Functional landscape of SARS-CoV-2 cellular restriction

Abstract

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Keywords: BST2; ISG; Orf7a; SARS-CoV-2; innate immunity; interferon; viral evasion.

Graphical abstract

Figure 1

IFN-mediated restriction of SARS-CoV-2 relies on a limited subset of ISGs (A) Schematic representation of the gain-of-function screen to identify ISGs that inhibit SARS-CoV-2 replication. (B) Ranked log2FC of the percentage of infected cells (SARS-CoV-2 N⁺ cells, blue shading) and normalized cell number (pink shading) after individual overexpression of 399 human ISGs and controls. Values are relative to the negative control CAT. Dashed lines illustrate cut offs for antiviral ISG hit calling strategy; the dotted blue line indicates log2FC infection = 4 × standard deviations (SDs) log2FC of CAT log2FC, and the dotted pink line indicates cell number = 70% of CAT. Controls are shown (CAT, negative; LY6E, positive). (C) Correlation plots between screens. r, Pearson correlation coefficient. (D) 293T-ACE2 cells transduced with lentiviruses carrying each of the identified ISGs were infected with SARS-CoV-2 (MOI = 0.25) for 40 h prior to immunostaining for viral SARS-CoV-2 nucleoprotein (N). Data represent mean log2FC values (percentage of N⁺ cells relative to parental control wells) from three independent experiments (n = 3). (E) Representative images are shown. Scale bars, 10 μm. (F) Calu-3 cells transduced with lentiviruses encoding the indicated ISGs were infected with SARS-CoV-2 (MOI =1.5) for 48 h prior to immunostaining for viral N protein. Data show mean ± SEM normalized infection (percentage of infected cells relative to parental control wells) from one representative experiment in quadruplicate (n = 4). (G) Differentiated HTBE cells stably expressing the indicating ISGs or negative control GFP were infected with SARS-CoV-2 (MOI = 1) on the apical side. At 18 h post-infection, supernatants were collected and the amount of SARS-CoV-2 focus-forming units per milliliter (FFU/mL) analyzed using Vero E6 cells. Data show mean ± SD and are representative from two sets of HTBE cells per ISG (n = 2). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparison post hoc test (D) or one-way ANOVA with Dunnett’s post hoc test (F and G).

Figure 2

Network model of SARS-CoV-2 antiviral effectors (A) The network containing the 65 identified antiviral ISGs was propagated to include a total of 343 high-confidence protein interactors (score > 0.7 STRING) and subjected to supervised community detection (Carlin et al., 2017; Shannon et al., 2003). The resultant hierarchy is shown. Here, each node represents a community of densely interconnected proteins, and each edge (arrow) denotes containment of one community (edge target) by another (edge source). Enriched biological processes are indicated. The percentage of each community that corresponds to the 65 antiviral ISGs is shown in dark blue. (B–E) Zoom-in insets from selected protein communities are indicated with an asterisk (^∗) in the hierarchy. Nodes indicate proteins, and edges indicate interactions from STRING. Blue nodes indicate ISGs that restricted SARS-CoV-2 replication.

Figure 3

SARS-CoV-2 antiviral effectors inhibit discrete viral replication steps (A–C) 293T-ACE2 cells stably expressing each of the indicated ISGs were subjected to (A) infection with SARS-CoV-2 pseudotyped VSV luciferase virus for 16 h prior to measurement of luciferase signal. In parallel, cells were subjected to synchronized infection with SARS-CoV-2 (MOI = 4) for 6 h prior to measurement of viral RNA (B), or supernatants at 18 h post-infection were used to infect naive Vero E6 cells. The percentage of infected cells was then determined at 18 h post-infection using immunostaining for viral N protein (C). In parallel to these experiments, the impact of these ISGs on SARS-CoV-2 replication at 24 h post-infection was evaluated (full life cycle). Results are summarized in the heatmap and show the mean (n = 2) of relative activities compared to parental cells.

Figure 4

Integrated model of SARS-CoV-2 cellular restriction mechanisms ISGs that inhibited SARS-CoV-2 replication were placed at specific positions along the viral infectious cycle based on experimental data generated in Figure 3 in conjunction with Gene Ontology, KEGG, and Reactome databases and the literature (see STAR Methods). Human ISGs are represented in blue circles and SARS-CoV-2 proteins in yellow circles. ISGs in bold indicate those ISGs that were validated using lentiviral transduction (Figure 1D). Dashed lines (edges) represent indirect interactions between these ISGs and the indicated viral proteins based on reported ISG interactors (Hubel et al., 2019) and SARS-CoV-2 interactors (Gordon et al., 2020).

Figure 5

BST2 inhibits release of SARS-CoV-2 and is antagonized by Orf7a (A and B) 293T and Huh7 cells transfected with BST2 along with ACE2 and TMPRSS2 were infected with SARS-CoV-2 at the indicated MOIs for 48 h prior to immunostaining for viral N protein. Shown is mean ± SEM normalized infection (percentage of SARS-CoV-2 N⁺ cells) relative to empty vector control from three independent experiments (n = 3) (A) and representative images of Huh7 cells infected at MOI 0.03 (B). (C) HeLa-ACE2 parental or BST2 KO cells were infected with SARS-CoV-2 (MOI = 2). At the indicated hours post-infection, supernatants were collected and analyzed by plaque assay in Vero E6 cells. LoD, limit of detection. Data show mean ± SD from one representative experiment in triplicate (n = 3) of two independent experiments. (D) Calu-3 cells were transfected with siRNAs targeting BST2 or IRF9 or the negative control scrambled siRNA. At 48 h post-transfection, cells were treated with 18 IU/mL IFN for 6 h and then infected with SARS-CoV-2 (MOI = 0.75). At indicated times post-infection, supernatants were collected and analyzed by plaque assay in Vero E6 cells. Data show mean ± SD and are representative from one experiment in quadruplicate (n = 4). (E) 293T cells were transfected with the indicated constructs for 24 h. Cell lysates and supernatants (VLPs) were then analyzed using SDS-PAGE and immunoblotted with indicated antibodies. Only blot lanes 2–4 and 6 (shown in boxes) are relevant to this experiment and included in the figure. (F) HeLa-ACE2 cells transfected with M-FLAG, E, and N were subjected to immunostaining for BST2 and FLAG, as indicated. Shown are deconvolved widefield microscopic images revealing colocalization of BST2 and M (arrows). Scale bars, 10 μm. (G) HeLa-ACE2 cells transfected with Orf7a-FLAG were subjected to immunostaining for BST2 and FLAG, as indicated. Shown are confocal images revealing colocalization of BST2 and Orf7a (arrows). Scale bars, 10 μm. (H) Top two ClusPro docking models of Orf7a interaction with the BST-2 ectodomain dimer. BST-2 is shown in gray, and the two Orf7a molecules are green and yellow. (I) Close-up of the interface circled in H. Left: electrostatic surface of BST2 (blue, positive; red, negative). Beta strands A and G of Orf7a (green) are involved in the interaction. Selected interaction residues are labeled. Right: electrostatic surface of Orf7a. The beta strands A and G form an electrostatic ridge on the surface (boxed). (J) 293T cells were transfected with BST2 expression plasmid (WT or N2Q mutant) and empty plasmid, Orf7a-FLAG, or HIV-1 Vpu-FLAG expression plasmid. Immunoprecipitation (IP) was carried out using FLAG M2 magnetic beads. Inputs and IP samples were analyzed by SDS-PAGE using indicated antibodies. Blot is representative from two independent experiments. The asterisks (^∗) denote the 25-kDa anti-FLAG (M2) light chain. (K) Parental 293T-ACE2 or BST2 stable cells were infected with WT or dOrf7a (MOI = 1). At indicated times post-infection, supernatants were collected and analyzed by plaque assay in VeroE6 cells. Data show mean ± SD from one representative experiment in triplicate (n = 3) of two independent experiments. Statistical significance was calculated using one-way ANOVA with Dunnett's post hoc (A and D), Student’s t test (C), or Tukey’s multicomparison test (K).

Figure 6

Comparative antiviral activities of SARS-CoV-2 restriction factors Heatmap showing normalized infection upon overexpression of indicated ISGs across 21 viruses. Data for SARS-CoV-2 were generated within this study (see Table S2). Data for the remaining 20 viruses were obtained from previously published work (Schoggins et al., 2011, 2014). Data for SARS-CoV-1 were generated by infecting 293T-ACE2 stably expressing each of the indicated ISGs with SARS-CoV-1 (MOI = 0.01). At 48 h post-infection, supernatants were collected and used to calculate the median tissue culture infectious dose (TCID50). Data show TCID50/mL relative to parental control wells. Data show mean ± SD from one representative experiment in triplicate (n = 3) of two independent experiments. Virus families are indicated. Virus full names and abbreviations are described in STAR Methods.