Human gene therapy vol:19 issue:10 pages:1193-1194
ESGCT Meeting location:Brugge, Belgium date:13-16 November 2008
Lentiviruses stably introduce their viral genome into the host cell chromosome. The transcriptional co-activator LEDGF/p75 is the dominant cellular co-factor for HIV-1 integrase (IN), tethering the preintegration complex to chromatin; however, the exact orchestration of this process remains unknown. LEDGF/p75 organizes the nuclear accumulation and chromosomal localization of IN via its N-terminal domain containing an ensemble of chromatin binding domains. IN interacts with the IN binding domain (IBD, aa 347-429) at the C-terminal end of LEDGF/p75. Stable overexpression of the IBD or the C-terminal end of LEDGF/p75 (Δ325, aa 325-530) strongly inhibit HIV replication by competition for interaction with IN.
We generated strong LEDGF/p75 knock-down lines using miRNA-based hairpins. These cells were stably backcomplemented with chimeric proteins, where we replaced the N-terminal chromatin binding components of LEDGF/p75 (aa 1-324) with alternative DNA binding domains (AR-DBD, H1, H2B, or hepatoma derived growth factor (HDGF)). In addition, we generated chimeric proteins, by replacing the LEDGF PWWP domain with that of HDGF or Hrp2.
Upon knock-down, LEDGF/p75 protein was depleted as shown by Western and Q-PCR (>93% knock-down for the polyclonal cells and >97% for the most potent monoclonal lines). When judged by immunocytochemistry, LEDGF/p75 was depleted from the nucleus. All backcomplementation cell lines were tested for lentiviral vector transduction and HIV-1 infection (NL4.3 ΔNefΔEnv-fLuc virus or wt NL4.3). Hooking the AR DBD to the LEDGF/p75 C-terminus could not rescue HIV replication. However, with the other DNA-binding protein fusions both LV transduction and HIV infection were rescued. Whereas fusion of Δ325 with full length H1 or H2B resulted in moderate rescue, fusion with full length HDGF or the chimeric PWWP-replacements rescued HIV replication and vector transduction to even wild-type levels.
Chimeric LEDGF proteins containing alternative DNA binding domains can be substituted for LEDGF, corroborating the tethering role of LEDGF/p75 in viral vector transduction and HIV replication. Our results facilitate the design of safer vectors for gene therapy by fusing the IBD of LEDGF with sequence specific DNA binding motifs.