scholarly journals Drug Repurposing Campaigns for Human Cytomegalovirus Identify a Natural Compound Targeting the Immediate-Early 2 (IE2) Protein: A Comment on “The Natural Flavonoid Compound Deguelin Inhibits HCMV Lytic Replication within Fibroblasts”

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 117 ◽  
Author(s):  
Beatrice Mercorelli ◽  
Anna Luganini ◽  
Giorgio Palù ◽  
Giorgio Gribaudo ◽  
Arianna Loregian
Keyword(s):  
Human Cytomegalovirus ◽  
Natural Compound ◽  
Protein A ◽  
Drug Repurposing ◽  
Lytic Replication ◽  
Immediate Early ◽  
Flavonoid Compound

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that establishes a lifelong persistence in the host through both chronic and latent states of infection [...]

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.

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.

Design, Synthesis, and Evaluation of WC5 Analogues as Inhibitors of Human Cytomegalovirus Immediate-Early 2 Protein, a Promising Target for Anti-HCMV Treatment

ChemMedChem ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. 1403-1414 ◽  
Author(s):  
Serena Massari ◽  
Beatrice Mercorelli ◽  
Luca Sancineto ◽  
Stefano Sabatini ◽  
Violetta Cecchetti ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Protein A ◽  
Immediate Early ◽  
Design Synthesis ◽  
Promising Target

scholarly journals The Human Cytomegalovirus UL35 Gene Encodes Two Proteins with Different Functions

2002 ◽  
Vol 76 (5) ◽  
pp. 2460-2468 ◽  
Author(s):  
Yingguang Liu ◽  
Bonita J. Biegalke
Keyword(s):  
Human Cytomegalovirus ◽  
Complex Structure ◽  
Protein A ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Terminal Portion ◽  
Virion Protein ◽  
Early Promoter ◽  
Major Immediate Early Promoter

ABSTRACT The human cytomegalovirus (HCMV) virion is a complex structure that contains at least 30 proteins, many of which have been identified. We determined that the HCMV UL35 gene encodes two proteins, including a previously unidentified virion protein. A 22-kDa phosphoprotein (ppUL35A) was translated from a 1.2-kb UL35 transcript by 4 h postinfection; a second phosphoprotein of 75 kDa (ppUL35) was translated from a 2.2-kb transcript predominantly late in infection. The 22-kDa protein localized to the nucleus, while the 75-kDa protein localized to the juxtanuclear compartment and was packaged into virion particles. The 22-kDa protein was identical to the COOH-terminal end of the 75-kDa protein but was not found in virions, thus defining the NH2-terminal portion of the 75-kDa protein as essential for packaging. Expression of the 22-kDa protein inhibited activation of the major immediate-early promoter by ppUL82 (pp71), suggesting that the UL35 22-kDa protein may modulate expression of the major immediate-early gene.

scholarly journals Characterization of an Antisense Transcript Spanning the UL81-82 Locus of Human Cytomegalovirus

2005 ◽  
Vol 79 (17) ◽  
pp. 11022-11034 ◽  
Author(s):  
Mariana Bego ◽  
J. Maciejewski ◽  
S. Khaiboullina ◽  
G. Pari ◽  
S. St. Jeor
Keyword(s):  
Cdna Library ◽  
Human Cytomegalovirus ◽  
Antisense Transcript ◽  
Human Fibroblasts ◽  
Protein Product ◽  
Lytic Replication ◽  
Immediate Early ◽  
Lambda Phage

ABSTRACT In this study we present the characterization of a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR, UL81-82ast was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found. UL81-82ast was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.

scholarly journals Characteristics of Immediate-Early 2 (IE2) and UL84 Proteins in UL84-Independent Strains of Human Cytomegalovirus (HCMV)

2021 ◽  
Author(s):  
Salome Manska ◽  
Cyprian C. Rossetto
Keyword(s):  
Birth Defects ◽  
Human Cytomegalovirus ◽  
Latent Infection ◽  
Lytic Replication ◽  
Morbidity And Mortality ◽  
Immediate Early ◽  
Initial Infection ◽  
Congenital Birth Defects

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication.

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 Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

2016 ◽  
Vol 90 (19) ◽  
pp. 8855-8865 ◽  
Author(s):  
Kyle C. Arend ◽  
Benjamin Ziehr ◽  
Heather A. Vincent ◽  
Nathaniel J. Moorman
Keyword(s):  
Human Cytomegalovirus ◽  
Core Promoter ◽  
Viral Latency ◽  
Lytic Replication ◽  
Lytic Infection ◽  
Full Length ◽  
Immediate Early ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Infected Cells

ABSTRACTExpression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5′ untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.IMPORTANCEThe HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

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.

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