scholarly journals Alternative Promoters Drive Human Cytomegalovirus Reactivation from Latency

2018 ◽  
Author(s):  
Donna Collins-McMillen ◽  
Mike Rak ◽  
Jason Buehler ◽  
Suzu Igarashi-Hayes ◽  
Jeremy Kamil ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Viral Gene ◽  
Alternative Promoters ◽  
Cytomegalovirus Reactivation ◽  
Viral Transactivators ◽  
Context Specific ◽  
Major Immediate Early Promoter

ABSTRACTReactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP is poorly active in HPCs, it is unclear how viral transactivators are expressed during reactivation. We demonstrate that transcripts originating from alternative promoters within the canonical major immediate early locus are abundantly expressed upon reactivation, whereas MIEP-derived transcripts remain undetectable. Further, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings change the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

scholarly journals Alternative promoters drive human cytomegalovirus reactivation from latency

2019 ◽  
Vol 116 (35) ◽  
pp. 17492-17497 ◽  
Author(s):  
Donna Collins-McMillen ◽  
Mike Rak ◽  
Jason C. Buehler ◽  
Suzu Igarashi-Hayes ◽  
Jeremy P. Kamil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Viral Gene ◽  
Genome Replication ◽  
Alternative Promoters ◽  
Cytomegalovirus Reactivation ◽  
Context Specific

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

scholarly journals General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism

2011 ◽  
Vol 92 (12) ◽  
pp. 2757-2769 ◽  
Author(s):  
Martin Zydek ◽  
Ralf Uecker ◽  
Nina Tavalai ◽  
Thomas Stamminger ◽  
Christian Hagemeier ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Nuclear Genome ◽  
Viral Gene Expression ◽  
Lytic Replication ◽  
Immediate Early ◽  
Viral Gene ◽  
Level Control ◽  
G2 Phase

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36–38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

scholarly journals Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic ‘pre-immediate-early’ repression of viral gene expression mediated by histone post-translational modification

2009 ◽  
Vol 90 (10) ◽  
pp. 2364-2374 ◽  
Author(s):  
Ian J. Groves ◽  
Matthew B. Reeves ◽  
John H. Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Viral Gene Expression ◽  
Lytic Infection ◽  
Immediate Early ◽  
Late Gene ◽  
Viral Gene ◽  
Gene Promoters ◽  
Late Gene Expression

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at ‘pre-IE’ times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

scholarly journals Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

2002 ◽  
Vol 76 (1) ◽  
pp. 313-326 ◽  
Author(s):  
Jeffery L. Meier ◽  
Michael J. Keller ◽  
James J. McCoy
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Gene Transcription ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Viral Gene ◽  
Distal Enhancer ◽  
Cis Acting ◽  
Hcmv Replication

ABSTRACT We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

scholarly journals Small Internal Deletions in the Human Cytomegalovirus IE2 Gene Result in Nonviable Recombinant Viruses with Differential Defects in Viral Gene Expression

2004 ◽  
Vol 78 (4) ◽  
pp. 1817-1830 ◽  
Author(s):  
Elizabeth A. White ◽  
Charles L. Clark ◽  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Early Gene ◽  
Immediate Early ◽  
Viral Gene ◽  
Recombinant Viruses ◽  
Late Genes

ABSTRACT The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86Δ356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86Δ427-435 and IE2 86Δ505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.

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 Separate DNA Elements Containing ATF/CREB and IE86 Binding Sites Differentially Regulate the Human Cytomegalovirus UL112-113 Promoter at Early and Late Times in the Infection

1998 ◽  
Vol 72 (4) ◽  
pp. 2697-2707 ◽  
Author(s):  
Steven M. Rodems ◽  
Charles L. Clark ◽  
Deborah H. Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Viral Gene ◽  
Dependent Manner ◽  
Start Site ◽  
Temporal Regulation ◽  
Early Protein ◽  
Dna Elements ◽  
Late Transcription

ABSTRACT The human cytomegalovirus (HCMV) UL112-113 promoter represents a useful model for studying temporal regulation of viral gene expression. Stimulation of this promoter by the 86-kDa immediate-early protein (IE86) is controlled by sequences between nucleotides −113 and −59, which include both an ATF/CREB and an IE86 binding site. In transient assays, the ATF/CREB site is essential, and the IE86 site, although nonessential, can enhance transcription. With recombinant viruses, we have assessed the function of these promoter elements in the context of the viral genome. Transcription from the inserted UL112-113 promoter shows the same temporal pattern as the endogenous promoter, including the switch to an upstream RNA start site late in infection. Deletion of sequences containing the IE86 site results in a decrease in the level of early transcription and elimination of late transcription. In contrast, when the ATF/CREB site is deleted, early RNA synthesis is almost completely abolished, but late transcription is comparable to that of the wild type, with repositioning of the RNA start site downstream by the number of nucleotides deleted. Replacement of sequences between −108 and −95 with the HCMVcis-repression signal from the major immediate-early promoter had no effect on the level of late RNAs but resulted in the repositioning of the RNA start site 39 nucleotides upstream. These results suggest that the ATF/CREB site is functional only at early times, while sequences containing the IE86 site modulate the level of early RNAs and may be required for activating late transcription in a distance-dependent manner.

scholarly journals Role of Noncovalent SUMO Binding by the Human Cytomegalovirus IE2 Transactivator in Lytic Growth

2010 ◽  
Vol 84 (16) ◽  
pp. 8111-8123 ◽  
Author(s):  
Eui Tae Kim ◽  
Young-Eui Kim ◽  
Yong Ho Huh ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Growth Curves ◽  
Viral Gene Expression ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Dependent Manner ◽  
Viral Growth ◽  
Transactivation Activity

ABSTRACT The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.

scholarly journals Human cytomegalovirus immediate-early gene expression is restricted by the nuclear domain 10 component Sp100

2011 ◽  
Vol 92 (7) ◽  
pp. 1532-1538 ◽  
Author(s):  
Martina Adler ◽  
Nina Tavalai ◽  
Regina Müller ◽  
Thomas Stamminger
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Immediate Early ◽  
Viral Gene ◽  
Major Protein ◽  
Hcmv Replication ◽  
Protein Components ◽  
Nuclear Domains

Nuclear domains 10 (ND10s) are discrete subnuclear structures that contain the three major protein components promyelocytic leukaemia protein (PML), hDaxx and Sp100. Previous studies identified the ND10-components PML and hDaxx as cellular restriction factors that independently counteract human cytomegalovirus (HCMV) infection via the repression of viral immediate-early (IE) gene expression. Consequently, we asked whether Sp100 is likewise involved in this repressive activity. Infection of Sp100 knockdown (kd) cells with HCMV resulted in a significantly increased plaque-forming ability. In addition, ablation of Sp100 led to a considerable increase in the number of IE1-expressing cells, indicating that Sp100 suppresses the initiation of viral gene expression. Next, double-kd cells, lacking either Sp100/hDaxx or Sp100/PML, were generated. Here, infection resulted in an additional enhancement in HCMV replication efficacy compared with the single-kd cells. Thus, our results further strengthen the concept that the three major ND10-components independently contribute to the cellular restriction of HCMV replication.

scholarly journals Autorepression of the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer at Late Times of Infection Is Mediated by the Recruitment of Chromatin Remodeling Enzymes by IE86

2006 ◽  
Vol 80 (20) ◽  
pp. 9998-10009 ◽  
Author(s):  
Matthew Reeves ◽  
Jane Murphy ◽  
Richard Greaves ◽  
Jennifer Fairley ◽  
Alex Brehm ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Chromatin Remodeling ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Immediate Early ◽  
Viral Gene ◽  
Early Promoter ◽  
Early Protein ◽  
Major Immediate Early Promoter

ABSTRACT The human cytomegalovirus major immediate-early protein IE86 is pivotal for coordinated regulation of viral gene expression throughout infection. A relatively promiscuous transactivator of viral early and late gene transcription, IE86 also acts during infection to negatively regulate its own promoter via direct binding to a 14-bp palindromic IE86-binding site, the cis repression sequence (crs), located between the major immediate-early promoter (MIEP) TATA box and the start of transcription. Although such autoregulation does not involve changes in the binding of basal transcription factors to the MIEP in vitro, it does appear to involve selective inhibition of RNA polymerase II recruitment. However, how this occurs is unclear. We show that autorepression by IE86 at late times of infection correlates with changes in chromatin structure around the MIEP during the course of infection and that this is likely to result from physical and functional interactions between IE86 and chromatin remodeling enzymes normally associated with transcriptional repression of cellular promoters. Firstly, we show that IE86-mediated autorepression is inhibited by histone deacetylase inhibitors. We also show that IE86 interacts, in vitro and in vivo, with the histone deacetylase HDAC1 and histone methyltransferases G9a and Suvar(3-9)H1 and that coexpression of these chromatin remodeling enzymes with IE86 increases autorepression of the MIEP. Finally, we show that mutation of the crs in the context of the virus abrogates the transcriptionally repressive chromatin phenotype normally found around the MIEP at late times of infection, suggesting that negative autoregulation by IE86 results, at least in part, from IE86-mediated changes in chromatin structure of the viral MIEP.

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