| Interferon-gamma (IFN-gamma) is critical in eliminating viruses from the central nervous system. IFN-gamma up-regulates Nitric Oxide Synthase-1 (NOS-1) protein level posttranscriptionally. This increases nitric oxide (NO) production, which is essential in the clearance of Vesicular Stomatitis Virus (VSV) from neurons. In this study, we are focusing on how the proteasome-ubiquitin pathway and NOS-1 interacting proteins, including Protein Inhibitor of NOS-1 (PIN), NOS Interaction Protein (NOSIP) and C-terminal PDZ ligand of NOS-1 (CAPON), may regulate the IFN-gamma mediated antiviral response in the CNS.;The proteasome is a proteolytic enzyme complex that degrades intracellular proteins targeted to it by poly-ubiquitination. Pharmacologic inhibition of the proteasome resulted in increased NOS-1 protein levels and increased NO production in neuronal cells. This correlated with an increased antiviral effect of IFN-gamma in vitro. Protein inhibitor of nNOS (PIN), which prevents formation of the functional NOS-1 homodimer, was down-regulated by IFN-gamma. More ubiquitin-modified PIN and faster degradation of PIN was found in IFN-gamma treated neuronal cells. Our results indicate that IFN-gamma treatment of neurons increases PIN ubiquitination and degradation, resulting in an increase in activation of NOS-1. Importantly, we observed that ubiquitin associated degradation was not involved in IFN-gamma induced inhibition of VSV protein synthesis. This hypothesis was tested by overexpression and RNAi of PIN, confirming the involvement of PIN in the IFN-gamma mediated accumulation and activation of NOS-1. Following treatment with IFN-gamma, we also identified the colocalization pattern of NOS-1 and PIN, NOS-1 and NOSIP, as well as NOS-1 and CAPON in polarized neuronal cells and primary neurons. |
