Investigating the Role of PARP-1 in HIV-1 Replication
Poly (ADP-ribose) Polymerase 1 (PARP-1) is a cellular protein that that has multiple roles in the cell including DNA repair and transcription. PARP-1 has also proven by our laboratory to impair HIV-1 replication in T cells infected with HIV-1 strains entering via CD4/CXCR4. The goal of my thesis is to increase our understanding of the mechanism implicated in the anti-HIV-1 activity of PARP-1. Initially, I focused my research in deciphering the impact of PARP-1 on the replication of HIV-1 strains entering the cell through the alternative pathway CD4/CCR5. However, technical difficulties prevented answering this question. Nevertheless, aided by the technical training gained in the first project I investigated the implication of PARP-1 chromatin binding activity and subcellular distribution in its anti-HIV-1 activity. These studies were additionally relevant because the mechanism of chromatin binding of PARP-1 in cells is ill-defined, despite of the central role of this function in all PARP-1 cellular roles. Using cell fractionation and immunochemistry techniques I found that PARP-1 is tightly bound to chromatin under basal conditions. This binding required the N-terminal region of PARP-1 that was also sufficient. Importantly, the N- but not the C-terminus exhibited anti-HIV-1 activity, establishing correlation between anti-viral effects and chromatin binding. Mapping in further details N-terminal domains mediating chromatin binding, I found that deletion of the BRCT domain, nuclear localization signal, zinc fingers I or II did not affect PARP-1 chromatin binding. Zinc finger domains are essential for DNA binding therefore my data showed that PARP-1 in cells mainly interacts with chromatin through binding to chromatin-associated proteins rather than to DNA. To distinguish the implication of DNA-binding activity of PARP-1 in its anti-HIV-1 activity, we used a PARP-1 inhibitor that ejects zinc from zinc fingers impairing DNA binding. This inhibitor blocked the anti-HIV-1 activity of PARP-1 but did not impair its chromatin binding, highlighting the relevance of DNA binding, but not association with chromatin-bound proteins, in the anti-HIV-1 mechanism. I also found no correlation between subcellular distribution, determined by immunofluorescence, and anti-HIV-1 activity. In summary, my results indicate that the anti-HIV-1 activity of PARP-1 requires DNA binding rather than association to chromatin-bound proteins or nuclear residency.
Garcia, Sara Raquel, "Investigating the Role of PARP-1 in HIV-1 Replication" (2018). ETD Collection for University of Texas, El Paso. AAI10823314.