The long term goal of this proposal is to provide the applicant with a solid base in molecular biology. This foundation will enable him to address fundamental issues in neuroimmunology. The candidate will meet these objectives by continuing his involvement in determining molecular mechanisms of gene regulation by interfereons. This work, ongoing for three years, has consisted in two complementary projects. In the first, we investigated the mechanisms by which influenza virus replication is inhibited in interferon-treated cells. We found that viral mRNA transcription was abolished in MDBK cells treated with human alpha interferon. This observation led to further experiments using nuclear extracts of influenzainfected HeLa cells as in in vitro viral transcription system. Requiring a source of purified interferon-induced proteins, we constructed a cDNA library from interferon-treated MDBK cells in lambda GT 11. This library will be screened for clones representing interferon-induced mRNAs. Clones will be usd in hybrid selection/cell-free translation/in vitro viral transcription experiments, to detect clones encoding proteins which mediate inhibition of influenza transcription. These experiments will lead directly to characterization of the interferon-induced activity which results in imparied influenza virus transcription. The second project concerns the molecular regulation of expression of Ia (immune-associated) genes in human astorcytes. We have identified an astocytic tumor line, in which constitutive Ia expression is markedly enhanced by recombinant gamma interferon. Preliminary experiments indicate that this effect is specific for gamma interferon, and that mRNAs encoding Ia polypeptides accumulate to high levels in a dose-dependent, co- ordinate fashion ininterferon-treated cells. In these astrocytic cells and in a monocytic cell line, U 937, we will characterize kinetics of induction of Ia-encoding mRNAs and determine whether accumulation of these mRNAs is under transcriptional control. We will then clarify whether Ia expression on human astrocytes can e inhibited by other interferons or prostaglandins. These experiments will address issues of fundamental and clinical relevance in intrathecal immune responses.
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