Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells

Abstract

Small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), virus-derived sncRNAs, and more recently identified tRNA-derived RNA fragments, are critical to posttranscriptional control of genes. Upon viral infection, host cells alter their sncRNA expression as a defense mechanism, while viruses can circumvent host defenses and promote their own propagation by affecting host cellular sncRNA expression or by expressing viral sncRNAs. Therefore, characterizing sncRNA profiles in response to viral infection is an important tool for understanding host-virus interaction, and for antiviral strategy development. Human metapneumovirus (hMPV), a recently identified pathogen, is a major cause of lower respiratory tract infections in infants and children. To investigate whether sncRNAs play a role in hMPV infection, we analyzed the changes in sncRNA profiles of airway epithelial cells in response to hMPV infection using ultrahigh-throughput sequencing. Of the cloned sncRNAs, miRNA was dominant in A549 cells, with the percentage of miRNA increasing in a time-dependent manner after the infection. In addition, several hMPV-derived sncRNAs and corresponding ribonucleases for their biogenesis were identified. hMPV M2-2 protein was revealed to be a key viral protein regulating miRNA expression. In summary, this study revealed several novel aspects of hMPV-mediated sncRNA expression, providing a new perspective on hMPV-host interactions.

Figure 1

Pipeline of analyses of Illumina high-throughput sequencing data. Flowchart of the sequencing data analyses is depicted. hMPV, human metapneumovirus; miRNA, microRNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA.

Figure 2

Kinetics of changes in sncRNA expression. (a) Human genome–derived small RNAs were sorted according to their origins, and their relative abundance was calculated by dividing their frequency numbers by total read numbers, and depicted in pie charts. (b) miRNAs, their abundance were changed with a fold change of ≥1.5 (upregulated) or ≤0.7 (downregulated), compared to baseline (uninfected), are represented at various times postinfection. Fold change is calculated on relative abundance for each time point compared to the baseline in mock infected cells. miRNAs whose expression was not changed are also represented. hMPV, human metapneumovirus; miRNA, microRNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA.

Figure 3

Experimental validation of sncRNA expression. (a) miRNA validation. Total RNA from indicated treatments in A549 cells was harvested at indicated time points postinfection, followed by reverse transcription using a TaqMan microRNA reverse transcription kit (Life Technologies). Quantitative real-time polymerase chain reaction was used to validate the expression of representative miRNAs. U6 snRNA was used as an internal control. All reactions were done in a 10-μl reaction volume in triplicate. Data are representative of two to three independent experiments. *P < 0.05, **P < 0.01, relative to the first bar in each group. (b). hMPV-derived sncRNA expression. A549 cells, mock- or hMPV-infected, were harvested at 15 hours postinfection, followed by total RNA extraction and loading to a denaturing polyacrylamide gel for northern hybridization using indicated probes. 5S rRNA is shown for equal loading. Data are representative of two to three independent experiments. hMPV, human metapneumovirus; miRNA, microRNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA.

Figure 4

Let-7f inhibits human metapneumovirus (hMPV) replication. (a) Knockdown of let-7f inhibits hMPV replication. Let-7f inhibitor was transfected into A549 cells using lipofectamine 2000. At 24 hours post-transfection, cells were mock infected or infected with hMPV at multiplicity of infection (MOI) of 2. Cells were harvested at 6 or15 hours postinfection, and viral infectious particles were measured by immunostaining. (b) Let-7f mimic were transfected into A549 cell. At 2 hours post-transfection, cells were mock infected or infected with hMPV at MOI of 2. At 6 or 15 hours postinfection, viral titration was assayed. Data are representative of two to three independent experiments. *P < 0.05, relative to the white bar at each time point postinfection as indicated. pfu, plaque-forming unit.

Figure 5

M2-2 regulates microRNA (miRNA) expression. (a) A549 cells were transfected with plasmid expressing M2-2 or its control (0.1 µg/well). At 30 hours post-transfection, the cells were mock infected or infected with rhMPV, wild type (WT), or ΔM2-2, at multiplicity of infection (MOI) of 2. At 15 hours postinfection, total RNA was prepared followed by quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of miR-30a and miR-16. (b) U4A cells were infected with rhMPV at MOI of 2 for 15 hours, followed by total RNA preparation and qRT-PCR to measure the expression of miR-30a and miR-16. U6 snRNA was used as an equal loading internal control. Data are representative of two to three independent experiments. *P < 0.05, **P < 0.01, relative to the first bar in each group. (c) The total protein from U4A cells was also subjected to Western blot to assess the viral gene expression. hMPV, human metapneumovirus.

Figure 6

Human metapneumovirus (hMPV)-derived sncRNA is generated by Dicer and RXN1. (a) A549 cells were transfected with 120 nmol/l of siRNA against indicated proteins or control siRNA as a negative control. At 48 hours post-transfection, the cells were mock- or hMPV-infected for 24 hours. Total RNAs were then subjected to northern hybridization as described in Figure 3b; 5S rRNA expression is shown for equal loading. (b) The suppression of target mRNAs by each siRNA was confirmed by quantitative real-time polymerase chain reaction measurement. The values are the relative expression levels of each mRNA, with the value of control siRNA set as one. Data are the representative of two independent experiments.