Title: Rescue of HIV integration via LEDGF/p75 fused to alternative DNA binding domains
Authors: Gijsbers, Rik ×
Vets, Sofie
McNeely, Melissa
De Rijck, Jan
Debyser, Zeger #
Issue Date: 2008
Conference: International Conference on Retroviral Integrase edition:3 location:Woods Hole, Boston, USA date:14-18 September 2008
Abstract: The transcriptional co-activator LEDGF 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 orchestrates the nuclear accumulation and chromosomal localization of HIV-1 IN via an N-terminal domain containing an ensemble of chromatin binding domains (PWWP, A/T hook, charged regions) and a NLS. HIV IN interacts with the C-terminal end of LEDGF/p75, specifically with the IN binding domain (IBD, aa 347-429). Whereas the pivotal role of LEDGF/p75 in HIV replication was initially demonstrated by RNA interference, we have also shown that HIV replication could be blocked upon stable overexpression of the IBD or the C-terminal end of LEDGF/p75 (Δ325, aa 325-530).
We generated a strong knock-down cell line using miRNA based hairpins specifically directed against LEDGF/p75. HeLaP4 cells were stably transduced with either an HIV-based or a MLV-based retroviral vector and monoclonal cell lines were selected. No LEDGF/p75 protein could be detected by Western analysis, and Q-PCR revealed >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. The results obtained with the lentiviral constructs were corroborated in the cells generated with the MLV-based vectors. The latter cell lines allow detailed analysis of HIV-1 viral and HIV-based lentiviral vector integration sites.
In parallel, we replaced the N-terminal chromatin binding components of LEDGF/p75 (aa 1-324) with alternative DNA binding domains, such as the androgen-receptor DNA binding domain (AR-DBD), or chromatin binding proteins, such as human histone H1, histone 2B (H2B) or hepatoma derived growth factor (HDGF). In addition, we generated chimeric proteins, by replacing the PWWP domain of LEDGF with that of HDGF or Hrp2. Together with RNAi-resistant wild-type LEDGF/p75, all these constructs were used to backcomplement LEDGF/p75-deficient cells using MLV-based vectors, allowing integration site analysis of HIV-based vectors. All chimeric proteins were readily detected in the nucleus and migrated at the expected size as detected by Western analysis. Subsequently, all cell lines were tested for lentiviral vector transduction and HIV-1 infection (single round HIV NL4.3 ΔNefΔEnv-fLuc virus, or wt NL4.3). Hooking the AR DBD to the LEDGF/p75 C-terminus could not rescue HIV replication. With the other DNA-binding protein fusions however, 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 could rescue HIV replication and vector transduction to even wild-type levels, corroborating the tethering role of LEDGF/p75 in HIV replication.
Publication status: published
KU Leuven publication type: IMa
Appears in Collections:Molecular Virology and Gene Therapy
× corresponding author
# (joint) last author

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