The overall goal of our research is to delineate the pathogenic mechanism(s) involved in retrovirus-induced immunodeficiencies and neurologic disorders. We have been using Moloney murine leukemia virus (MoMuLY) and its mutants for these studies. One of these mutants, is ts1, is unique among murine retroviruses because like HIV, it destroys T cells and neurons. Although ts1 appears to destroy T cell directly, the neuronal death induced by ts1 appears to be indirect. The primary objectives of this proposal will attempt to characterize the mechanisms involved in these two types of cell death induced by ts1. It is hoped that this investigation will enhance our understanding of the pathogenic processes relevant to retrovirus-induced cell destruction.
Our specific aims are: 1. To determine the possible mechanisms involved in T-cell killing by ts1: A. To investigate the sequence of events leading to program cell death (PCD) by addressing the following questions: 1) Does the binding of ts1 envelope protein to thymocyte surface (receptor) trigger imbalanced signal transduction? If so, does this involve in PCD?: ii) Is there an increase in the amount of unintegrated viral DNA in ts1 infected T cells? If so, does this occur before endonuclease(s) activation and nuclear damage? iii) Does intracytoplasmic accumulation of viral env protein in ts1 infected T cells occur before nuclear or DNA damage? If so, is activation of ts1 envelope gene alone from a latent integrated form sufficient to induce PCD:7; iv) Does alteration in cytokines precede the nuclear or DNA damage: Is so, can this cytotoxic effect be blocked by specific antibodies or antagonists to these cytokines 7; v) Is suppression of oncogenes such as bcl-2 involved in ts1- induced PCD? Is so, when does this occur before PCD. B. To determine whether the events observed in the above in vitro studies can be reproduced ex vivo using thymic and splenic T cells obtained on a temporal basis from ts1 infected mice. 2. To determine the possible mechanisms involved in neuronal cell death induced y ts1 infection. A. To identify and assess the frequency of infection in different cell types of the nervous system, and to determine the intracellular location of the envelope proteins in infected mice by using the following approaches: i) Immunohistochemistry; ii) Electron microscopy, iii) In situ hybridization; and iv) Confocal microscopy. B. To evaluate the role of excitatory amino acids in ts1- induced neuronal degeneration by using the following methods; i) Amino acid analysis; ii) In situ localization of glutamate and aspartate; and iii) In vivo pharmacological experiments with antagonist to the N- methyl-D-aspartate (NMDA) receptor. C. To evaluate the role of cytokines in ts1-induced neuronal death.
