A Short
            <i>cis</i>
            -Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication

Perez, Kareni J.; Martínez, Francisco Puerta; Cosme-Cruz, Ruth; Perez-Crespo, Neysa M.; Tang, Qiyi

doi:10.1128/jvi.03171-12

Cited by 9 publications

(22 citation statements)

References 44 publications

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“…Our data could have considerable implications for several studies assigning defined MCMV phenotypes and functions to pIE3 (11,(26)(27)(28), e.g., the analysis of cell cycle regulation. Although wt MCMV infection results in G 1 and G 2 arrest and the ⌬ie3 virus has lost this ability, ectopically expressed pIE611 exhibits cell cycle arrest activity only in G 1 and not G 2 (29), exemplifying the occasion for reevaluation.…”

Section: Discussionmentioning

confidence: 99%

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

Rattay

Trilling

Megger

et al. 2015

J Virol

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Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. IMPORTANCECytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Human cytomegalovirus (HCMV) is a ubiquitous humanpathogenic herpesvirus. Primary infection is mostly subclinical and leads to lifelong persistence of the viral genome in a latent state from which reactivation occurs under immune stress or immunosuppression. HCMV reactivation is a major cause of morbidity and mortality in AIDS patients and other immunocompromised individuals (1). Like HCMV, mouse cytomegalovirus (MCMV) represents a prototypical member of the family of betaherpesviruses, and it has been extensively used as a model system to study common aspects of CM...

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Section: Discussionmentioning

confidence: 99%

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

Rattay

Trilling

Megger

et al. 2015

J Virol

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“…The plasmid pBB2.9, containing the whole M112-113 gene and the promoter, has been previously described [21]; pET (expressing GFP) and pgfpie3 (expressing GFP-tagged IE3) have been previously reported [22]. A diagram of part of the M112-113 gene and the constructed M112-113-expressing vectors is shown in Figure 1A.…”

Section: Plasmids and Molecular Cloningmentioning

confidence: 99%

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

Cruz-Cosme

Armstrong

Tang

2020

Viruses

Self Cite

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The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.

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“…Since ie3 is required for productive virus infection and early CMV gene expression [34][35][36] , the virus cannot easily circumvent this requirement, making Clr-b a pattern recognition axis and the NKR-P1B receptor a selfspecific pattern recognition receptor. IE3 is known to promote cell cycle arrest in G 1 /G 0 and interact in MCMV replication compartments with PML, Daxx, and ND10 complex proteins involved in viral restriction, and the HCMV homolog IE2 functions to repress important host transcription factors (including HDAC and TBP/TFIID) [37][38][39][40] and it is thought to function as a transcriptionassociated factor [41] . This is interesting because the Clrb promoter contains an inverted TATA motif and may function as a TBP-dependent promoter.…”

Section: Discussionmentioning

confidence: 99%

Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition

Kirkham

Aguilar²,

Yu³

et al. 2017

J Innate Immun

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Natural killer (NK) cells are innate lymphocytes that aid in self-nonself discrimination by recognizing cells undergoing pathological alterations. The NKR-P1B inhibitory receptor recognizes Clr-b, a self-encoded marker of cell health downregulated during viral infection. Here, we show that Clr-b loss during mouse cytomegalovirus (MCMV) infection is predicated by a loss of Clr-b (Clec2d) promoter activity and nascent transcripts, driven in part by MCMV ie3 (M122) activity. In contrast, uninfected bystander cells near MCMV-infected fibroblasts reciprocally upregulate Clr-b expression due to paracrine type-I interferon (IFN) signaling. Exposure of fibroblasts to type-I IFN augments Clec2d promoter activity and nascent Clr-b transcripts, dependent upon a cluster of IRF3/7/9 motifs located ∼200 bp upstream of the transcriptional start site. Cells deficient in type-I IFN signaling components revealed IRF9 and STAT1 as key transcription factors involved in Clr-b upregulation. In chromatin immunoprecipitation experiments, the Clec2d IRF cluster recruited STAT2 upon IFN-α exposure, confirming the involvement of ISGF3 (IRF9/STAT1/STAT2) in positively regulating the Clec2d promoter. These findings demonstrate that Clr-b is an IFN-stimulated gene on healthy bystander cells, in addition to a missing-self marker on MCMV-infected cells, and thereby enhances the dynamic range of innate self-nonself discrimination by NK cells.

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A Short cis -Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication

Cited by 9 publications

References 44 publications

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition

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