Title: The domesticated transposase POGz is a cellular binding partner of LEGDF/p75
Authors: Bartholomeeusen, Koen
Gijsbers, Rik
De Rijck, Jan
Emiliani, S
Benarous, R
Christ, Frauke
Debyser, Zeger #
Issue Date: 2008
Conference: Retroviral Integrase, Molecular Biology and Pharmacology location:Woods Hole, Boston, USA date:14-18 September 2008
Abstract: The transcriptional co-activator LEDGF/p75 is believed to be the dominant factor that tethers the HIV-1 preintegration complex to the chromatin through a strong interaction with HIV-1 integrase (IN). The IN-LEDGF/p75 interaction is now extensively studied in the context of antiviral drug therapy. In addition, detailed studies on the cellular role of LEDGF/p75 and its cellular binding partners are also necessary.
Using a Y2H screen we identified pogZ, pogo transposable element with ZnF domain, as a new cellular interaction partner of LEDGF/p75. Co-localization and co-immunoprecipitation studies confirmed the interaction of pogZ with LEDGF/p75 in HeLaP4 cells and have revealed the interaction of pogZ with LEDGF/p75 to be mediated by the C-terminal region of pogZ with the integrase binding domain (IBD) of LEDGF/p75. Conserved domain analysis of pogZ predicts a DDE-domain at the C-terminus. In addition to primary sequence homology with TIGD transposases, the predicted secondary structure shows significant homology of the DDE domain with the known structure of the mariner-like-transposase, mos-1. The recombinantly expressed C-terminus of pogZ (aa 1117-1410) was purified and its binding characteristics to WT LEDGF/p75 and IBD mutants were analyzed in a direct interaction AlphaScreen assay. JPO2, a previously identified interaction partner of LEDGF/p75, and HIV-1 IN were also included. I365A, V370A and F406A mutations in LEDGF/p75 IBD were able to either abrogate or severely inhibit the interaction with both pogZ and HIV-1 IN, whereas the interaction with JPO2 was affected only moderately, suggesting an overlapping interaction site in LEDGF/p75 for HIV-1 IN and pogZ. Interestingly, the D366A and V408A mutations severely influenced the interaction of LEDGF/p75 with HIV-1 IN but did not affect the pogZ interaction. Relative Kd values of JPO2, pogZ and HIV-1 IN core for LEDGF/p75 were determined at 62, 62.5 and 28.5 nM, respectively. In addition, competition assays revealed that the pogZ-LEDGF/p75 interaction is efficiently outcompeted by purified HIV-1 IN core domain whereas the JPO2-LEDGF/p75 interaction is only affected moderately.
In conclusion, we have shown that pogZ, next to JPO2, is a second cellular interaction partner of LEDGF/p75. Considering that pogZ might have evolved from an ancient transposon, this is the first indication that lentiviruses might not be the only mobile genetic elements that engage LEDGF/p75 for chromosomal tethering.
Publication status: published
KU Leuven publication type: IMa
Appears in Collections:Molecular Virology and Gene Therapy
Biochemistry, Kulak (-)
# (joint) last author

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