SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells

Figure 1. Generating a plasmid construct to study the function of ORF8SARS-CoV-2 in human A549 lung epithelial cells. (A) Cloning strategy to generate the pEGFP-ORF8SARS-CoV-2 (ORF8-GFP) construct. (B) Verification of ORF8-GFP expression in A549 lung epithelial cells transfected with pEGFP-ORF8SARS-CoV-2 by immunoblot analysis using anti-GFP antibody. Whole cell lysates were collected from cells transfected with empty vector (pEGFP-N1; EV) or ORF8-GFP at 24 hours post-transfection. (C) Characterization of the transfection efficiency of ORF8-GFP in A549 lung epithelial cells using FACS analysis. Cells were subjected to flow cytometry 24 hours after transfection with pEGFP-N1 (EV) or ORF8-GFP. The experiments were repeated twice with triplicate samples.

Figure 2. ORF8SARS-CoV-2 forms intracellular aggregates in human lung epithelial cells. (A-C) Aggregation of ORF8SARS-CoV-2 protein in A549 cells viewed by fluorescent microscopy. Human lung epithelial A549 cells were transfected with pEGFP-N1 (empty vector; EV) or pEGFP-ORF8SARS-CoV-2. After 24 h, fluorescent microscopy was performed on either live cells (A) or paraformaldehyde-fixed cells that were stained with anti-GFP antibody (B). In the anti-GFP antibody-stained cells, quantitative analysis of nuclear-cytoplasmic distribution pattern of aggregated ORF8SARS-CoV-2 was performed (C). (D, E) Prediction of aggregation based on the ORF8SARS-CoV-2 protein primary structure using the PASTA 2.0 algorithm (http://protein.bio.unipd.it/pasta2/). Aggregation disorder profile (D) and aggregation-free energy profile (E) showing that N-terminal residues 1-18 have the lowest aggregation free energy and thus is the most aggregation-stabilizing region underlying the propensity for ORF8SARS-CoV-2 aggregates formation. (F, G) The intracellular distribution pattern of ORF8SARS-CoV-2 aggregates is not affected by a poly (I:C)-induced inflammatory response. A549 cells were treated with poly (I:C) during…

Figure 3. Expression of ORF8SARS-CoV-2 does not affect lung epithelial cell proliferation or cell cycle progression. Human A549 lung epithelial cells were transfected with pEGFP-N1 (empty vector, EV) or pEGFP-ORF8SARS-CoV-2 and treated with or without poly (I:C). At 24 hours post-transfection, cells were processed for in vitro proliferation assay (A) and flow cytometry-based cell cycle analysis (B). n = 3 for each group.

Figure 4. Characterization of the role of ORF8SARS-CoV-2 on expression of antiviral immunity-associated genes in lung epithelial cells. A549 cells were transfected with pEGFP-N1 (empty vector, EV) or pEGFP-ORF8SARS-CoV-2. At 24 hours post-transfection, cells were processed for RNA extraction and RT-qPCR to determine the expression levels of indicated immune response genes. (A) ORF8-inhibited genes. (B) Non-responsive genes. n = 3-5, *p < 0.05 was considered as statistically significant.

Figure 5. Characterization of the role of ORF8SARS-CoV-2 on regulation of type I and II interferon (IFN)-induced expression of antiviral molecules in human lung epithelial cells. Human A549 lung epithelial cells were transfected with pEGFP-N1 (empty vector, EV) or pEGFP-ORF8SARS-CoV-2. The transfected cells were treated with medium containing IFNβ (100 ng/mL) (A) or IFNγ (100 ng/mL) (B) for 24 h followed by RNA extraction and RT-qPCR analysis of expression of the indicated genes. n = 3-5. *p < 0.05 was considered as statistically significant.