The analysis of interferon-tau activity using a molecular genetic approach.

摘要

Interferon-tau is a type I interferon (IFN) produced by sheep, cows, and other ruminants. It is secreted in large amounts by the conceptus and is responsible for the maternal recognition of pregnancy in these animals. It also possesses classical type I IFN properties, including antiviral, antiproliferative, and immunomodulatory functions.; Like other type I IFNs, its structure is a five helical bundle separated by loop regions. Using this model for all type I IFNs, this work focused on the role of the amino terminus, specifically regions of Helix A and the AB loop, on IFN-tau's antiviral and antiproliferative activities, as well as on binding to its receptor complex. Six IFN-tau proteins were produced, each with a single amino acid substitution. Three of the changes (13E:R, 16K:M, and 19D:A) were in Helix A, and three (24L:I, 26P:L, and 34K:H) were in the AB loop. The ability of mutated IFN-tau molecules to protect against virus induced cytopathic effect and to decrease cell proliferation was assessed. All mutants, except IFN-tau26P:L, showed decreased antiviral activity compared to wildtype IFN-tau and IFN-alphaA but maintained the ability to decrease cell growth on Daudi cells, the cell line most sensitive to the effects of IFN-tau, and the antiproliferative activity of IFN-tau26P:L was similar or better than IFN-tau. No mutant proteins showed toxicity at high concentrations when exposed to cells of human origin.; IFN-tau26P:L was able to compete with IFN-tau for binding to the type I IFN receptor complex and was a better competitor than the wildtype protein itself. Consistent with strong binding, IFN-tau26P:L maintained production of STAT1, a signaling protein necessary for IFN response in a similar fashion to IFN-tau and IFN-alphaA. This was seen only with this mutant and IFN-tau16K:M.; The mutant protein IFN-tau13E:R displayed the greatest loss of antiviral activity. Its circular dichroism spectrum displayed some loss in helical structure as well. In spite of its loss in activity and possible structural change, IFN-tau13E:R was also able to compete with wildtype IFN-tau for binding the type I IFN receptor.; This work has proven that specific amino acids within the N-terminus of IFN-tau affect activity, with the antiviral activity being particularly sensitive to changes within the region. It has also shown that the disparity in activity involves differences in receptor binding, and subsequent signaling through the known IFN signaling pathway. In addition, this work has identified one IFN-tau protein, IFN-tau26P:L, with enhanced antiproliferative activity and no decrease in antiviral activity compared to wildtype IFN-tau. This protein deserves further attention, as it may potentially be beneficial as a therapeutic agent.