Herpes virus transformation

David Hartley

Appointment Period: 1996-1997 / Grant Year: [12]

Herpesvirus saimiri (HVS) is part of the gamma-subgroup of Herpesviridae. Members of this subgroup, which includes Epstein-Barr virus, have a lymphoid tissue tropism. Strains of HVS are further subdivided into three subgroups on the basis of their transforming potential. Group C strains are the mosoncogenic. Strains 484 and 488, members of group C, will induce polyclonal lymphomas and leukemias in many primates, as well as New Zealand white rabbits. In vitro infection with HVS will also render human peripheral blood lymphocytes (PBLs) capable of continued growth in vitro in the absence of either interleukin-2 (IL-2) or periodic restimulation with antigen. Transformed cells have been found to stably maintain functional signal transduction capability and surface expression of most receptors, including the T cell antigen receptor (TCR).

HVS transformed cells express only two viral proteins, both derived from a single bi-cistronic mRNA (open reading frames 1 and 2 of 75 ORFs in the viral genome), and four small nuclear RNAs. Studies have shown that both ORFs are required for transformation, but that the small RNAs are not. In strain 484 of HVS, ORF-2 codes for a 32 kDa protein termed Tip, for Tyrosine kinase-Interacting Protein. This protein binds to the T cell specific tyrosine kinase p56lck and is a substrate of p56lck. ORF-1 of strain 488 is called STP, for Saimiri Transforming Protein, and codes for a 20 kDa protein that has the capability of transforming certain murine epithelial cell types in the absence of other viral proteins, apparently through an association with cellular p21ras.

The association of Tip with p56lck is of significance because p56lck is required for the early steps in T cell receptor (TCR) signaling that lead to cytokine production. HVS transformed cells have been shown to produce both IL-2 and IL-4 spontaneously. It is likely, therefore, that the interaction of Tip with p56lck is instrumental in the growth and proliferation of HVS transformed cells and of vital importance to the leukemogenesis induced by the virus. A primary focus of this proposal is to study the effect of Tip on p56lck both in vitro and in vivo.

Primary T cell activation requires two signals for induction of proliferation; one through the TCR/CD3 complex, and a second signal through the co-stimulatory molecule CD28. Failure to receive both signals can result in apoptosis (cell death), or anergy. Transformation by HVS leads to sustained T cell growth without the need for either antigenic stimulation, or co-stimulation. However, HVS transformed cells will respond to antigen receptor stimulation alone with increased proliferation. In contrast, many T cell hybridomas, leukemias, and ex vivo proliferating primary T cells undergo growth arrest and subsequent death following antigen receptor stimulation alone. Therefore, the expression of Tip and STP in transformed cells will both promote growth and prevent some forms of cell death.

The experiments supported by this training grant will provide an insight into both the mechanisms of HVS induced leukemogenesis, and the role p56lck plays in the activation of normal T cells. In addition, these studies will help to understand the regulatory steps involved in the initial activation and expansion of naive primary T cells. Although STP can transform certain cell types without the aid of Tip, lymphocytes possess unique regulation over their growth and activation that requires additional disruption of normal cellular processes. Understanding how the HVS proteins overcome this regulation will also provide understanding of how this occurs in vivo to mount a primary immune response.

PUBLICATIONS (resulting from this training)

Hartley DA, Hurley TR, Hardwick JS, Lund TC, Medveczky PG, Sefton BM. (1999) Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation. J Biol Chem. 274:20056-9.

Hartley DA, Cooper GM. (2000) Direct binding and activation of STAT transcription factors by the herpesvirus saimiri protein tip. J Biol Chem. 275:16925-32.

Hartley DA, Amdjadi K, Hurley TR, Lund TC, Medveczky PG, Sefton BM. (2000) Activation of the Lck tyrosine protein kinase by the Herpesvirus saimiri tip protein involves two binding interactions. Virology. 276:339-48.