a. Integration of DNAs with aberrant ends. These experiments grew out of experiments in which we determined how mutations either in RT or in the viral genome led to the generation of linear viral DNAs with a high proportion of aberrant ends. We used the RCAS vector system to show that (1) integration is not necessarily 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 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. We are adapting the HIV vectors for analyses of the effects of the IN inhibitors on integration.b. HIV-1 integrase strand transfer inhibitor (INSTI) resistance and the development of new INSTIs. This collaborative effort combines studies of INSTIs resistance with a plan to use that information to develop more effective INSTIs. Dr. Yves Pommier is testing compounds developed by Dr. Terrence Burke, Jr. 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, we had no structural information to guide the development of HIV-1 IN inhibitors. However, Dr. Peter Cherepanov obtained high-resolution structures of full-length prototype foamy virus (PFV) IN in complexes with both DNA and anti-IN drugs. Dr. Cherepanov has joined our collaboration and has solved structures of PFV IN in complexes with some of the compounds developed by Dr. Burke;we have developed a model to predict INSTI binding to WT and mutant HIV-1 INs.c. Integration of HIV-1 DNA. Redirecting HIV-1 DNA integration has the potential to help make gene therapy safer;if the integration sites can be appropriately restricted, it may help solve the problems associated with the insertional activation of oncogenes. In addition, redirected HIV-1 DNA integration 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, and 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 investigate how multiple CBDs interact to define the specificity with which proteins bind chromatin. We are testing whether capsid (CA) mutants, either alone or in conjunction with Nup mutants, redirect HIV-1 integration. We have obtained LEDGF knockout mice from our collaborator Dr. Alan Engelman, and will use cells/tissues from these mice to determine how differentiation and transformation affect the distribution of some well-characterized CBD-IBD fusions. We are developing parallel in vitro systems to study the behavior and specificity of integrations directed by LEDGF fusion proteins. We will investigate the distribution of integration sites in patients before and after successful highly active antiretroviral therapy (HAART) therapy.[Corresponds to Hughes Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011426-01
Application #
8553177
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2012
Total Cost
$1,190,784
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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Noh, Kyung-Min; Wang, Haibo; Kim, Hyunjae R et al. (2015) Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs. Mol Cell 59:89-103
Zhao, Xue Zhi; Smith, Steven J; Metifiot, Mathieu et al. (2014) Bicyclic 1-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxamide-containing HIV-1 integrase inhibitors having high antiviral potency against cells harboring raltegravir-resistant integrase mutants. J Med Chem 57:1573-82

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