ILRUN Downregulates ACE2 Expression and Blocks Infection of Human Cells by SARS-CoV-2

Abstract

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.

Keywords: COVID-19; ILRUN; RNA virus; SARS-CoV-2; cell biology.

FIG 1

Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2^−ΔΔCT relative to GAPDH) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). n = 4 biological replicates. (B) Caco-2 cells were transfected with siRNAs (40 nM, 72 h) and then treated with transfected poly(I·C) (10 μg/ml, 6 h). Cells were then harvested and analyzed for mRNA expression of the listed cytokines by qRT-PCR, normalized to GAPDH. n ≥ 5 biological replicates. (C) TCID₅₀ measurements of virus titers. n = 3 biological replicates. (D) qRT-PCR measurements of intracellular viral RNA represented by 2^−ΔΔCT normalized first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1. n = 3 biological replicates. (E) Percent infected cells in Caco-2 cells infected with SARS-CoV-2 compared to inoculum (MOI, 0.1). n = 3 biological replicates. Graphs represent means ± standard deviations (SD). (F) Representative field of view of immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Caco-2 cells infected with SARS-CoV-2 (MOI, 0.1; 24 h). Cell nuclei were stained using DAPI (blue). n = 6 biological replicates.

FIG 2

Host pathways regulated by ILRUN in Caco-2 cells. (A) Unsupervised variance analysis of 5 biological replicate samples of Caco-2 cells transfected with 40 nM siNEG or siILRUN for 96 h and analyzed by RNA-seq. Samples plotted based on principle component 1 (PC1) and PC2 dimensions. (B) Volcano plot showing global transcriptional changes of samples described in panel A. A total of 457 transcripts (shown in red) were differentially expressed by ILRUN knockdown based on the cutoff P value of <0.05, log₂ fold change (FC) of >1, and baseMean of >5. Genes that did not exceed the fold change threshold are shown in blue. Genes of interest related to antiviral immunity are labeled. (C) Functional annotation clustering of the 457 differentially expressed genes in host pathways (BIOCARTA and KEGG) are plotted against the fold enrichment and ordered based on P value.

FIG 3

ILRUN regulates expression of key RAAS genes. The impact on siILRUN (40 nM, 96 h) on the RAAS was investigated by quantitatively coloring the “Renin-Angiotensin System” KEGG pathway map (https://www.genome.jp/kegg/) using a diverging color palette to show fold change effects across the entire pathway based on our transcriptomics analysis. Shades of red indicate upregulation, while shades of blue indicate downregulation. Genes that were not detected in this study are shown in green.

FIG 4

ILRUN suppresses SARS-CoV-2 infection and downregulates host genes essential for SARS-CoV-2 entry. (A) RNA profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h postinfection (MOI, 0.1) and analyzed by RNA-seq. n = 5 biological replicates. (B) Caco-2 cells were transfected with indicated siRNAs (40 nM, 72 h) or expression plasmids (500 ng, 48 h) and infected with SARS-CoV-2 (24 h; MOI, 0.1). Cells were then harvested and analyzed for viral RNA by qRT-PCR, normalized to GAPDH. n ≥ 3 biological replicates. (C) SARS-CoV-2 titers of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h; MOI, 0.3) posttransfection with indicated siRNAs (40 nM, 72 h) or expression plasmids (100 ng, 48 h). n = 5 biological replicates. (D) Normalized RNA-seq counts measuring expression of ACE2, TMPRSS2, and CTSL in Caco-2 cells transfected with indicated siRNAs (40 nM, 72 h) with or without infection with SARS-CoV-2 (24 h; MOI, 0.1). n = 5 biological replicates. (E) Percent ACE2-positive Caco-2 cells following transfection with indicated siRNAs (40 nM, 72 h) and staining with anti-ACE2 antibody and Alexa488-tagged secondary antibody and analyzed by flow cytometry. n = 3 biological replicates. (F) ILRUN, ACE2, and AGT expression in primary human bronchial epithelial cells stimulated with Th2 cytokines (30 ng/ml of IL-4 and IL-13) for 24 h (data set from publicly available Gene Expression Omnibus series GSE113185 [21]). n = 3 biological replicates. *, P < 0.05; **, P < 0.01. Graphs represent means ± SD.