scholarly journals FOXO Transcription Factors Activate Alternative Major Immediate Early Promoters to Induce Human Cytomegalovirus Reactivation

2020 ◽  
Author(s):  
Andrew E Hale ◽  
Donna Collins-McMillen ◽  
Erik M Lenarcic ◽  
Jeremy P Kamil ◽  
Felicia Goodrum ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Human Cytomegalovirus ◽  
Disease Risk ◽  
Lytic Replication ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Viral Genes ◽  
Latently Infected ◽  
Foxo Transcription Factors

AbstractA key step during viral reactivation from latency is the re-expression of viral genes. Hematopoietic progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is re-expression of viral genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.SignificanceHuman cytomegalovirus infection is lifelong and persistent. Periodic reactivation of cytomegalovirus poses serious disease risk for immune-compromised patients. A critical driver of reactivation is expression of viral genes from the major immediate early locus. Recent paradigm-shifting evidence shows that reactivation is driven from promoters distinct from those that drive replication in permissive cells. Understanding the contextual control of these promoters and how they specifically respond to cellular cues that drive reactivation is critical for establishing future therapies that prevent reactivation. Our findings mechanistically define a previously enigmatic relationship between differentiation and reactivation, and provide potential targets for therapeutic intervention to prevent HCMV reactivation and disease.

scholarly journals FOXO transcription factors activate alternative major immediate early promoters to induce human cytomegalovirus reactivation

2020 ◽  
Vol 117 (31) ◽  
pp. 18764-18770 ◽  
Author(s):  
Andrew E. Hale ◽  
Donna Collins-McMillen ◽  
Erik M. Lenarcic ◽  
Suzu Igarashi ◽  
Jeremy P. Kamil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Human Cytomegalovirus ◽  
Lytic Replication ◽  
Immediate Early ◽  
Alternative Promoters ◽  
Cytomegalovirus Reactivation ◽  
Latently Infected ◽  
Infected Cells ◽  
Foxo Transcription Factors

Human progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.

scholarly journals The AP-1 Transcription Factor Is a Key Determinant of Human Cytomegalovirus Latency and Reactivation

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 127
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor
Keyword(s):  
Transcription Factor ◽  
Human Cytomegalovirus ◽  
Infected Individual ◽  
Virus Production ◽  
Mapk Signaling ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Activator Protein 1 ◽  
Latently Infected ◽  
Signaling Axis

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic progenitor cells (HPCs). Individuals with a competent immune system are, for the most part, asymptomatic for the disease. However when a latently infected individual becomes immunosuppressed, HCMV can reactivate, causing severe morbidity and mortality. While much of the viral genome is transcriptionally silenced during latency, some genes are expressed, including the HCMV-encoded G-protein coupled receptor US28. We showed that US28 expression is required for latency, as it suppressed the activator protein-1 (AP-1) transcription factor by attenuating the AP-1 subunit, fos. In turn, this prevents AP-1 from binding and activating the major immediate early promoter (MIEP), the key promoter regulating the latent-to-lytic transcriptional “switch”. Our new data suggest that US28-mediated signaling during latency attenuates the Src-MAPK signaling axis to regulate AP-1. We find that US28 expression suppresses Src, MEK, and ERK, as well as fos phosphorylation and AP-1 binding to the MIEP. Conversely, the pharmacological inhibition of Src, MEK, or ERK in US28Δ-latently infected HPCs suppresses infectious virus production, demonstrating the important role for this signaling axis during latency. Our recent data also reveal that regulating AP-1 is a key determinant in balancing HCMV latency and reactivation. Infection with a virus in which we disrupted the proximal AP-1 binding site in the MIEP (AP-1Δp) leads to reduced AP-1 binding and inefficient viral reactivation compared to wild type. Furthermore, AP-1 is critical for the de-repression of MIEP-driven transcripts and downstream early and late genes, while other immediate early genes remain unaffected. Collectively, these data suggest that AP-1 binding to the MIEP is suppressed during latency, but is required for the efficient transactivation of the MIEP during reactivation. We are currently elucidating US28’s involvement in recruiting AP-1 to the MIEP during reactivation.

scholarly journals Human Cytomegalovirus Gene Expression Is Silenced by Daxx-Mediated Intrinsic Immune Defense in Model Latent Infections Established In Vitro

2007 ◽  
Vol 81 (17) ◽  
pp. 9109-9120 ◽  
Author(s):  
Ryan T. Saffert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Lytic Replication ◽  
Immune Defense ◽  
Latent Infections ◽  
Abortive Infection ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT In addition to productive lytic infections, herpesviruses such as human cytomegalovirus (HCMV) establish a reservoir of latently infected cells that permit lifelong colonization of the host. When latency is established, the viral immediate-early (IE) genes that initiate the lytic replication cycle are not expressed. HCMV IE gene expression at the start of a lytic infection is facilitated by the viral pp71 protein, which is delivered to cells by infectious viral particles. pp71 neutralizes the Daxx-mediated cellular intrinsic immune defense that silences IE gene expression by generating a repressive chromatin structure on the viral major IE promoter (MIEP). In naturally latently infected cells and in cells latently infected in vitro, the MIEP also adopts a similar silenced chromatin structure. Here we analyze the role of Daxx in quiescent HCMV infections in vitro that mimic some, but not all, of the characteristics of natural latency. We show that in these “latent-like” infections, the Daxx-mediated defense that represses viral gene expression is not disabled because pp71 and Daxx localize to different cellular compartments. We demonstrate that Daxx is required to establish quiescent HCMV infections in vitro because in cells that would normally foster the establishment of these latent-like infections, the loss of Daxx causes the lytic replication cycle to be initiated. Importantly, the lytic cycle is inefficiently completed, which results in an abortive infection. Our work demonstrates that, in certain cell types, HCMV must silence its own gene expression to establish quiescence and prevent abortive infection and that the virus usurps a Daxx-mediated cellular intrinsic immune defense mechanism to do so. This identifies Daxx as one of the likely multiple viral and cellular determinants in the pathway of HCMV quiescence in vitro, and perhaps in natural latent infections as well.

scholarly journals Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells

2005 ◽  
Vol 86 (3) ◽  
pp. 535-544 ◽  
Author(s):  
Edward Wright ◽  
Mark Bain ◽  
Linda Teague ◽  
Jane Murphy ◽  
John Sinclair
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Histone Deacetylase ◽  
Human Cytomegalovirus ◽  
Histone Deacetylation ◽  
Early Gene ◽  
Immediate Early ◽  
Physical Interaction ◽  
Cellular Transcription

Previous work from this laboratory has shown that expression of human cytomegalovirus (HCMV) immediate-early (IE) genes from the major immediate-early promoter (MIEP) is likely to be regulated by chromatin remodelling around the promoter affecting the acetylation state of core histone tails. The HCMV MIEP contains sequences that bind cellular transcription factors responsible for its negative regulation in undifferentiated, non-permissive cells. Ets-2 repressor factor (ERF) is one such factor that binds to such sequences and represses IE gene expression. Although it is not known how cellular transcription factors such as ERF mediate transcriptional repression of the MIEP, it is likely to involve differentiation-specific co-factors. In this study, the mechanism by which ERF represses HCMV IE gene expression was analysed. ERF physically interacts with the histone deacetylase, HDAC1, both in vitro and in vivo and this physical interaction between ERF and HDAC1 mediates repression of the MIEP. This suggests that silencing of viral IE gene expression, associated with histone deacetylation events around the MIEP, is mediated by differentiation-dependent cellular factors such as ERF, which specifically recruit chromatin remodellers to the MIEP in non-permissive cells.

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

1992 ◽  
Vol 66 (1) ◽  
pp. 95-105 ◽  
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

scholarly journals Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timo W. M. De Groof ◽  
Elizabeth G. Elder ◽  
Eleanor Y. Lim ◽  
Raimond Heukers ◽  
Nick D. Bergkamp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected ◽  
Infected Cells

AbstractLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

scholarly journals 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

2002 ◽  
Vol 76 (11) ◽  
pp. 5369-5379 ◽  
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.

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

2018 ◽  
Vol 92 (7) ◽  
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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