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.

Meaghan H Hancock,Lauren M Hook,Jay A Nelson,Jennifer Mitchell

doi:10.1128/mbio.00109-17

Abstract

ABSTRACTEmerging 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.

Highlights

Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs
We have previously shown that miR-UL112-3p targets the Toll-like receptor 2 (TLR2) transcript to limit signaling through the TLR2-IRAK1 axis [45], suggesting that NF-␬B signaling may be a target of Human cytomegalovirus (HCMV) miRNAs
HCMV miRNAs partially block signaling mediated by IL-1␤ and TNF-␣

Summary

Introduction

Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. We show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the I␬B kinase (IKK) complex components IKK␣ and IKK␤ to limit production of proinflammatory cytokines in response to interleukin 1␤ (IL-1␤) and tumor necrosis factor alpha (TNF-␣). Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKK␣ and IKK␤ proteins, an impaired ability to control NF-␬B 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. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, downregulate IKK␣ and IKK␤ signaling factors necessary to propagate NF-␬B signaling and subsequent IL-6, CCL5, and TNF-␣ production Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host. The timely activation of the IKK complex downstream of TNF-␣ and IL-1␤ receptor binding is the single most important regulatory step in the induction of NF-␬B-mediated transcription

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Conclusion