Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication

Eilbrecht, Mareike; Le‐Trilling, Vu Thuy Khanh; Trilling, Mirko

doi:10.3389/fcimb.2020.00171

Cited by 3 publications

(4 citation statements)

References 40 publications

(65 reference statements)

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“…However, we found UL34 to be augmenting, as ΔUL34 infections were able to spread but produced 100-fold less extracellular virus in cell culture ( Figure 2 D,E). Our results were obtained using a ΔUL34 virus stock derived from a UL34 complementing cell line and are consistent with similar results recently reported for M34 in MCMV [ 60 ]. Our results were obtained using the serially passaged AD169 strain in fibroblasts and await further validation in other HCMV strains and cell types.…”

Section: Discussionsupporting

confidence: 93%

“…Our results were obtained using the serially passaged AD169 strain in fibroblasts and await further validation in other HCMV strains and cell types. We observed robust expression and intranuclear localisation of UL34 from 48 HPI ( Figure 3 A,B and Figure S3A ), consistent with viral RCs [ 60 , 61 , 62 ]. UL34 has been previously suggested to impact viral DNA replication efficiency [ 62 ].…”

Section: Discussionsupporting

confidence: 65%

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UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

Turner

Templin

Barugahare

et al. 2022

IJMS

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Over 50% of the world’s population is infected with Human Cytomegalovirus (HCMV). HCMV is responsible for serious complications in the immuno-compromised and is a leading cause of congenital birth defects. The molecular function of many HCMV proteins remains unknown, and a deeper understanding of the viral effectors that modulate virion maturation is required. In this study, we observed that UL34 is a viral protein expressed with leaky late kinetics that localises to the nucleus during infection. Deletion of UL34 from the HCMV genome (ΔUL34) did not abolish the spread of HCMV. Instead, over >100-fold fewer infectious virions were produced, so we report that UL34 is an augmenting gene. We found that ΔUL34 is dispensable for viral DNA replication, and its absence did not alter the expression of IE1, MCP, gB, UL26, UL83, or UL99 proteins. In addition, ΔUL34 infections were able to progress through the replication cycle to form a viral assembly compartment; however, virion maturation in the cytoplasm was abrogated. Further examination of the nucleus in ΔUL34 infections revealed replication compartments with aberrant morphology, containing significantly less assembled capsids, with almost none undergoing subsequent maturation. Therefore, this work lays the foundation for UL34 to be further investigated in the context of nuclear organization and capsid maturation during HCMV infection.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 65%

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

Turner

Templin

Barugahare

et al. 2022

IJMS

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“…2C ), supporting a homotypic interaction. In contrast, M35-HAHA did not coprecipitate with a different nuclear V5/His-tagged protein of MCMV, M34 ( 65 ). Further, analysis of M35 in HEK293T lysates by native PAGE and immunoblot showed that M35 forms one defined species ( Fig.…”

Section: Resultsmentioning

confidence: 83%

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes

Schwanke

Magalhães

Schmelz

et al. 2023

J Virol

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“…Co-expression of M35-V5/His and C-terminally HAHA-tagged M35 (M35-HAHA) in HEK293T cells followed by immunoprecipitation for the V5 epitope showed that M35-HAHA readily co-precipitated with M35-V5/His (Figure 2C), supporting a homotypic interaction. In contrast, M35-HAHA did not co-precipitate with a different nuclear V5/His-tagged protein of MCMV, M34 (71). Further, analysis of M35 in HEK293T lysates by native PAGE and immunoblot showed that M35 forms one defined species (Figure 2D).…”

Section: Structure Determination Reveals Formation Of M35 Homodimersmentioning

confidence: 94%

The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

Schwanke

Magalhães

Schmelz

et al. 2023

Preprint

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Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Using electrophoretic mobility shift assays (EMSA), we demonstrate that purified M35 protein specifically binds to the regulatory DNA element that governs transcription of the first type I IFN gene induced in non-immune cells, Ifnb1. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35's immunomodulatory activity. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signalling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signalling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labelled transcripts (SLAM-seq), and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically down-regulated basal expression of IRF3-dependent genes, and during MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonises gene induction by IRF3 and impairs the antiviral response more broadly than formerly recognised. Importance Replication of the ubiquitous human cytomegalovirus (CMV) in healthy individuals mostly goes unnoticed, but can impair foetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes. Unrelated proteins from other herpesviruses employ the same mechanism, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

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Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication

Cited by 3 publications

References 40 publications

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes

The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

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