Involvement of nuclear factor I-binding sites in control of Akv virus gene expression

The U3 region of Akv murine leukemia virus carries a 99-base-pair repeat that is associated with transcriptional enhancement in murine NIH 3T3 cells. Deletion analysis points to a critical function of a region within the repeat unit related to the recognition sequences for nuclear factor I proteins but distinct from the sites previously analyzed in related viruses. Nuclear proteins binding to the critical site were detected in NIH 3T3 cells and in mouse livers. A protein fraction binding to this site was purified from mouse livers by ion-exchange and DNA affinity chromatography and shown to have nuclear factor I properties. Mutations that caused a partial or complete reduction of the in vitro binding were introduced into an Akv long terminal repeat with one 99-base-pair repeat copy driving a reporter gene, and the expression activities of the mutants in NIH 3T3 cells were found to correspond to their in vitro binding activities. This correlation strongly supports the role of nuclear factor I proteins in Akv expression. Residual expression activity was, however, detected in mutants devoid of in vitro binding. This residual activity may relate to the presence of additional sequences with homology to nuclear factor I binding sites both within and outside the repeat region. The ability of these sites to bind crude and purified protein fractions with nuclear factor I activity was analyzed, and the role of the sites within and outside the repeat region for control of gene expression of Akv and related viruses is discussed.