a. Integration of DNAs with aberrant ends. These experiments grew out of experiments designed to examine how mutations either in RT or in the viral genome altered the process of reverse transcription;this led to the generation of linear viral DNAs with a high proportion of aberrant ends. Having characterized the nature of the errors at the ends of the viral DNAs, we then examined how the aberrant ends affected viral DNA integration. Using the RCAS vector system to facilitate the rescue of aberrant-end viral DNAs, we found that (1) integration is not concerted;if a viral DNA has a good end and an aberrant end, the good end integrates using viral integrase (IN) and the aberrant end is integrated with high efficiency, apparently by host enzymes. (2) In most, but not all, cases, microhomology is involved in the host-mediated integration reaction. (3) The host integration reaction frequently causes a relatively large duplication (hundreds to thousands of nucleotides), and less frequently a deletion of host sequences. (4) The host joining reaction usually, but not always, involves the loss of viral sequences. (5) More rarely, complex events occur that involve duplications of viral sequences, inversion of host sequences, or the insertion of sequences from different chromosomes. These experiments may have implications for the treatment of patients with suboptimal doses of IN inhibitors;suboptimal therapy may lead to aberrant integrations. b. Developing IN inhibitors. Dr. Y. Pommier is testing compounds developed by Dr. T. Burke for their ability to inhibit HIV integration in vitro (using purified recombinant IN);we are testing their effect on viral replication and their toxicity in cultured cells. Until quite recently, it was not possible to use structural information to guide the development of IN inhibitors. However, Dr. P. Cherepanov and his colleagues have obtained high-resolution structures of full-length foamy virus (FV) IN in complexes with both DNA substrates and anti-IN drugs. Dr. Cherepanov has joined our collaboration and will solve the structures of FV IN in complex with some of the more promising compounds developed by Dr. Burke. The FV active site is similar, but not identical, to the HIV-1 active site, so the FV structures should be useful. However, it should be possible to mutate the region around the FV active site to make it more similar to HIV-1 IN, which should provide better guidance for design/development of more effective inhibitors. c. Redirecting the integration of HIV-1 DNA. Redirecting HIV-1 DNA integration has the potential to help make gene therapy safer because it may help solve the problems associated with the insertional activation of oncogenes;in addition, the technology can be used to determine where on the genome proteins/domains bind to chromatin. Lens epithelium derived growth factor (LEDGF) interacts with HIV-1 IN, which directs HIV-1 DNA integration to the bodies of expressed genes. The C-terminus of LEDGF contains an IN-binding domain and the N-terminus binds chromatin. We and others showed that replacing the N-terminus of LEDGF with chromatin-binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. The initial experiments were done either with single CBDs, or, in one case, two domains from a larger protein. We will do a number of additional experiments: (1) We will investigate how multiple CBDs interact to define the specificity with which proteins bind chromatin. Ultimately, we will determine how whole proteins bind chromatin. We will obtain well-characterized domains/proteins from the laboratory of our collaborator Dr. C.D. Allis. (2) All the domains we have analyzed so far bind marks on histone tails. We have started to apply the technology to proteins that bind specific DNA sequences, and will investigate domains/proteins that interact with chromatin in other ways. (3) Genes are expressed differently in different tissues and in certain disease states, like cancer. This implies a change in the chromatin. We have just obtained, from our collaborator Dr. A. Engelman, LEDGF knockout mice, and will use cells/tissues from these mice to determine how differentiation and transformation affect the distribution of some well-characterized CBD-IBD fusions. (4) We will adapt the technology to human cells. [Corresponds to Hughes Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]

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Zhao, Xue Zhi; Maddali, Kasthuraiah; Metifiot, Mathieu et al. (2011) Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors. Bioorg Med Chem Lett 21:2986-90
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