Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction

Aiyer, Sriram; Swapna, G.V.T.; Malani, Nirav; Aramini, James M.; Schneider, William M.; Plumb, Matthew R.; Ghanem, Mustafa H.; Larue, Ross C.; Sharma, Amit; Studamire, Barbara; Kvaratskhelia, Mamuka; Bushman, Frederic D.; Montelione, Gaetano T.; Roth, Monica J.

doi:10.1093/nar/gku175

Cited by 69 publications

(146 citation statements)

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“…S3B). Interestingly, both the Brd4 ET domain loop region and the C-terminal tail of MLV integrase are poorly structured on their own (23,26). Thus, in the context of the isolated domains, induced fit folding of both components is required to form the interface, which partly explains the large chemical shift perturbations observed within this region of the Brd4 ET domain (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…The NMR studies reveal that these components interact via binding-coupled folding, in which regions that were poorly structured in isolation (23,26) fold to form a new three-stranded β sheet that generates complementary pairing of charged and hydrophobic residues on both molecules. In cells, interactions between Brd4 and MLV IN are critical for the recruitment of the MLV preintegration complex (PIC) to host chromatin during integration.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis experiments have shown that a conserved sequence motif from the extreme C terminus of MLV IN is responsible for specific tethering to the ET domain (Fig. 1B, yellow) (12,23), and NMR studies of the MLV IN C-terminal domain revealed that this region is unstructured, suggesting a "fly-casting" mechanism for BET protein binding (23). Modifications to this region compromise MLV IN binding to BET proteins and alter MLV integration patterns (12,(23)(24)(25).…”

Section: Significancementioning

confidence: 99%

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Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

Crowe

Larue

Yuan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a proteinprotein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a wellstructured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.T he bromodomain and extraterminal domain (BET) family of proteins (Brd2, 3, 4, and T) play central roles in regulating cell fate (1). They have been implicated in diverse cellular phenomena, including inflammation, obesity, spermatogenesis, cancer, DNA damage repair, viral latency, and, more recently, γ-retroviral integration site selection (2-8). Most of these functions have been associated with its dual role as epigenetic reader and transcriptional activator (1-4).The BET family of proteins, as well as the extended BET family (Brd1,7,8,and 9), is characterized by two N-terminal bromodomains and the extraterminal (ET) domain, for which the proteins are named; Brd4 is known to occur in two different isoforms, with the longer variant containing a C-terminal motif (9) (Fig. 1A). The dual bromodomains recognize and bind acetylated lysines on histone H3 and H4 tails on chromatin (9-11), thereby tethering the protein and any associated factors to those histone posttranslational modifications (PTMs). Whereas recognition of select histone marks is achieved through the bromodomains, the overall high-affinity binding to chromatin is the result of cooperative binding to both its cognate PTMs and nonspecific binding to DNA wrapped around mononucleosomes through interaction with the conserved motifs A and B (12, 13). The C-terminal domain of the long Brd4 isoform functions in activation of RNA polymerase II via interactions with pTEFb (positive transcription elo...

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Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

Crowe

Larue

Yuan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, cellular chromatin-associated protein lens epithelial-derived growth factor (LEDGF/p75) interacts with HIV-1 IN and directs lentiviral integration into actively transcribed genes (11)(12)(13). Similarly, BET (bromodomain and extraterminal domain) proteins have been shown to interact with MLV IN and target MLV integration to transcription start sites, enhancers, and gene regulatory regions (14)(15)(16)(17)(18). These host cell factors bind their cognate viral IN and selected histone marks to act as a bimodal tether to recruit the preintegration complex to specific genomic regions surrounding the host factor binding sites (8-10, 19, 20).…”

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confidence: 99%

The FACT Complex Promotes Avian Leukosis Virus DNA Integration

Winans

Larue

Abraham

et al. 2017

J Virol

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All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro. Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells. IMPORTANCEThe majority of human gene therapy approaches utilize HIV-1-or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells.

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“…p12 accomplishes this by binding the viral preintegration complex (PIC) with its N terminus (3) and nuclear chromatin with its C terminus (2)(3)(4). This p12 chromatin binding motif was shown to not affect MLV integration targeting to transcriptional start sites (4), and integrase (IN) binding to BET proteins was found to achieve the integration targeting (5)(6)(7)(8). Cumulatively, these results suggest that p12 releases the chromatin to allow for subsequent IN-BET interactions and integration.…”

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confidence: 99%

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12

Brzezinski

Modi

Liu

et al. 2016

J Virol

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Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60 PSPMA 65 ), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25 DLLTEDPPPY 34 , which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70 RREPP 74 ) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70 AAAAA 74 ) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p12 70 -74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61 SPIASRLRGRR 71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25 DLLTEDPPPY 34 motif in the N terminus suppresses the ability to tether. IMPORTANCEThis study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p12 61-71 . Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.T he p12 structural protein of murine leukemia virus (MLV) was recently identified as a critical chromatin tether for the early viral life cycle. MLV requires cells to undergo mitosis to gain nuclear entry and requires a mechanism to remain nuclear after mitosis is complete, since integration occurs after exit from mitosis (1, 2). p12 accomplishes this by binding the viral preintegration complex (PIC) with its N terminus (3) and nuclear chromatin with its C terminus (2-4). This p12 chromatin binding motif was shown to not affect MLV integration targeting to transcriptional start sites (4), and inte...

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Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction

Cited by 69 publications

References 51 publications

Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

The FACT Complex Promotes Avian Leukosis Virus DNA Integration

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12

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