CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and 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.

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Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

Viruses ◽

10.3390/v12010110 ◽

2020 ◽

Vol 12 (1) ◽

pp. 110 ◽

Cited By ~ 5

Author(s):

Catherine S. Adamson ◽

Michael M. Nevels

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Protein Function ◽

Severe Disease ◽

Early Gene ◽

Immediate Early ◽

Congenital Defects ◽

Immunosuppressed Patients ◽

Experimental Approaches ◽

Hcmv Replication

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. “Bright and early” events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.

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

Journal of General Virology ◽

10.1099/vir.0.030981-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1532-1538 ◽

Cited By ~ 44

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.

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Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

Journal of Virology ◽

10.1128/jvi.66.1.95-105.1992 ◽

1992 ◽

Vol 66 (1) ◽

pp. 95-105 ◽

Cited By ~ 61

Author(s):

A M Colberg-Poley ◽

L D Santomenna ◽

P P Harlow ◽

P A Benfield ◽

D J Tenney

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Immediate Early ◽

Regulate Gene Expression ◽

Early Proteins ◽

Immediate Early Proteins ◽

Regulate Gene

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Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication

Journal of Virology ◽

10.1128/jvi.79.15.9597-9607.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9597-9607 ◽

Cited By ~ 49

Author(s):

Hiroki Isomura ◽

Mark F. Stinski ◽

Ayumi Kudoh ◽

Tohru Daikoku ◽

Noriko Shirata ◽

...

Keyword(s):

Viral Replication ◽

Human Cytomegalovirus ◽

Significant Role ◽

Binding Sites ◽

Human Fibroblast ◽

Immediate Early ◽

Fibroblast Cells ◽

Recombinant Viruses ◽

Human Fibroblast Cells ◽

Hcmv Replication

ABSTRACT We previously demonstrated that the major immediate early (MIE) proximal enhancer containing one GC box and the TATA box containing promoter are minimal elements required for transcription and viral replication in human fibroblast cells (H. Isomura, T. Tsurumi, M. F. Stinski, J. Virol. 78:12788-12799, 2004). After infection, the level of Sp1 increased while Sp3 remained constant. Here we report that either Sp1 or Sp3 transcription factors bind to the GC boxes located at approximately positions −55 and −75 relative to the transcription start site (+1). Both the Sp1 and Sp3 binding sites have a positive and synergistic effect on the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. There was little to no change in MIE transcription or viral replication for recombinant viruses with one or the other Sp1 or Sp3 binding site mutated. In contrast, mutation of both the Sp1 and Sp3 binding sites caused inefficient MIE transcription and viral replication. These data indicate that the Sp1 and Sp3 binding sites have a significant role in HCMV replication in human fibroblast cells.

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The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

Journal of Clinical Investigation ◽

10.1172/jci116995 ◽

1994 ◽

Vol 93 (2) ◽

pp. 474-478 ◽

Cited By ~ 64

Author(s):

L J Geist ◽

M M Monick ◽

M F Stinski ◽

G W Hunninghake

Keyword(s):

Gene Expression ◽

Tumor Necrosis Factor ◽

Tumor Necrosis Factor Alpha ◽

Human Cytomegalovirus ◽

Tumor Necrosis ◽

Immediate Early ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Alpha Gene ◽

Necrosis Factor

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Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome

Journal of Virology ◽

10.1128/jvi.76.11.5369-5379.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5369-5379 ◽

Cited By ~ 54

Author(s):

Elizabeth A. Fortunato ◽

Veronica Sanchez ◽

Judy Y. Yen ◽

Deborah H. Spector

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Human Cytomegalovirus ◽

Dna Content ◽

Laser Scanning ◽

Small Population ◽

S Phase ◽

Early Gene ◽

Immediate Early ◽

Laser Scanning Cytometry

ABSTRACT Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G0 using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE+ in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE+ cells that gradually accumulated within the S and G2/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU+ cells were IE− and had progressed into G2/M or back to G1. The majority of the IE+ cells in S and G2/M were BrdU−. Only a few cells were IE+ BrdU+, and they resided in G2/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.

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The 5′ Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication

Journal of Virology ◽

10.1128/jvi.02128-17 ◽

2018 ◽

Vol 92 (7) ◽

Cited By ~ 6

Author(s):

Kyle C. Arend ◽

Erik M. Lenarcic ◽

Nathaniel J. Moorman

Keyword(s):

Protein Expression ◽

Human Cytomegalovirus ◽

Untranslated Region ◽

Mrna Translation ◽

Lytic Replication ◽

Immediate Early ◽

Translation Control ◽

Positive Role ◽

Free System ◽

Hcmv Replication

ABSTRACTThe human cytomegalovirus (HCMV) immediate early 1 (IE1) and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the posttranscriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5′ untranslated region (5′ UTR) plays an important role in regulating mRNA translation. Therefore, to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared 5′ UTR of the IE1 and IE2 mRNAs (MIE 5′ UTR) in regulating translation. In a cell-free system, the MIE 5′ UTR repressed translation, as predicted based on its length and sequence composition. However, in transfected cells we found that the MIE 5′ UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5′ UTR has a positive role in translation control. We also found that the MIE 5′ UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5′ UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild-type MIE 5′-UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5′ UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.IMPORTANCEThe HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5′ UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date, most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore, this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation and thus inhibit lytic replication or prevent HCMV reactivation.

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Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2004 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2004-2011 ◽

Cited By ~ 97

Author(s):

K P Anderson ◽

M C Fox ◽

V Brown-Driver ◽

M J Martin ◽

R F Azad

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Antiviral Activity ◽

Protein Expression ◽

Human Cytomegalovirus ◽

Host Cells ◽

Early Gene ◽

Immediate Early ◽

Mrna Levels ◽

Virus Adsorption

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

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Phorbol Ester-Induced Human Cytomegalovirus Major Immediate-Early (MIE) Enhancer Activation through PKC-Delta, CREB, and NF-κB Desilences MIE Gene Expression in Quiescently Infected Human Pluripotent NTera2 Cells

Journal of Virology ◽

10.1128/jvi.00416-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8495-8508 ◽

Cited By ~ 22

Author(s):

Xiaoqiu Liu ◽

Jinxiang Yuan ◽

Allen W. Wu ◽

Patrick W. McGonagill ◽

Courtney S. Galle ◽

...

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Binding Sites ◽

Molecular Mechanisms ◽

Cell Model ◽

Gene Activation ◽

Dominant Negative ◽

Immediate Early ◽

Signaling Cascade ◽

Pkc Delta

ABSTRACT The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE+) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-κB (κB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and κB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-κB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and κB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-κB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

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