The overall objective of this project is to understand the molecular mechanism of MHC neuropathogenesis. Our major goal is to understand the molecular basis of viral tissue and cellular tropism, particularly concerning the mechanism and specificity of viral entry process. The four specific aims for this project are as follows: 1. To study the molecular basis of the specificity of the receptor utilization by MHV. We will determine the sequence of the receptor molecules responsible for the specificity of the virus-receptor interactions. We will measure the affinity of the virus-receptor interactions. We will measure the affinity of the virus-receptor interactions to determine whether the binding affinity determines the specificity of the virus receptor-interaction. 2. To characterize the molecular events subsequent to virus-receptor binding. We will explore the significance of the activation of calcium- dependent phospholipase A-2 (PLA2) in the virus entry process. My laboratory has shown that this enzyme is activated transiently and immediately following virus binding to the target cells. We will study whether it is necessary for virus entry and which domain of the receptor molecule is involved in its activation. Finally, we will investigate whether it is associated with endocytosis or virus-cell fusion. 3. To identify and characterize additional cellular factors for virus entry. We will use a phage display library screening method to identify the possible cellular factors required for virus entry. We will also use an alternative approach, a cDNA library transfection/infection procedure, to complement the phage display library technique. 4. To generate pseudotype viruses containing different S proteins, using the MHV-defective-interfering (DI) RNA as a expression vector. These viruses will be used to study the potential function of the S protein in viral neuropathogenesis.
