lymphoid cells and that an activated form of ras does so more efficiently than the normal cellular form of ras.

FIG. 1

Schematic diagram to construct the recombinant HVS containing c-ras or v-ras. The detailed procedure has been described previously (16). pA, polyadenylation site.

FIG. 2

Analysis of surface expression of lymphocyte antigens on wt HVS-, HVSΔSTP/c-ras-, or HVSΔSTP/v-ras-transformed cells. Surface phenotypes were determined by flow cytometry. Positive gates were established by using an isotype-matched control antibody.

FIG. 3

Morphological changes in common marmoset T cells transformed by HVSΔSTP/v-ras. Cells were photographed with a phase microscope at a magnification of ×100.

FIG. 4

Immunoblot analysis of HVSΔSTP/c-ras- or HVSΔSTP/v-ras-transformed cells. The same amounts of precleared lysates of 5 × 10⁶ cells were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with antibodies against STP-C488 or Ras. Reactivity was detected by enhanced chemiluminescence. Lane 1, wt HVS-transformed cells; lane 2, HVSΔSTP/c-ras-transformed cells; lane 3, HVSΔSTP/v-ras-transformed cells.

FIG. 5

Activation of AP-1 transcription factor activity in HVSΔSTP/v-ras-transformed cells. A total of 10⁷ cells transformed by wt HVS (□) or HVSΔSTP/v-ras (▪) were electroporated at 960 μF and 200 V with 30 μg of pGKβgal and 30 μg of reporter plasmid TRE-luc, NF-κB-driven 3X-κB-luc, NFAT-SEAP, or OCT-SEAP. At 24 h after transfection, cell supernatants and lysates were used for luciferase, alkaline phosphatase, and β-galactosidase assays. Luciferase and alkaline phosphatase activities were determined and normalized on the basis of β-galactosidase activities. Fold activation represents normalized luciferase and alkaline phosphatase activity relative to that of the wt HVS-transformed cells 24 h after transfection. Values represent averages of three independent experiments.

FIG. 6

Schematic diagram of upregulation of ras expression from STP promoter. The −91 basal transcriptional initiation site and −42 and −111 TPA-inducible transcriptional initiation sites are indicated with arrows as described previously (19).