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. 2017 Mar 7;8(2):e00109-17.
doi: 10.1128/mBio.00109-17.

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  1 , Lauren M Hook  1 , Jennifer Mitchell  1 , Jay A Nelson  2
Affiliations

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 et al. mBio. .

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.

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Figures

FIG 1
FIG 1
HCMV miR-US5-1 and miR-UL112-3p reduce proinflammatory cytokine transcripts in response to IL-1β and TNF-α. (A) HeLa cells were transfected with negative control (Neg) or double-stranded miR-US5-1 and miR-UL112-3p mimics alone or in combination or with a combination of siRNAs targeting the IKKα and IKKβ transcripts. Forty-eight hours after transfection, cells were treated with IL-1β or TNF-α for 16 h and then RNA was harvested for quantitative RT-PCR using primer/probe sets for IL-6. (B) A dual luciferase reporter containing the IL-6 3′ UTR was transfected into 293T cells along with negative control or double-stranded HCMV miRNA mimics. Twenty-four hours after transfection, luciferase expression was assayed. (C) The experiment was performed as in panel A using a CCL5 primer/probe set. (D) The experiment was performed as in panel B using a dual luciferase vector containing the CCL5 3′ UTR. (E) HeLa cells were transfected with negative control, miR-US5-1 or miR-UL112-3p double-stranded mimics, or a combination of siRNAs targeting the IKKα and IKKβ transcripts for 48 h and then treated with IL-1β or TNF-α for 10 min. Lysates were harvested and blotted for phospho- and total IκBα. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the negative control and presented numerically beneath each lane. *, P  < 0.05.
FIG 2
FIG 2
miR-US5-1 and miR-UL112-3p directly target IKKα through sites within the 3′ UTR. (A) A dual luciferase reporter containing the 3′ UTR of IKKα was transfected into 293T cells along with negative control (Neg) or double-stranded HCMV miRNA mimics. Luciferase expression was assessed 24 h posttransfection. (B) Luciferase assays were performed as in panel A using dual luciferase vectors containing either WT IKKα 3′ UTR or a mutation in the putative miR-US5-1 binding site. (C) Luciferase assays were performed as in panel A using dual luciferase vectors containing either WT IKKα 3′ UTR or a mutation in the putative miR-UL112-3p binding site. (D) 293T cells were transfected with increasing amounts of negative control or miR-US5-1 double-stranded mimic for 48 h, after which lysates were harvested and blotted for IKKα and GAPDH. (E) 293T cells were transfected with increasing amounts of negative control or miR-UL112-3p mimic for 48 h, after which lysates were harvested and blotted for IKKα and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the “0” negative control and presented numerically beneath each lane. *, P < 0.05.
FIG 3
FIG 3
miR-US5-1 and miR-UL112-3p directly target IKKβ through sites within the 3′ UTR. (A) A dual luciferase reporter containing the 3′ UTR of IKKβ was transfected into 293T cells along with negative control (Neg) or double-stranded HCMV miRNA mimics. Luciferase expression was assessed 24 h posttransfection. (B) Luciferase assays were performed as in panel A using dual luciferase vectors containing either WT IKKβ 3′ UTR or mutations in the putative miR-US5-1 binding sites alone or in combination. (C) Luciferase assays were performed as in panel A using dual luciferase vectors containing either WT IKKβ 3′ UTR or a mutation in the putative miR-UL112-3p binding site. (D) 293T cells were transfected with increasing amounts of negative control or miR-US5-1 double-stranded mimic for 48 h, after which lysates were harvested and blotted for IKKβ and GAPDH. (E) 293T cells were transfected with increasing amounts of negative control or miR-UL112-3p double-stranded mimic for 48 h, after which lysates were harvested and blotted for IKKβ and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the “0” negative control and presented numerically beneath each lane. *, P < 0.05.
FIG 4
FIG 4
IKKα and IKKβ protein levels are increased during infection with a miR-US5-1/miR-UL112-3p double mutant virus. (A) NHDF were infected with WT and miR-US5-1/miR-UL112-3p double mutant (Mut) virus for 48 and 72 h, after which lysates were harvested and immunoblotted for IKKα, IKKβ, and GAPDH. (B) NHDF were transfected with negative control (Neg), miR-US5-1 and miR-UL112-3p double-stranded mimics, or siRNAs targeting the IKKα and IKKβ transcripts. Forty-eight hours after transfection, cells were infected with WT or miR-US5-1/miR-UL112-3p double mutant viruses for 72 h and then protein lysates were harvested and immunoblotted for IKKβ and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the 48-h WT sample (in panel A) or mock (in panel B) and presented numerically beneath each lane.
FIG 5
FIG 5
miR-US5-1 and miR-UL112-3p are involved in the late block to NF-κB signaling observed in HCMV-infected fibroblasts. (A) Human fibroblasts expressing a luciferase reporter under the control of the minimal NF-κB promoter were infected with WT or miR-US5-1/miR-UL112-3p double mutant virus (Mut), and protein lysates were harvested at the indicated times. Lysates were immunoblotted for luciferase and GAPDH. (B) NHDF were mock infected or infected with WT or miR-US5-1/miR-UL112-3p double mutant virus for 72 h. After this time, cells were treated with IL-1β, and protein lysates were harvested at the indicated times and immunoblotted for IκBα or GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the uninfected, untreated sample and presented numerically beneath each lane. (C) The experiment was performed as in panel B with the exception that the NHDF were first transfected with negative control or miR-US5-1 and miR-UL112-3p mimics for 48 h prior to infection. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to each negative-control, untreated sample and presented numerically beneath each lane.
FIG 6
FIG 6
Proinflammatory cytokine transcript levels are elevated during infection with the miR-US5-1/miR-UL112-3p double mutant virus. (A) NHDF were infected with WT and miR-US5-1/miR-UL112-3p double mutant virus, and then RNA was harvested at 48 and 72 h postinfection and subjected to quantitative RT-PCR for IL-6. (B) The experiment was performed as in panel A, analyzing CCL5 transcript levels. (C) NHDF were transfected with negative control (Neg), miR-US5-1 and miR-UL112-3p double-stranded mimics, or siRNAs targeting the IKKα and IKKβ transcripts. Forty-eight hours posttransfection, the cells were infected with WT or miR-US5-1/miR-UL112-3p double mutant virus. RNA was harvested 72 h postinfection and analyzed for IL-6 transcript levels using quantitative RT-PCR. (D) The experiment was performed as in panel C, analyzing CCL5 transcript levels. *, P < 0.05.
FIG 7
FIG 7
Expression of IKKα and IKKβ shRNAs in the context of the miR-US5-1/miR-UL112-3p double mutant virus complements the defect in NF-κB signaling. (A) NHDF were infected with WT, miR-US5-1/miR-UL112-3p double mutant, or miR-US5-1/shRNA/miR-UL112-3p virus (shRNA hairpins targeting the IKKα and IKKβ transcripts in place of the miR-US5-1 hairpin). Seventy-two hours after infection, RNA was harvested and quantitative RT-PCR for HCMV miR-US5-1, miR-US25-1, miR-UL112-3p, IKKα, or IKKβ was performed. Expression levels were normalized to miR-16 and compared to WT-infected cells. (B) NHDF were infected with WT and miR-US5-1/miR-UL112-3p and miR-US5-1/shRNA/miR-UL112-3p mutant viruses, and protein was harvested at the indicated times and immunoblotted for IKKα, IKKβ, HCMV IE86, and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to mock (or 48-h WT in the case of IE86) sample and presented numerically beneath each lane. (C) NHDF were infected as in panel B, and RNA was harvested at 48 and 72 h postinfection. Quantitative RT-PCR was performed using primer/probe sets for IL-6 and U6 (as a normalization control). (D) The experiment was performed as in panel C using a primer/probe set for CCL5. *, P < 0.05.
FIG 8
FIG 8
miR-US5-1 and miR-UL112-3p alone block proinflammatory cytokine production in cell types relevant to HCMV infection. (A) NHDF. (B) hAEC. (C) PMA-treated THP-1 cells. Cells were transfected with negative control (Neg), miR-US5-1 and miR-UL112-3p double-stranded mimics, or siRNAs targeting the IKKα and IKKβ transcripts. (i) Protein lysates were harvested 72 h posttransfection and immunoblotted for IKKα, IKKβ, and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the negative-control sample and presented numerically beneath each lane. (ii) Cells were transfected for 72 h and then treated with IL-1β for 8 h. Supernatants were harvested and analyzed for IL-6 protein levels. (iii) Supernatants were harvested and analyzed for CCL5 protein levels. (iv) TNF-α levels were assessed in transfected THP-1 cells. *, P < 0.05.
FIG 9
FIG 9
miR-US5-1 and miR-UL112-3p block proinflammatory cytokine production during HCMV infection. (A) NHDF. (B) hAEC. (C) PMA-treated THP-1 cells. Cells were infected with WT or miR-US5-1/miR-UL112-3p or miR-US5-1/shRNA/miR-UL112-3p mutant viruses. (i) Protein lysates were harvested 72 h postinfection and immunoblotted for IKKα, IKKβ, and GAPDH. Relative band intensity was determined by dividing the intensity of the band by GAPDH followed by normalization to the negative-control sample and presented numerically beneath each lane. (ii) Supernatants were harvested at 48 and 72 h postinfection and analyzed for IL-6 protein levels. (iii) Supernatants were harvested and analyzed for CCL5 protein levels. (iv) TNF-α levels were assessed in infected THP-1 cells. *, P < 0.05.
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