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Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

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.

SUBMITTER: Larue RC

PROVIDER: S-EPMC4005663 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

Larue Ross C RC Plumb Matthew R MR Crowe Brandon L BL Shkriabai Nikoloz N Sharma Amit A DiFiore Julia J Malani Nirav N Aiyer Sriram S SS Roth Monica J MJ Bushman Frederic D FD Foster Mark P MP Kvaratskhelia Mamuka M

Nucleic acids research 20140211 8

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, establis ...[more]

PMID: 24520112

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Project description:A critical step for retroviral replication is the stable integration of the provirus into the genome of its host. The viral integrase protein is key in this essential step of the retroviral life cycle. Although the basic mechanism of integration by mammalian retroviruses has been well characterized, the factors determining how viral integration events are targeted to particular regions of the genome or to regions of a particular DNA structure remain poorly defined. Significant questions remain regarding the influence of host proteins on the selection of target sites, on the repair of integration intermediates, and on the efficiency of integration.We describe the results of a yeast two-hybrid screen using Moloney murine leukemia virus integrase as bait to screen murine cDNA libraries for host proteins that interact with the integrase. We identified 27 proteins that interacted with different integrase fusion proteins. The identified proteins include chromatin remodeling, DNA repair and transcription factors (13 proteins); translational regulation factors, helicases, splicing factors and other RNA binding proteins (10 proteins); and transporters or miscellaneous factors (4 proteins). We confirmed the interaction of these proteins with integrase by testing them in the context of other yeast strains with GAL4-DNA binding domain-integrase fusions, and by in vitro binding assays between recombinant proteins. Subsequent analyses revealed that a number of the proteins identified as Mo-MLV integrase interactors also interact with HIV-1 integrase both in yeast and in vitro.We identify several proteins interacting directly with both MoMLV and HIV-1 integrases that may be common to the integration reaction pathways of both viruses. Many of the proteins identified in the screen are logical interaction partners for integrase, and the validity of a number of the interactions are supported by other studies. In addition, we observe that some of the proteins have documented interactions with other viruses, raising the intriguing possibility that there may be common host proteins used by different viruses. We undertook this screen to identify host factors that might affect integration target site selection, and find that our screens have generated a wealth of putative interacting proteins that merit further investigation.

Project description:BACKGROUND:One of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus. RESULTS:We introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs. CONCLUSIONS:M-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.

Dataset Information

Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

Publications

Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

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