Studies central to the life cycle of HIV virus --the structure of HIV DNA as chromatin will continue. Our working hypothesis is that to understand the control of HIV gene expression and where on the HIV genome there may be open windows for anti-viral agents, one must understand how HIV DNA is arranged by the histones, topoisomerases, and other proteins into chromatin in the infected cell. This data is critical for defining optimal drug target sites since the assembly of a potential target site into a nucleosome would mask it from attack. Thus a major goal is to locate gaps in the HIV chromatin sheath. Using high resolution electron microscopy (EM) and molecular tools, and building on our in vitro maps of nucleosome assembly and DNA bending for HIV, we will map sites of nucleosome assembly and exclusion on HIV DNA in vivo. This will utilize the ACH-2 CD4+ T cell line harboring a single integrated HIV genome, and an HIV-ebv minichromosome that we will construct from plasmids containing the EBV plasmid origin plus EBNA-1 coding sequences into which the HIV genome will be inserted for growth in human T cells. Using DnaseI and chemical probes, sites of nuclease hypersensitivity (nucleosome-free sites) will be mapped both on the integrated HIV genome in ACH-2 cells and on the unintegrated episomal HIV genomes of the HIV-ebv minichromosomes. This mapping will use amplified radiolabeled primer extension and gel electrophoresis. Topoisomerase II (topoII) cleavage and binding sites will be mapped on HIV DNA, in vitro, using EM and gel electrophoresis. Using the methods employed for mapping nucleosome positioning, topoII cleavage sites will be mapped on HIV chromatin in vivo using the ACH-2 cells and the HIV-ebv minichromosomes. EM studies will examine the folding of the HIV 5' LTR in isolated HIV-ebv minichromosomes and reconstituted with cellular LTR binding proteins. Future extension of these approaches could provide a means of inactivating oncogenes in neoplastic cells.
