This grant proposal will investigate the role of cellular DNA repair proteins in retroviral DNA integration. The working hypothesis for these studies is: retroviral DNA integration triggers specific cellular DNA repair systems that play a role in the repair and/or chromatin remodeling at integration sites. This host response is required for the successful integration of retroviral DNA into host DNA.
Specific Aim 1 will analyze the contribution of cellular non-homologous end joining (NHEJ) proteins to retroviral DNA integration. It is proposed that these proteins play a role in the repair step of integration that involves the 5'-end joining of viral DNA to the host DNA, or in the chromatin remodeling that may follow this process. These steps are investigated in normal cells and in cells deficient in NHEJ proteins. The investigation of 5'-end joining employs techniques that combine S1 endonuclease treatment with the Southern blot analysis and Alu-PCR. In addition to this technique, an alternative technique will be used that relies on a specifically designed retroviral mutant that allows examination of the 5'-end joining event. Chromatin remodeling will then be examined by advanced immunofluorescence methods combined with FISH (immuno-FISH), and by chromatin immunoprecipitation (ChIP). To determine if NHEJ proteins accumulate at integration sites and participate closely in integration, or are involved in integration indirectly, possibly by signaling to other proteins, co-immunoprecipitation and ChIP methods will be used.
Specific Aim 2 will employ similar methods as Specific Aim 1, to investigate the role of another protein, the ATR kinase, in retroviral DNA integration. The role of ATR in the 5'-end joining step of integration will be investigated using techniques described above. To characterize the molecular mechanisms that underlie ATR function in retroviral DNA integration, downstream targets of ATR will be examined. Western blotting will determine if modifications of these target proteins by ATR are triggered by retroviral DNA integration. In addition immuno-FISH and ChIP will be used to determine if ATR accumulates at integration sites. Results from these studies should deepen our understanding of the role that NHEJ and ATR proteins play to facilitate retroviral DNA integration. They should also significantly enhance our understanding of the function of these important cellular pathways. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01CA098090-01A1
Application #
6680768
Study Section
Subcommittee G - Education (NCI)
Program Officer
Eckstein, David J
Project Start
2003-09-30
Project End
2008-08-31
Budget Start
2003-09-30
Budget End
2004-08-31
Support Year
1
Fiscal Year
2003
Total Cost
$117,279
Indirect Cost
Name
Thomas Jefferson University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Nunnari, Giuseppe; Smith, Johanna A; Daniel, Rene (2008) HIV-1 Tat and AIDS-associated cancer: targeting the cellular anti-cancer barrier? J Exp Clin Cancer Res 27:3
Daniel, Rene; Smith, Johanna A (2008) Integration site selection by retroviral vectors: molecular mechanism and clinical consequences. Hum Gene Ther 19:557-68
Smith, Johanna A; Wang, Feng-Xiang; Zhang, Hui et al. (2008) Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellul Virol J 5:11
Smith, Johanna A; Nunnari, Giuseppe; Preuss, Mirjam et al. (2007) Pentoxifylline suppresses transduction by HIV-1-based vectors. Intervirology 50:377-86
Daniel, Rene (2006) DNA repair in HIV-1 infection: a case for inhibitors of cellular co-factors? Curr HIV Res 4:411-21
Smith, Johanna A; Daniel, Rene (2006) Following the path of the virus: the exploitation of host DNA repair mechanisms by retroviruses. ACS Chem Biol 1:217-26
Nunnari, Giuseppe; Argyris, Elias; Fang, Jianhua et al. (2005) Inhibition of HIV-1 replication by caffeine and caffeine-related methylxanthines. Virology 335:177-84
Daniel, Rene; Marusich, Elena; Argyris, Elias et al. (2005) Caffeine inhibits human immunodeficiency virus type 1 transduction of nondividing cells. J Virol 79:2058-65
Daniel, Rene; Greger, James G; Katz, Richard A et al. (2004) Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway. J Virol 78:8573-81
Daniel, Rene; Ramcharan, Joseph; Rogakou, Emmy et al. (2004) Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair. J Biol Chem 279:45810-4