Abstract
The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1–720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.
Highlights
In order to replicate, retroviruses must integrate reverse transcribed viral DNA into the host chromosome
We have recently reported that the addition of 0.5 mM Brd4(1–720) stimulated the strand-transfer reaction catalyzed by murine leukemia virus (MLV) IN in vitro [21]
We examined the effects of addition of 0.25, 0.5, 1.0 and 2.0 mM Brd4(1–720), nBrd4 and Brd4 ET on MLV IN (0.3 mM) strand transfer activities
Summary
Retroviruses must integrate reverse transcribed viral DNA into the host chromosome. The primary viral factor controlling the distribution of retroviral integration sites is integrase (IN), whose key role was demonstrated using a chimeric HIV-1 virus with its IN sequence replaced with the MLV counterpart [3]. Integration sites of the chimeric virus significantly changed from active genes towards transcription start sites and trended closer to MLV than HIV-1. The LEDGF/p75 C-terminal region, termed the Integrase Binding Domain or IBD, binds lentiviral INs [10,11]. The N-terminal portion of LEDGF/p75, which contains a PWWP domain, nuclear-localization signal, AT hooks and highly charged regions, selectively associates with chromatin. We have recently shown that the PWWP domain cooperatively engages both the trimethylated H3 tail (H3K36me3), a hallmark of active genes and DNA wrapped around nucleosomes to ensure high-affinity binding of LEDGF/p75 with chromatin [12]
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