Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ

ABSTRACT Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines w...

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Autores principales:	Meaghan H. Hancock, Lauren M. Hook, Jennifer Mitchell, Jay A. Nelson
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bc4277620a9546ccad70a6b7949f0798
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spelling	oai:doaj.org-article:bc4277620a9546ccad70a6b7949f07982021-11-15T15:50:59ZHuman Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ10.1128/mBio.00109-172150-7511https://doaj.org/article/bc4277620a9546ccad70a6b7949f07982017-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00109-17https://doaj.org/toc/2150-7511ABSTRACT Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the IκB kinase (IKK) complex components IKKα and IKKβ to limit production of proinflammatory cytokines in response to interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKKα and IKKβ protein levels, and site-directed mutagenesis of the 3′ untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKKα and IKKβ proteins, an impaired ability to control NF-κB signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infection in vivo. These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKKα and IKKβ, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment. IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-κB signaling, which culminates in the production of IL-6, CCL5, and TNF-α. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKKα and IKKβ signaling factors necessary to propagate NF-κB signaling and subsequent IL-6, CCL5, and TNF-α production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.Meaghan H. HancockLauren M. HookJennifer MitchellJay A. NelsonAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Meaghan H. Hancock Lauren M. Hook Jennifer Mitchell Jay A. Nelson Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
description	ABSTRACT Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the IκB kinase (IKK) complex components IKKα and IKKβ to limit production of proinflammatory cytokines in response to interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKKα and IKKβ protein levels, and site-directed mutagenesis of the 3′ untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKKα and IKKβ proteins, an impaired ability to control NF-κB signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infection in vivo. These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKKα and IKKβ, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment. IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-κB signaling, which culminates in the production of IL-6, CCL5, and TNF-α. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKKα and IKKβ signaling factors necessary to propagate NF-κB signaling and subsequent IL-6, CCL5, and TNF-α production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.
format	article
author	Meaghan H. Hancock Lauren M. Hook Jennifer Mitchell Jay A. Nelson
author_facet	Meaghan H. Hancock Lauren M. Hook Jennifer Mitchell Jay A. Nelson
author_sort	Meaghan H. Hancock
title	Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
title_short	Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
title_full	Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
title_fullStr	Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
title_full_unstemmed	Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ
title_sort	human cytomegalovirus micrornas mir-us5-1 and mir-ul112-3p block proinflammatory cytokine production in response to nf-κb-activating factors through direct downregulation of ikkα and ikkβ
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/bc4277620a9546ccad70a6b7949f0798
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Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKβ

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