Inducible DNA-binding proteins have been detected that interact with the sequence (A/G)GGGACTTTCC in the human immunodeficiency (HIV) enhancer and with the sequence GGGGGATTCCCC in the class I major histocompatibility (MHC) enhancer. Two of these proteins (SIF-1 and SIF-2) are inducible by serum treatment of quiescent fibroblasts and exhibit high affinity for the related HIV and class I MHC enhancer sites. Another is MBP-1, a protein encoded by a cDNA that we recently cloned (Singh et al., 1988). MBP-1 is characterized by the inducibility of its mRNA in both mitogen stimulated Jurkat T cells and in serum stimulated fibroblasts (Baldwin et al., 1990). Furthermore, MBP-1 has two pairs of zinc fingers, each of which interacts with the related HIV and MHC enhancer elements. Finally, we have identified a factor whose DNA-binding activity is elevated to high levels in HIV tat expressing cells and that exhibits high affinity for the class I MHC enhancer site. The broad objectives are to biochemically characterize these inducible factors, determine if they specifically regulate HIV and MHC gene expression and determine if they are involved in control of cellular proliferation and differentiation.
The specific aims to accomplish these objectives are (1) to characterize SIF-1 and SIF-2 activities biochemically, clone their cDNAs and develop antibodies to determine unequivocally if these activities are distinct from other MHC/HIV enhancer binding proteins (including NF-kappaB and MBP-1), (2) to determine the kinetics of SIF-1 and SIF-2 inducibility, their activities in the cell cycle and if they are expressed in cells that are infected by HIV, (3) to characterize the HIV tat inducible factor(s), (4) to use DNA transfection and expression analysis to determine if the HIV and MHC enhancer sites respond transcriptionally to these inducible DNA-binding proteins, (5) to analyze MBP-1 mRNA in regards to its induction kinetics and its cell type distribution, (6) to determine by cotransfection experiments if MBP-1 can transcriptionally regulate gene expression through interactions with class I MHC and HIV enhancer sites, and (7) to determine if antisense oligomers will inhibit MBP-1 mRNA levels and, if so, determine if MBP-1 is required for cellular proliferation and for regulated gene expression (including HIV gene expression) in activated T cells. The experiments proposed here relate to diseases caused by HIV, of which the most prevalent is AIDS. Furthermore, the inducible factors may control some aspect of cellular proliferation and may, therefore, relate to the induction of cancer. These inducible DNA-binding proteins appear to be novel HIV enhancer binding proteins that likely play a role in control of viral gene expression in non-lymphoid as well as lymphoid cells and may contribute to the activation of viral gene expression in latently infected cells. Finally, these studies may provide insight into mechanisms of signaling that link growth regulation with immunologic activation.
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