Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication

Abstract

West Nile virus (WNV) is a mosquito-transmitted flavivirus that naturally circulates between mosquitos and birds but can also infect humans, causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway; however, recent studies suggest that microRNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed small RNA sequencing (RNA-seq) analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin (WNVKUN), and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly upregulated in infected cells by quantitative reverse transcription (qRT)-PCR and Northern blot analyses, and one of them, miR-532-5p, exhibited a significant antiviral effect against WNVKUN infection. We have demonstrated that miR-532-5p targets and downregulates expression of the host genes SESTD1 and TAB3 in human cells. Small interfering RNA (siRNA) depletion studies showed that both SESTD1 and TAB3 were required for efficient WNVKUN replication. We also demonstrated upregulation of mir-532-5p expression and a corresponding decrease in the expression of its targets, SESTD1 and TAB3, in the brains of WNVKUN -infected mice. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent a host antiviral response aimed at limiting WNVKUN infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts.

Importance: West Nile virus (WNV) is a significant viral pathogen that poses a considerable threat to human health across the globe. There is no specific treatment or licensed vaccine available for WNV, and deeper insight into how the virus interacts with the host is required to facilitate their development. In this study, we addressed the role of host microRNAs (miRNAs) in antiviral response to WNV in human cells. We identified miR-532-5p as a novel antiviral miRNA and showed that it is upregulated in response to WNV infection and suppresses the expression of the host genes TAB3 and SESTD1 required for WNV replication. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent an antiviral response aimed at limiting WNV infection and highlight the important role of miRNAs in controlling virus infections in mammalian hosts.

FIG 1

WNV_KUN infection alters expression of host miRNAs in HEK293 cells. (A) Heat map representation of miRNA and pre-miRNA expression in HEK293 cells at 24 h and 48 h after WNV_KUN infection. miRNAs are ranked in descending order based on the fold change in mature miRNA levels in infected cells compared to the uninfected control. Only miRNAs representing more than 0.1% of the total mappable miRNA reads are shown. (B) Quantitative RT-PCR analysis of miRNA expression in HEK293 cells infected with WNV_KUN expressed as relative fold changes compared to mock infection. The values represent the means of 3 biological replicates, with the error bars showing standard deviations. *, P < 0.05; ****, P < 0.0001. (C) Northern blot analysis of miRNA expression in HEK293 cells before (M) and at 24 h and 48 h after WNV_KUN infection. Ethidium bromide-stained tRNA (upper gels) and 5S rRNA (lower gels) served as RNA loading controls and indicators of RNA integrity in the samples. L, molecular weight ladder.

FIG 2

Effects of miRNA mimics and inhibitors on WNV_KUN replication. (A and B) Transfections with FAM-labeled miRNA mimic (A) and inhibitor (B) were performed to estimate transfection efficiency and to monitor for the presence of RNA oligonucleotides in the cells in time course experiments. (C to E) HEK293 cells were transfected with synthetic mimics or inhibitors specific to miR-532-5p (C), mir-1271-5p (D), or miR-1307-3p (E). Cells transfected with nonspecific mimic or inhibitor were used as a negative control (NC). At 24 h posttransfection, the cells were infected with WNV_KUN at an MOI of 1, and virus titers in the culture fluids at the indicated times postinfection (hpi) were determined by plaque assay on BHK-21 cells. Titers are shown as means of biological replicates, with the error bars showing standard deviations. *, P < 0.05; ****, P < 0.0001.

FIG 3

WNV_KUN genomic RNA is not the target of miR-532-5p. (A) Predicted interactions between miR-532-5p and WNV_KUN genomic RNA. (B) Effect of miR-532-5p mimic on expression of the firefly luciferase gene (f-luc) from miRNA sensor constructs. Sequences of predicted target sites in the WNV_KUN genome (putative target 1 or 2) were inserted into the 3′ UTR of the f-luc gene in the pmirGLO dual-luciferase reporter vector. Sequences with full complementarity to miR-532-5p sequence or with scrambled (random nucleotides) sequence were inserted into the same region to create positive and negative (scrambled) control vectors. HEK293 cells were cotransfected with reporter constructs and the miR-532-5p or nonspecific miRNA mimics. The values are expressed as relative f-luc expression calculated as a ratio of f-luc to r-luc (Renilla luciferase) activity (RLU, relative light units). The values are the means of three independent experiments, with the error bars showing standard deviations. **, P < 0.01.

FIG 4

Experimental validation of predicted miR-532-5p targets. (A) Expression of predicted miR-532-5p targets in WNV_KUN-infected HEK293 cells determined by qRT-PCR. Relative expression is represented as log₂-fold change compared to an uninfected control. The means of three biological replicates with technical triplicates are shown. The error bars show standard deviations. *, P < 0.05. (B) Western blot detection of TAB3 and SESTD1 in HEK293 cells at 24 h, 48 h, and 72 h after infection with WNV_KUN at an MOI of 1. The GAPDH blot was used as a loading control, and WNV_KUN E protein antibodies were used to detect virus infection. (C) Effects of miR-532-5p mimic and inhibitor on expression of TAB3 and SESTD1 in mock- and WNV_KUN-infected cells. HEK293 cells were transfected with miR-532-5p mimic (532 mim.), miR-532-5p inhibitor (532 inhib.), or nonspecific control RNA mimic (NS mim.) or inhibitor (NS inhib.). At 24 h posttransfection, the cells transfected with mimics were lysed and the cells transfected with inhibitors were infected with WNV_KUN at an MOI of 1 and then lysed at 36 h postinfection. Expression of TAB3, SESTD1, GAPDH (loading control), and WNV_KUN E protein (control for infection) was determined by Western blotting with the corresponding antibodies.

FIG 5

MiR-532-5p interacts with predicted target sites in the 3′ UTRs of TAB3 and SESTD1 mRNAs. (A) Predicted target sites for miR-532-5p in the 3′ UTRs of TAB3 and SESTD1. Nucleotide positions in the corresponding 3′ UTRs that are complementary to the miR-532-5p seed region are indicated. Vertical lines show complementary interactions. (B) Effect of miR-532-5p mimic on expression of the firefly luciferase gene (f-luc) from reporter constructs containing in the f-luc 3′ UTR the putative miR-532-5p binding sites from the TAB3 and SESTD1 3′ UTRs. Constructs with scrambled TAB3 and SESTD1 sequences inserted in the 3′ UTR of f-luc were used as negative controls. The values are the means of the results of three independent experiments, with the error bars showing standard deviations. *, P < 0.05.

FIG 6

Effect of SESTD1 or TAB3 depletion on WNV_KUN replication. (A and B) Knockdown of SESTD1 (A) and TAB3 (B) in HEK293 cells by siRNAs. Nonspecific (NS) siRNAs were used as negative controls. (C and D) Effects of SESTD1 (C) and TAB3 (D) knockdown on WNV_KUN replication. At 24 h after transfection with SESTD1 (B) or TAB3 (D) siRNA or NS siRNA, cells were infected with WNV_KUN at an MOI of 0.1, and virus titers in the culture fluid collected at the indicated times after infection were determined by plaque assay on BHK-21 cells. The values in panels C and D are means of three biological replicates with technical duplicates. The error bars show standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Effects of WNV_KUN infection on expression of miR-532, TAB3, and SESTD1 in mouse brains. Relative levels of RNAs in the total brain RNA of infected mice were determined by the ΔΔC_T method with comparison to the levels in the brains of uninfected animals. Normalized levels of RNAs in samples from individual animals and median levels in the groups are shown. (A) Levels of miR-532 were determined by SL-qRT-PCR and normalized to the miR-30e-3p level. (B to D) Levels of WNV genomic RNA (B), TAB3 mRNA (C), and SESTD1 mRNA (D) were determined by qRT-PCR with normalization to the TBP mRNA levels. All experiments included duplicates for RT reactions and technical triplicates for qPCR.