Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the number of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling.

FIGURE 1:

Huh-7 cells and SK-N-SH cells are permissive to SARS-CoV-2 infection. (A) Huh-7 cells and SK-N-SH cells were seeded into 96-well tissue culture plates and infected with SARS-CoV-2 at a MOI of 1, or were mock-infected. Viability of SARS-CoV-2-infected cells at 24, 48, and 72 hpi was determined using the CellTiter-Glo Luminescent Cell Viability Assay kit and was normalized to the viability of mock-infected cells. The results of three independent experiments are shown. Bars depict mean ± SEM. (B) Huh-7 cells and SK-N-SH cells were infected with SARS-CoV-2 at a MOI of 1 or mock-infected. Total RNA was isolated at 24, 48 and 72 hpi and analyzed by qRT-PCR. Viral S-protein RNA was quantified using the 2^-ΔΔC_T method (Livak and Schmittgen, 2001); that is, the threshold cycle (CT) values for S-protein RNA were normalized against transcript levels of the human ß-actin gene (ACTB; ΔCT) and further normalized against the ΔCT values for S-protein RNA in mock-infected cells (ΔΔCT). The results of three independent experiments are displayed. Bars depict mean ± SEM. (C) Protein lysates were prepared from mock-infected cells and cells infected with SARS-CoV-2 for 24, 48, and 72 h, separated by SDS-PAGE, and subjected to immunoblotting with antibodies to nucleocapsid, PMP70, and actin. Actin served as a control for protein loading. Representative immunoblots from one of three independent experiments are shown. (D) Huh-7 cells and SK-N-SH cells were grown on coverslips, infected with SARS-CoV-2 at a MOI of 1 or mock-infected, and fixed at 24, 48, and 72 hpi. Cells were immunolabeled with antibodies against nucleocapsid and PMP70. Cell nuclei were stained with DAPI. Cells were observed by confocal fluorescence microscopy. Images presented are maximum-intensity projections and representative of three independent experiments. Squares in panels at left are shown at higher magnification in panels at right. Bars: 10 μm (left panels), 2 μm (right panels).

FIGURE 2:

Peroxisomal membranes undergo rearrangement in SARS-CoV-2-infected Huh-7 cells. Cells were grown on coverslips, infected with SARS-CoV-2 at a MOI of 1 or mock-infected, and fixed at 24, 48, and 72 hpi. Cells were labeled with antibodies against PMP70 and PEX14. Cell nuclei were stained with DAPI. Cells were observed by IF confocal microscopy. Images are maximum intensity projections. Black and white images show single labeling by PMP70 or PEX14 antibodies. Squares in a, e, I, and m are at higher magnification in d, h, l, and p, respectively. Bars: 10 μm (a–c, e–g, i–k, m–o), 2 μm (d, h, l, p). Also see Supplemental Video 1.

FIGURE 3:

SARS-CoV-2-infected Huh-7 cells exhibit a redistribution of peroxisomal matrix proteins to the cytosol and a reduction in peroxisome numbers. (A) Cells were grown on coverslips, infected with SARS-CoV-2 at a MOI of 1 or mock-infected, and fixed at 24, 48, or 72 hpi. Cells were labeled with antibodies against PMP70 and PTS1. Cell nuclei were stained with DAPI. Cells were observed by IF confocal microscopy. Images are maximum-intensity projections. Black and white images show single labeling by PMP70 or PTS1 antibodies. Squares in a, e, I, and m are at higher magnification in d, h, l, and p, respectively. Bars: 10 μm (a–c, e–g, i–k, m–o), 2 μm (d, h, l, p). (B) Mature peroxisomes defined as “PTS1-labeled puncta” were quantified using the Spot function of Imaris software. Points show peroxisome counts per μm³ of cytoplasm from three independent experiments, in which a minimum of 50 cells were analyzed. Bars depict mean ± SEM. Peroxisome counts were significantly different in SARS-CoV-2-infected cells from those in mock-treated cells, as determined by one-way ANOVA (p < 0.0001).

FIGURE 4:

SARS-CoV-2-infected cells show a redistribution of the PTS2-containing peroxisomal matrix protein thiolase to the cytosol. Cells were grown on coverslips, infected with SARS-CoV-2 at a MOI of 1 or mock-infected, and fixed at 24, 48, or 72 hpi. Cells were labeled with antibodies against PMP70 and thiolase. Cell nuclei were stained with DAPI. Cells were observed by IF confocal microscopy. Images are maximum-intensity projections. Black and white images show single labeling by PMP70 or thiolase antibodies. Squares in a, e, I, and m are at higher magnification in d, h, l, and p, respectively. Bars: 10 μm (a–c, e–g, i–k, m–o), 2 μm (d, h, l, p).

FIGURE 5:

SARS-CoV-2 ORF14 protein binds human PEX14. MBP alone or an MBP–ORF14 protein fusion made in E. coli was immobilized on amylose resin and incubated with E. coli extracts containing GST alone or a GST–PEX14 protein fusion. Bound protein was detected by immunoblotting with anti-GST antibody. Total MBP and MBP–ORF14 were detected by immunoblotting with anti-MBP antibody. Numbers at left denote migrations of molecular mass markers. One of five independent experiments is presented.