The long-term objective of this application is an understanding of the mechanism of action of interferons (IFN). Three main lines of research are proposed. One line of work will examine the antiviral activity of proteins coded for by IFN-induced mRNAs of unknown function. The cDNAs corresponding to several IFN- induced mRNAs have recently been cloned. These cDNAs will be inserted into a shuttle expression vector in an antisense orientation, in order to produce anti-mRNA which can specifically anneal with mRNA and suppress its expression. A vector containing elements of the BK virus and a neomycin gene under control of an SV-40 promoter has been constructed and used to test this method of suppressing the expression of an IFN-induced mRNA. Human cells transfected with DNA of the vector containing 2-5A synthetase cDNA under the control of the VP promoter, show very low levels of this enzymatic activity after treatment with IFN. These cells lack resistance to EMCV infection. Other cDNAs will be inserted into separate vectors and the resistance to infection by different viruses will be tested in cells transfected with vector DNA. If resistance to a specific virus is decreased in cells incapable of producing an IFN-induced mRNA it will be concluded that the corresponding protein plays some role in the antiviral mechanisms which affords protection against that virus. Another line of research will characterize 2- 5A metabolites, which are inhibitory for the endonuclease (RNase L) activated by 2-5A. These compounds have been detected in IFN-treated cells incubated with poly(I:C) and in cell extracts incubated either with this dsRNA or with synthetic 2-5A. The RNase L inhibitors may play an important role in preventing indiscriminate RNA cleavage in IFN-treated virus-infected cells. The inhibitors will be fractionated by HPLC and characterized by a combination of enzymatic degradations and chemical analysis. Finally, a third line of research will examine the effect of IFN administered together with surfactants on the nasal mucosa. Preliminary experiments have shown that few IFN receptors are present on this mucosa. However, membranes prepared from nasal mucosa have abundant receptors, presumably accessible to circulating IFN. Some surfactant agents promote passage of insulin across the nasal mucosa and their effect on IFN transport will be assayed by measuring 2-5A synthetase activity in the mucosal layer of turbinates. These experiments will be carried out in the sheep, as an animal model in the IFN prophylaxis against the common cold.
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