Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway

Abstract

Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.

Fig 1. TLR2 3’UTR is targeted by HCMV miRNAs.

(A) Dual luciferase reporter assay suggests that TLR2 3’UTR is targeted by miR-UL112-3p and miR-US25-2. Two independent pSICHECK2-TLR2 3’UTR clones were tested; stars indicate potential Renilla luciferase down-regulation. Putative and confirmed HCMV miRNAs are indicated in abbreviated form (see detailed list in legend of S1 Fig) (B) TLR2 3’UTR is down-regulated by miR-UL112-3p. HEK293 cells were transfected with pSICHECK2-TLR2 3’UTR and the indicated miRNA mimics alone or in combination. (C) Two potential miR-UL112-3p target site are present near the 5’end of TLR2 3’UTR (bold characters). Wobble G-U pairs are in italics; seed sequences are indicated. (D) Both target sites are required for full miR-UL112-3p down-regulation of TLR2. pSICHECK2 vectors containing mutations in target sites #1 and #2 (mut1 and mut2, respectively) were assayed by luciferase assay with the indicated miRNAs. (E) A miR-UL112-3p mimic down-regulates endogenous TLR2 in THP1 cells. TPA-differentiated THP1 cells left untransfected (Ø) or transfected as indicated were harvested 2 days post-transfection for IB analysis.

Fig 2. TLR2 is down-regulated during HCMV infection.

(A) TLR2 follows a biphasic expression pattern during HCMV AD169 infection of NHDF fibroblasts with early induction and late down-regulation. Numbers below the TLR2 blot represent quantification of the protein signal. IB, immunoblot. TRAM1 was used as loading control. (B) miR-UL112-3p accumulates at late time points during AD169 infection of NHDF cells. miR-UL112-3p copy number was determined by RT-PCR at various points during the infection of NHDF fibroblasts with HCMV AD169 (MOI: 3). (C) TLR2 is down-regulated late during TB40E infection of THP-1 monocytic cells. (D) miR-UL112-3p accumulates at late time points during TB40E infection of differentiated THP-1 cells (MOI: 3).

Fig 3. miR-UL112-3p targets endogenous TLR2 during HCMV infection.

(A) A miR-UL112-3p deficient AD169 virus fails to down-regulate endogenous TLR2 during infection. NHDF cells infected (MOI: 1) with AD169 WT or a miR-UL112-3p mutant that does not express miR-UL112-3p were harvested at various times post-infection for IB analysis. (B) Transfection of a miR-UL112-3p mimic down-regulates TLR2 in NHDF cells infected with a miR-UL112-3p-deficient virus. NHDF cells were infected with AD169 112 KO (MOI: 3), left untransfected (Ø) or transfected 2 hrs later with various siRNA and miRNA mimics and harvested 2 dpi for IB analysis.

Fig 4. miR-UL112-3p inhibits TLR2-dependent activation of the NFκB pathway in THP1 cells.

(A) Schematic of cell treatment. THP1 or NFκB luciferase reporter THP1 cells (THP1-NFκBRE-luc) were transfected with siRNA or miRNA mimics after being differentiated for 24hrs with TPA. Cells were stimulated 48hrs post-transfection with the TLR2/TLR6 agonist FSL-1 and harvested either 30 min or 5 hrs later for IB analysis or luciferase assay, respectively. (B) miR-UL112-3p inhibits NFκBRE-driven luciferase activity stimulated by the TLR2 agonist FSL-1 with similar efficacy as siTLR2. Each test was performed in quadruplicate. (C) miR-UL112-3p decreases TLR2 mRNA level (upper panel) and IL-1β, IL-6 and IL-8 mRNA expression stimulated by the TLR2 agonist FSL-1 (three lower panels) with similar efficacy as siTLR2. Each test was performed in duplicate. **, P-value <0.0001 (sample vs. NEG).

Fig 5. miR-UL112-3p inhibits TLR2-dependent activation of IRAK1.

(A) Schematic of the upper section of the TLR2/NFκB pathway with known factors and post-translational modifications of IRAK4 and IRAK1 upon stimulation with TLR2 agonists. Grey ellipse, agonist; P, phosphorylation; K63, Lys63 poly-ubiquitin chain. (B) IRAK1 undergoes post-translational modifications upon stimulation with a TLR2/TLR1 agonist (PAM3CSK4, 100ng/ml) but not with a TLR4 agonist (LPS). TPA-differentiated THP-1 cells were treated as described in panel A. Unmodified and post-translationally modified forms of IRAK1 were detected by IB. (C) miR-UL112-3p inhibits FSL-1 induced IRAK1 post-translational modifications. TLR2, β-actin and IRAK1 unmodified and modified forms were detected by IB. Numbers below the IRAK1 blot represent quantification (in relative units) of the unmodified protein signal.