scholarly journals Establishment of Murine Cytomegalovirus Latency In Vivo Is Associated with Changes in Histone Modifications and Recruitment of Transcriptional Repressors to the Major Immediate-Early Promoter

2008 ◽  
Vol 82 (21) ◽  
pp. 10922-10931 ◽  
Author(s):  
Xue-feng Liu ◽  
Shixian Yan ◽  
Michael Abecassis ◽  
Mary Hummel
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Latent Infection ◽  
Control Point ◽  
Immediate Early ◽  
Productive Infection ◽  
Murine Cytomegalovirus ◽  
Critical Control Point ◽  
Latently Infected

ABSTRACT Human cytomegalovirus (CMV) is a ubiquitous herpesvirus with the ability to establish a lifelong latent infection. The mechanism by which this occurs is not well understood. Regulation of, for example, immediate-early (IE) gene expression is thought to be a critical control point in transcriptional control of the switch between latency and reactivation. Here, we present evidence that supports previous studies showing that the majority of genomes are quiescent with respect to gene expression. To study the possible role of epigenetic factors that may be involved in repression of ie gene expression in latency, we have analyzed changes in the patterns of modifications of histones bound to the major IE promoter (MIEP) in the kidneys of acutely and latently infected mice. Our studies show that, like herpes simplex virus, murine CMV genomes become relatively enriched in histones in latent infection. There are dramatic changes in modifications of histones associated with the MIEP when latency is established: H3 and H4 become hypoacetylated and H3 is hypomethylated at lysine 4, while H3 lysine 9 is hypermethylated in latently infected mice. These changes are accompanied by a relative loss of RNA polymerase and gain of heterochromatin protein 1γ and Yin-Yang 1 bound to the MIEP. Our studies suggest that, in the majority of cells, CMV establishes a true latent infection, defined as the lack of expression of genes associated with productive infection, and that this occurs through changes in histone modifications and recruitment of transcriptional silencing factors to the MIEP.

scholarly journals The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs

2009 ◽  
Vol 83 (9) ◽  
pp. 4030-4038 ◽  
Author(s):  
Andreas Busche ◽  
Anja Marquardt ◽  
Andre Bleich ◽  
Peter Ghazal ◽  
Ana Angulo ◽  
...  
Keyword(s):  
Latent Infection ◽  
Acute Infection ◽  
Lytic Replication ◽  
Immediate Early ◽  
Productive Infection ◽  
Viral Gene ◽  
Mouse Cytomegalovirus ◽  
Early Protein ◽  
Latently Infected

ABSTRACT The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Δie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Δie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Δie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Δie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.

scholarly journals In Vivo Replication of Recombinant Murine Cytomegalovirus Driven by the Paralogous Major Immediate-Early Promoter-Enhancer of Human Cytomegalovirus

1999 ◽  
Vol 73 (6) ◽  
pp. 5043-5055 ◽  
Author(s):  
Natascha K. A. Grzimek ◽  
Jürgen Podlech ◽  
Hans-Peter Steffens ◽  
Rafaela Holtappels ◽  
Susanne Schmalz ◽  
...  
Keyword(s):  
Core Promoter ◽  
Control Point ◽  
Regulatory Elements ◽  
Immediate Early ◽  
Productive Infection ◽  
Murine Cytomegalovirus ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Human Cmv

ABSTRACT Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502–8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.

scholarly journals Transcriptional reactivation of murine cytomegalovirus ie gene expression by 5-aza-2′-deoxycytidine and trichostatin A in latently infected cells despite lack of methylation of the major immediate-early promoter

2007 ◽  
Vol 88 (4) ◽  
pp. 1097-1102 ◽  
Author(s):  
Mary Hummel ◽  
Shixian Yan ◽  
Zhigao Li ◽  
Thomas K. Varghese ◽  
Michael Abecassis
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Trichostatin A ◽  
Immediate Early ◽  
Productive Infection ◽  
Murine Cytomegalovirus ◽  
Virus Reactivation ◽  
Early Promoter ◽  
Latently Infected ◽  
Major Immediate Early Promoter

We have used a spleen explant model to investigate mechanisms of murine cytomegalovirus latency and reactivation. Induction of immediate-early (ie) gene expression occurs in explants after approximately 9 days in culture and virus reactivation follows induction of ie gene expression with kinetics similar to that of productive infection in vitro. This occurs independently of TNF receptor signalling. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A results in more rapid induction of ie gene expression and reactivation of virus. Despite these results, which suggest a role for DNA methylation in maintenance of viral latency, we find that the major immediate-early promoter/enhancer is not methylated in latently infected mice. Our results support the hypothesis that latency is maintained by epigenetic control of ie gene expression, and that induction of ie gene expression leads to reactivation of virus, but suggest that these are not controlled by DNA methylation.

scholarly journals Biphasic Recruitment of Transcriptional Repressors to the Murine Cytomegalovirus Major Immediate-Early Promoter during the Course of Infection In Vivo

2010 ◽  
Vol 84 (7) ◽  
pp. 3631-3643 ◽  
Author(s):  
Xue-feng Liu ◽  
Shixian Yan ◽  
Michael Abecassis ◽  
Mary Hummel
Keyword(s):  
Gene Expression ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Viral Gene ◽  
Transcriptional Repressors ◽  
Early Promoter ◽  
Viral Promoters ◽  
Major Immediate Early Promoter

ABSTRACT Our previous studies showed that establishment of murine cytomegalovirus (MCMV) latency in vivo is associated with repression of immediate-early gene expression, deacetylation of histones bound to the major immediate-early promoter (MIEP), changes in patterns of methylation of histones, and recruitment of cellular repressors of transcription to the MIEP. Here, we have quantitatively analyzed the kinetics of changes in viral RNA expression, DNA copy number, and recruitment of repressors and activators of transcription to viral promoters during the course of infection. Our results show that changes in viral gene expression correlate with changes in recruitment of RNA polymerase and acetylated histones to viral promoters. Binding of the transcriptional repressors histone deacetylase type 2 (HDAC2), HDAC3, YY1, CBF-1/RBP-Jk, Daxx, and CIR to the MIEP and HDACs to other promoters showed a biphasic pattern: some binding was detectable prior to activation of viral gene expression, then decreased with the onset of transcription and increased again as repression of viral gene expression occurred. Potential binding sites for CBF-1/RBP-Jk and YY1 in the MIEP and for YY1 in the M100 promoter (M100P) were identified by in silico analysis. While recruitment of HDACs was not promoter specific, binding of CBF-1/RBP-Jk and YY1 was restricted to promoters with their cognate sites. Our results suggest that sequences within viral promoters may contribute to establishment of latency through recruitment of transcriptional repressors to these genes. The observation that repressors are bound to the MIEP and other promoters immediately upon infection suggests that latency may be established in some cells very early in infection.

scholarly journals ZIC2 Is Essential for Maintenance of Latency and Is a Target of an Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Yuanzhi Lyu ◽  
Kazushi Nakano ◽  
Ryan R. Davis ◽  
Clifford G. Tepper ◽  
Mel Campbell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Kaposi's Sarcoma ◽  
Latent Infection ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Immediate Early ◽  
Chromatin Domains ◽  
Infected Cells ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modifications toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication. IMPORTANCE Posttranslational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, a transcriptional factor essential for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts with and maintains polycomb repressor complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2-mediated local histone modification.

Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa

1992 ◽  
Vol 12 (4) ◽  
pp. 1568-1577
Author(s):  
J V Paietta
Keyword(s):  
Gene Expression ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Control Point ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Cys3 Protein

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

scholarly journals The gammaherpesvirus 68 viral cyclin facilitates reactivation by promoting latent gene expression

2019 ◽  
Author(s):  
Brian F Niemeyer ◽  
Joy E Gibson ◽  
Jennifer N Berger ◽  
Lauren M Oko ◽  
Eva Medina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Productive Infection ◽  
Viral Gene ◽  
Cellular Distribution ◽  
Viral Cyclin ◽  
Latently Infected ◽  
Infected Cells ◽  
Virally Encoded ◽  
The Impact

AbstractGammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into more active lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on latent gene expression, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of latent gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential latent gene expression, and that a primary function of the viral cyclin is to promote latent gene expression within infected cells in vivo.AUTHOR SUMMARYGammaherpesviruses are ubiquitous viruses with oncogenic potential that establish latency for the life of the host. These viruses can emerge from latency through reactivation, a process that is controlled by the immune system. Control of viral latency and reactivation is thought to be critical to prevent γHV-associated disease. This study focuses on a virally-encoded cyclin that is required for reactivation from latency. By characterizing how the viral cyclin influences latent infection in pure cell populations, we find that the viral cyclin has a vital role in promoting viral gene expression during latency. This work provides new insight into the function of a virally encoded cyclin in promoting reactivation from latency.

scholarly journals Expression of the Pseudorabies Virus Latency-Associated Transcript Gene during Productive Infection of Cultured Cells

1999 ◽  
Vol 73 (12) ◽  
pp. 9781-9788 ◽  
Author(s):  
Ling Jin ◽  
Gail Scherba
Keyword(s):  
Gene Expression ◽  
Pseudorabies Virus ◽  
Cultured Cells ◽  
Initiation Site ◽  
Early Gene ◽  
Productive Infection ◽  
Virus Latency ◽  
Infected Cells ◽  
Transcript Gene

ABSTRACT Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8.4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8.0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8.0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8′ and 8. Its synthesis was inhibited by cycloheximide but not by cytosine β-d-arabinofuranoside, which suggests that the 2.0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

scholarly journals The Cellular Coactivator HCF-1 Is Required for Glucocorticoid Receptor-Mediated Transcription of Bovine Herpesvirus 1 Immediate Early Genes

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Laximan Sawant ◽  
Insun Kook ◽  
Jodi L. Vogel ◽  
Thomas M. Kristie ◽  
Clinton Jones
Keyword(s):  
Gene Expression ◽  
Glucocorticoid Receptor ◽  
Bovine Herpesvirus ◽  
Transcription Unit ◽  
Lytic Infection ◽  
Immediate Early ◽  
Productive Infection ◽  
Core Element ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1

ABSTRACTFollowing productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatmentin vivo. As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1–GR mechanism in the absence of the viral IE activator VP16.IMPORTANCEBoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1–GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.

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