Cold Spring Harbor Laboratory meeting on Retroviruses location:Cold Spring Harbor, New York [state], USA date:19-24 May 2008
The transcriptional co-activator LEDGF/p75 is believed to be the dominant factor that tethers the HIV-1 preintegration complex to the chromatin through the interaction with HIV-1 integrase (IN). In parallel with exploiting integrase-LEDGF/p75 interaction for antiviral drug discovery, studies on the cell biology of LEDGF/p75 are required.
Using a Y2H screen we identified “pogo transposable element with ZNF domain” (POGz) 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 revealed the interaction of POGz with LEDGF/p75 to be mediated by the C-terminal part of POGz and the integrase binding domain of LEDGF/p75. Conserved domain analysis of the POGz protein predicts a DDE-domain present at the C-terminus. In addition to the amino acid sequence homology with TIGD transposases, a secondary structure prediction and alignment shows a significant homology of this DDE domain with the structure solved mariner-like-transposase, mos-1. A recombinant C-terminal fragment (amino acids 1117-1410) of POGz 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 included as well. I365A, V370A and F406A mutations in LEDGF/p75 were able to abrogate or severely inhibit the interaction with both POGz and HIV-1 IN while the interaction with JPO2 was only affected moderately.
Interestingly the D366A and V408A mutations severely affect the interaction of LEDGF/p75 with HIV-1 IN but have no effect on the interaction with POGz. Our data indicate a significantly overlapping interaction mechanism of HIV-1 IN and POGz with LEDGF/p75 as opposed to JPO2. Competition assays revealed that the interaction between POGz and LEDGF/p75 is efficiently outcompeted by purified HIV-1 IN core domain while the interaction between JPO2 and LEDGF/p75 is significantly less affected. These findings are reflected in the relative affinities of JPO2, pogZ and HIV-1 IN core for LEDGF/p75 and corroborate the high degree of overlap in pogZ and HIV-1 IN residues engaged in the IBD.
In conclusion, we show that POGz is next to JPO2 the second cellular interaction partner of the C-terminus of LEDGF/p75. Considering that POGz has 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.