MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA

Schneider, William M.; Wu, Daitze; Amin, Vaibhav; Aiyer, Sriram; Roth, Monica J.

doi:10.1016/j.virol.2012.01.034

Cited by 30 publications

(39 citation statements)

References 63 publications

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“…Cellular DNA was extracted via the DNeasy blood and tissue kit (catalog number 69581; Qiagen), and 100 ng was used directly for triplicate quantitative PCRs (qPCRs) with Power SYBR green PCR master mix (Life Technologies). Plus strand extension (PSE) primers have been previously described (28); RPPH1 primers are 5=-CGTGAGTCTGTTCCAA GCTC-3= and 5=-GGGAGGTGAGTTCCCAGAG-3. PSE cycle threshold (C T ) values were first subtracted from paired RPPH1 gene C T values to normalize samples for DNA extraction efficiency.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…To pinpoint the replication steps affected by JQ-1 treatment, we quantified viral DNA forms longitudinally, including the minusstrand strong-stop extension products (MSSEs), plus-strand extension products (PSEs), 2-LTR circles, and integrated proviruses (33). JQ-1 treatment did not alter MSSEs or PSEs (Fig.…”

Section: Antagonism Of Bet Proteins Reduces MLV Integration In Infectedmentioning

confidence: 99%

“…3D). These dead-end products serve as surrogate markers and are known to increase in quantity in the presence of defective integration (33). Quantitation of integrated proviruses by Alu-based quantitative (q) PCR revealed a significant reduction in integrated MLV proviruses upon treatment with JQ-1 (Fig.…”

Section: Antagonism Of Bet Proteins Reduces MLV Integration In Infectedmentioning

confidence: 99%

BET proteins promote efficient murine leukemia virus integration at transcription start sites

Sharma¹,

Larue²,

Plumb³

et al. 2013

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

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The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy. (1-4). The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus studied (5-7). For example, the γ-retroviruses favor integration near transcription start sites, whereas lentiviruses favor integration within transcription units. These observations have suggested that different cellularbinding partners of retroviral integrases are likely to be responsible for integration target-site selection. However, to date, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes (8-14). Cellular cofactors of other retroviral genera are currently unknown.The molecular mechanisms of γ-retroviral murine leukemia virus (MLV) integration are of particular significance because MLV-based vectors are used for human gene therapy. In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. 15-18). The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.In this report, we have identified the bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as the cellularbinding partners of MLV IN and demonstrate their significance for stimulating and targeting MLV integration at transcription start sites. (Table 1, Table S1, and Fig. S1). Of these, Brd4 and Brd3 were the top hits in NIH 3T3 and Sup-T1 cells, respectively. Differential pull-down levels of these proteins (Table 1) could be attributable to the varying expression le...

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“…Cells were also infected with HIV luc IN WT in the presence of the integrase inhibitor Raltegravir (60 nM). Due to the transient nature of the expression of unintegrated retroviral genomes (Butler et al, 2001;Kilzer et al, 2003;Schneider et al, 2012;Yu et al, 2008), luciferase was measured in cells infected with integrated retroviruses (HIV luc IN WT) 4 days post-infection, to allow elimination of the transgene expression from unintegrated viral genomes, and in cells infected with unintegrated retroviruses (HIV luc IN D64V or WT plus Raltegravir) 2 days post-infection, to prevent loss of the unintegrated viral genomes. In support of this rationale, luciferase expression in HIV luc IN D64V-infected cells decreased by more than 90 % by day 4, being undetectable by day 6 post-infection; this rate of decay was similar in cells expressing or not PARP-1 (data not shown).…”

mentioning

confidence: 99%

“…PARP-1 has been reported to silence integrated retroviruses and retrotransposons through epigenetic mechanisms implicated in the formation and maintenance of the host heterochromatin (Bueno et al, 2013;Kotova et al, 2010Kotova et al, , 2011Kotova et al, , 2011Tulin et al, 2002). Unintegrated and integrated retrovirus genomes are associated with chromatin (Kantor et al, 2009;Schneider et al, 2012;Sloan & Wainberg, 2011); therefore, PARP-1 could silence these through similar mechanisms. However, potential variations in the chromatin organized at these two forms of viral genome could determine differences in the mechanisms implicated.…”

mentioning

confidence: 99%

Poly(ADP-ribose) polymerase-1 silences retroviruses independently of viral DNA integration or heterochromatin formation

Gutierrez

Valdes

Serguera

et al. 2016

Journal of General Virology

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PARP-1 silences retrotransposons in Drosophila, through heterochromatin maintenance, and integrated retroviruses in chicken. Here, we determined the role of viral DNA integration and cellular heterochromatin in PARP-1-mediated retroviral silencing using HIV-1-derived lentiviral vectors and Rous-associated virus type 1 (RAV-1) as models. Analysis of the infection of PARP-1 knockout and control cells with HIV-1 harbouring WT integrase, in the presence or absence of an integrase inhibitor, or catalytic-dead mutant integrase indicated that silencing does not require viral DNA integration. The mechanism involves the catalytic activity of histone deacetylases but not that of PARP-1. In contrast to Drosophila, lack of PARP-1 in avian cells did not affect chromatin compaction globally or at the RAV-1 provirus, or the cellular levels of histone H3 N-terminal acetylated or Lys27 trimethylated, as indicated by micrococcal nuclease accessibility and immunoblot assays. Therefore, PARP-1 represses retroviruses prior to viral DNA integration by mechanisms involving histone deacetylases but not heterochromatin formation.The cellular response to invading genomes is evolutionarily conserved, suggesting an early origin of these mechanisms

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MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA

Cited by 30 publications

References 63 publications

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

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

BET proteins promote efficient murine leukemia virus integration at transcription start sites

Poly(ADP-ribose) polymerase-1 silences retroviruses independently of viral DNA integration or heterochromatin formation

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