: In order to replicate, a retrovirus such as HIV must integrate a cDNA copy of its RNA genome into a chromosome of the host. Although the cDNA integration system is a promising target for antiretroviral therapy, no clinically useful inhibitors have yet been developed. Our proposed studies are designed to provide mechanistic information useful in developing such inhibitors. Integration in vivo is carried out by a sub-viral particle containing the viral cDNA complexed with proteins. Such """"""""preintegration complexes"""""""" (PICs) can be isolated from infected cells and when presented with a target DNA in vitro can carry out integration. We have been engaged in a long term study of the composition, organization and activities of PICs. We propose to continue this work in the renewal and investigate the implications with studies of viral replication in cell culture. We propose three specific aims 1) we will further improve methods for the large-scale production of PICs and their biochemical analysis, particularly the detection and quantitation of complex components using LC-MS and other methods. 2) We will investigate the organization of PICs using chromatin immunoprecipitation (ChIP) assays, EM, and activity based tests. 3) We will investigate the function of PIC components using cells and viruses containing appropriate mutations. Virus growth will be monitored using quantitative PCR assays, including a new method we devised for quantitating integration in vivo. Highlight of our studies include the findings of three cellular proteins associated with PICs, the Ku heterodimer and the architectural DNA binding protein HMG I (Y). The discovery of Ku protein in PICs together with other work has lead to a new model for the role of the host cell non-homologous DNA end joining pathway in retroviral growth. Long term, these studies, together with data on protein structures, will provide a high-resolution picture of the organization and function of preintegration complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI034786-12
Application #
6846449
Study Section
Special Emphasis Panel (ZRG1-AARR-4 (02))
Program Officer
Sharma, Opendra K
Project Start
1993-07-01
Project End
2005-05-31
Budget Start
2003-12-01
Budget End
2004-05-31
Support Year
12
Fiscal Year
2003
Total Cost
$106,195
Indirect Cost
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Ciuffi, Angela; Diamond, Tracy L; Hwang, Young et al. (2006) Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor. Hum Gene Ther 17:960-7
Ciuffi, Angela; Bushman, Frederic D (2006) Retroviral DNA integration: HIV and the role of LEDGF/p75. Trends Genet 22:388-95
Lewinski, Mary K; Yamashita, Masahiro; Emerman, Michael et al. (2006) Retroviral DNA integration: viral and cellular determinants of target-site selection. PLoS Pathog 2:e60
Wang, Gary P; Bushman, Frederic D (2006) A statistical method for comparing viral growth curves. J Virol Methods 135:118-23
Diamond, Tracy L; Bushman, Frederic D (2005) Division of labor within human immunodeficiency virus integrase complexes: determinants of catalysis and target DNA capture. J Virol 79:15376-87
Barr, Stephen D; Leipzig, Jeremy; Shinn, Paul et al. (2005) Integration targeting by avian sarcoma-leukosis virus and human immunodeficiency virus in the chicken genome. J Virol 79:12035-44
Lewinski, M K; Bisgrove, D; Shinn, P et al. (2005) Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J Virol 79:6610-9
Groschel, Bettina; Bushman, Frederic (2005) Cell cycle arrest in G2/M promotes early steps of infection by human immunodeficiency virus. J Virol 79:5695-704
Ciuffi, Angela; Llano, Manuel; Poeschla, Eric et al. (2005) A role for LEDGF/p75 in targeting HIV DNA integration. Nat Med 11:1287-9
Gao, Kui; Wong, Steven; Bushman, Frederic (2004) Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro. J Virol 78:6715-22

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