High-frequency epigenetic repression and silencing of retroviruses can be antagonized by histone deacetylase inhibitors and transcriptional activators, but uniform reactivation in cell clones is restricted by additional mechanisms.

Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Journal of Virology (Impact Factor: 4.65). 04/2007; 81(6):2592-604. DOI: 10.1128/JVI.01643-06
Source: PubMed

ABSTRACT Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(-)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(-) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(-) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(-) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(-) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Retroviruses have evolved complex transcriptional enhancers and promoters that allow for their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses, and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES pluripotency. This overlap is not coincidental: retroviral-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 12/2014; 35(5). DOI:10.1128/MCB.01293-14 · 5.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene therapy using integrating retroviral vectors has proven its effectiveness in several clinical trials for the treatment of inherited diseases and cancer. However, vector-mediated adverse events related to insertional mutagenesis were also observed, emphasizing the need for safer therapeutic vectors. Paradoxically, alpharetroviruses, originally discovered as cancer-causing agents, have a more random and potentially safer integration pattern compared to gammaretro- and lentiviruses. In this review, we provide a short overview of the history of alpharetroviruses and explain how they can be converted into state-of-the-art gene delivery tools with improved safety features. We discuss development of alpharetroviral vectors in compliance with regulatory requirements for clinical translation, and provide an outlook on possible future gene therapy applications. Taken together, this review is a broad overview of alpharetroviral vectors spanning the bridge from their parental virus discovery to their potential applicability in clinical settings.
    Viruses 12/2014; 6(12):4811-4838. DOI:10.3390/v6124811 · 3.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Histone deacetylase inhibitors (HDACi) were recently identified as having significant clinical potential in reversing β-cell functional inhibition caused by inflammation, a shared precursor of Type 1 and Type 2 diabetes. However, HDACi are highly complex and little is known of their direct effect on important cell secretion pathways for blood glucose regulation. The aims of the present study were to investigate the effect of HDACi on insulin secretion from β-cells, GLP-1 secretion from L-cells, and recombinant insulin secretion from engineered L-cells. The β-cell line βTC-tet, L-cell line GLUTag, or recombinant insulin-secreting L-cell lines were exposed to Trichostatin A for 24 hours. Effects on insulin or GLP-1mRNA, intracellular protein content, processing efficiency, and secretion were measured by real-time PCR, ELISA, and radioimmunoassay. HDACi increased secretion per viable cell in a dose-dependent manner for all cell types. Effects on mRNA levels were variable, but enhanced intracellular polypeptide content and secretion were comparable among cell types. Enhanced recombinant insulin secretion was sustained for seven days in alginate microencapsulated L-cells. HDACi enhances β- and L-cell secretion fluxes in a way that could significantly improve blood glucose regulation in diabetes patients and holds potential as a novel method for enhancing insulin-secreting non-β or β-cell grafts.
    Experimental Cell Research 10/2014; 330(1). DOI:10.1016/j.yexcr.2014.09.031 · 3.37 Impact Factor

Full-text (2 Sources)

Available from
Jun 3, 2014