This laboratory has isolated murine cell lines which express a mutated form of the human interferon-gamma gene lacking a signal sequence. These cells synthesize human interferon-gamma but fail to secrete it and accumulate the protein intracellularly. Although the murine cells used are completely resistant to the effects of human interferon-gamma added to the culture medium, the cells expressing this heterologous interferon intracellularly are resistant to infection with vesicular stomatitis virus and Mengo virus and exhibit elevated levels of oligo 2,5 A synthetase and eIF-2 kinase, enzymes known to be induced by treatment with interferons. The cells also show enhanced levels of mRNA encoding major histocompatibility complex class II antigen. These results suggest the novel concept that interferon gamma can function intracellularly across a species barrier to cause specific gene activation. The proposed research is to analyze this phenomenon in detail to ensure that the observations are due directly to the intracellular interferon-gamma and not to production by the cells of a homologous antiviral agent. The generality of these observations will be tested by determining whether murine interferon-gamma expressed intracellularly in human cells produces similar effects. Since the results could be explained by the secretion of interferons or other agents with antiviral properties, assays for increased levels of mRNA for homologous interferons alpha, beta and gamma and tumor necrosis factor will be carried out by Northern blotting and polymerase chain reaction techniques. Specific antibodies will be used to test for the secretion of agents with antiviral properties in culture supernatants and by co-culture experiments. If intracellular interferon-gamma is found to activate expression of specific genes directly, the mechanism responsible for this effect will be sought. If intracellular interferon-gamma causes the production of a homologous agent with interferon-like properties it will be important to identify this substance and determine how interferon gamma can induce its production and whether such an effect plays a part in the normal response of cells to interferon-gamma. The biological response modifier, interferon-gamma has been the subject of much recent interest because of its antiviral action and its stimulatory effects on cells of the mammalian immune system. Current research is based on the assumption that this hormone-like substance acts as do many peptide hormones, by binding to a surface membrane receptor and, subsequently triggering a cascade of intracellular biochemical events. Dr. Lewis has preliminary results suggesting a novel, intracellular site of action for interferon-gamma. If this is the case it could represent a very important new mechanism of action, but much critical work must be done to establish unequivocally the basis for the preliminary observations. This grant will support the critical test of these potentially important observations.
