Mechanisms of Inactivation of T Cells by HSV

Fred Hutchinson Cancer Research Center, Seattle WA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The herpesviruses have evolved a variety of mechanisms to deal with the threat posed by the host immune response. In one of these, which we refer to as "inactivation", T cells that come in physical contact with HSV-infected cells develop a profound defect in their ability to respond to stimulation via the T cell receptor (TcR). Inactivated T cells do not perform granule-mediated cytotoxity, nor do they secrete cytokines after TcR stimulation. We have recently demonstrated two signaling defects within inactivated T cells, one related to the phosphorylation of HSP90, and one related to the dephosphoryation of linker for activation of T cells (LAT). However, the mechanism by which HSV-infected cells induce inactivation is not clear. To address this, we propose the following. Specific Aim 1. Determine whether delivery of viral protein by defective HSV particles is necessary for inactivation to occur. Based on our preliminary data, the most likely hypothesis is that inactivation occurs via delivery of protein to the T cells by defective viral particles lacking DNA. To evaluate this, we will use T cells transfected with a reporter construct driven by a viral transactivator abundantly packaged within the defective particles, to determine whether delivery of viral protein into T cells correlates with inactivation. Next, we will use purified particles to determine whether binding and fusion of these particles are sufficient to induce inactivation. Specific Aim 2. Determine the viral proteins necessary and sufficient for inactivation. We will use deletion mutants of HSV tegument and envelope proteins to determine which viral proteins are necessary for inactivation to occur. Once the necessary proteins are identified, we will express these proteins individually and in combination in T cells to determine whether any are sufficient to mediate inactivation. Specific Aim 3. Determine the cellular target for the inactivating stimulus. To begin to understand the intracellular events leading to phosphorylation of HSP90 and dephosphorylation of LAT, we will determine the initial cellular target for the inactivating signal. We will do this by identifying the cellular proteins interacting with the viral proteins found to be sufficient in aim 2, and by evaluating the effect of inactivation on cellular proteins known to interact with or modify HSP90 and LAT. The elucidaton of the mechanism by which HSV inactivates T cells will provide new insight into the regulation of T cells, and may suggest targets for pharmacologic manipulation. [unreadable] [unreadable] [unreadable]