Influenza A virus infection of human respiratory cells induces primary microRNA expression

Abstract

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. In addition to well established means of regulating antiviral gene expression, it has been demonstrated that RNA interference (RNAi) can play an important role in antiviral responses. Virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded microRNA (miRNA) species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates. Examination of miRNA abundance in human lung cell lines revealed endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains, A/Udorn/72 and A/WSN/33. Known antiviral response pathways, including Toll-like receptor, RIG-I-like receptor, and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems.

FIGURE 1.

Cellular response to influenza A virus infection. A549 and BEAS-2B cells were mock infected or infected with 1 pfu/cell A/Udorn/72 or A/WSN/33 for 8, 12, or 24 h. The relative abundance of antiviral mRNAs IFNβ, ISG15, CCL5, and the influenza virus mRNA PB1 was measured. p.i., postinfection.

FIGURE 2.

The abundance of selected miRNAs increases in response to influenza virus infection. A, A549 and BEAS-2B cells (10⁶ cells) were mock infected or infected with 1 pfu/cell A/Udorn/72 or A/WSN/33 for 8, 12, or 24 h. Heat maps indicate significant (p < 0.05) changes in expression of cellular microRNAs at all time points during influenza A virus infection in microarray experiments. B, A549 or BEAS-2B cells were treated as in A. Relative abundance of miRNAs miR-7, miR-132, miR-146a, miR-187, miR-200c, miR-1275, and miR-16 was measured.

FIGURE 3.

mRNA and miRNA abundance changes during influenza A virus infection in primary airway epithelial cells. HAE cells were mock infected or infected with 10⁶ pfu/cell culture chamber of A/Udorn/72 for 8 or 24 h. A, relative abundance of antiviral mRNAs IFNβ, ISG15, IL-28a, and the influenza virus PB1 mRNA were measured. B, relative abundance of miR-16, miR-7, miR-132, miR-146a, miR-200c, miR-1275, and miR-21 was measured. C, immunofluorescence of HAE cells that were mock infected or infected as above for 24 h. Cells were immunostained for the influenza nucleoprotein (NP). p.i., postinfection.

FIGURE 4.

Influenza virus is a more potent inducer of miRNAs. A549 cells were infected with 1 pfu/cell of the indicated virus. Total RNA was purified 10 h postinfection. Relative abundance of miR-146a, miR-187, miR-200c, miR-1275, miR-132, miR-7, miR-16, and CCL5 mRNA was measured. Inset graph shows smaller changes in miR-146a due to EMCV, SeV, PIV5, and VSV.

FIGURE 5.

Influenza virus induction of pri-miRNAs requires new protein synthesis. A–C, A549 cells were mock infected (M) or infected with 1 pfu/cell A/Udorn/72 for 2, 4, or 6 hpi in the absence or presence of cycloheximide (CHX, 100 mg/ml). A, relative abundance of primiR-146a, primiR-187, primiR-200c, and primiR-132 was determined. B, relative abundance of IL-28a, ISG15, CCL5, and PB1 mRNA was determined. C, relative abundance of mature miR-146a, miR-187, miR-200c, and miR-132 was determined. D, same as A–C, but infection was for 8 or 24 h, as indicated. Relative abundance of primiR-146a, primiR-187, primiR-200c, and primiR-132 and mature miR-146a, miR-187, miR-200c, and miR-132 was determined.

FIGURE 6.

Antiviral mediators are insufficient for miRNA induction. A, A549 cells were either untreated (UNT), treated with 1000 units/ml of IFNα or 500 ng/ml of IFNγ, 400 ng/ml of IL-6 and 500 ng/ml of soluble IL-6 receptor, 10 ng/ml of NFα, mock infected (mock), or infected with 1 pfu/cell A/Udorn/72 or A/WSN/33 for 10 h. Relative abundance of miR-146a, miR187, miR-200c, miR-1275, miR-132, miR7, and miR-16 was determined, inset graph shows smaller changes in miR-146a due to cytokine treatments. B, A549 cells were either mock transfected, or transfected with 5 μg of poly(I:C) per ml of media for 12 h. Relative abundance of miR-16, miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275 was determined. C, control experiments to verify the cellular response to cytokine treatments or poly(I:C) transfections as indicated. Relative abundance of ISG15, CCL5 mRNA was determined to verify response to IFNα, IFNγ and TNFα. IL-6 activity was confirmed using a GAS luciferase assay (4x-M67). To verify response to poly(I:C) transfection relative abundance of IFNβ and CCL5 mRNA was determined.

FIGURE 7.

Functional interactome analysis of miRNA targets. A, genes identified to be down-regulated in the presence of miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275 mimics were analyzed based on literature search terms. Twelve of the 26 down-regulated genes were functionally clustered. B, expanded view of the dense central interconnected node representing IRAK1, MAPK3, and STMN1.

FIGURE 8.

MAPK3 and IRAK1 protein levels are reduced during influenza virus infection. A549 cells were mock infected (M) or infected with A/WSN/33 (m.o.i. of 5 pfu/cell; 10 h) in the absence or presence of control (CTRL) or specific miRNA mimics for miR-132, miR-146a, or miR-1275 as indicated. 40 μg of total protein was separated by SDS-PAGE and probed with antisera for MAPK1 + MAPK3, IRAK1, influenza NP, and GAPDH.