Book of Abstracts: Albany 2007
June 19-23 2007
Identification and Functional Characterization of Cellular Proteins Interacting with HIV-1 Integrase and its Inhibition by Specific DNAzyme
The HIV Integrase is the key enzyme performing lot of function other than integration like disintegration, transport, virus maturation, and packaging of virus genome. The multifunctional role of this enzyme makes it a vulnerable target for antisense therapy. Integration of Human Immunodeficiency Virus type 1, proviral DNA in the nuclear genome is catalyzed by retroviral integrase. In addition to the retroviral integrase several cellular protein also assist in the integration event. The major reported players are HMG, Ku80, IN1, BAF, Ledgf/p75, Rad 51, Hsp60, SNF5, and ATM. In an attempt to search the HIV integrase interacting proteins we have developed an in vitro system to identify host cell protein. The Human T lymphocyte cell line, Jurkat was used for the host cell protein extraction. We could identify four proteins by co-immunoprecipitation followed by cross linking and Peptide mass fingerprinting. Out of them one is Polypyrimidine track binding protein associated splicing factor(PSF). The PSF protein has been reported to be DNA and RNA binding protein, required in the early spliceosome. The PSF is also reported to be involved in homologous DNA pairing. It promotes invasion of ss DNA into ds DNA and produces D-loop. The PSF-NONO heterodimer may be involved in DNA unwinding modulating the function of topoisomerase I and involved in DNA non homologous and end joining (NHEJ) repair and recombination. It also interacts with the Ku80/XRCC5 dimer to establish a functional preligation complex, which are in turn responsible in HIV genome integration.
Our laboratory currently investigating the exact role of PSF in assisting the HIV Integrase in integration event. In addition to it three DNAzymes were designed to cleave the HIV-Integrase RNA by in vitro transcription reaction and then evaluated the efficiency as well as did conformational analysis of DNAzymes for in vitro cleavage in the presence of different metal ions. The similar DNAzymes were transfected in cell culture and could achieve high degree of inhibition of integrase.
Chemical Biology Laboratory