Fig 2: SIRPA inhibits endosomal but not cell surface entry of SARS-CoV-2.A) 293T-ACE2 or Calu-3 cells were transfected with the indicated siRNAs and then infected with SARS-2-S or VSV G pseudoviruses. Luciferase assays were performed at 48 hpi. B) Calu-3 or 293T-ACE2 cells were treated with the indicated drugs (camostat inhibits TMPRSS2; E-64D inhibits endosomal Cat B/L; nanchangmycin is an endocytosis inhibitor) infected with SARS-2-S and VSV G pseudoviruses. Shown is the average ± SD of 3–6 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.02; **, P ≤ 0.001; ***, P ≤ 0.0003; ****, P ≤ 0.0001.

Fig 3: Interluekin-4 (IL-4) treatment upregulates SIRPA expression and decreases viral internalization.A) U2OS cells were pre-treated with the indicated concentrations of IL-4 for 48 hr and SIRPA expression levels were quantified by western blot. The MW of human SIRPA is 85kD. Shown is the average ± SD of 3 independent experiments. One-way ANOVA was used to determine significance. **, P ≤ 0.003. B) and C) U2OS cells were pre-treated with IL-4 and were subjected to VIAs (B) and infection for 24 hr. (C) with JUNV-C1 and LCMV. Shown is the average ± SD of 3–4 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.02; **, P ≤ 0.002; ***, P ≤ 0.0005.

Fig 4: SIRPA inhibits virus internalization.A) Viral internalization assays (VIA) were carried out with U2OS or NR-9456 cells transfected with the indicate siRNAs. Internalized viral RNA was analyzed by RT-qPCR. Shown is the average ± SD of 3 independent experiments. Student’s T test was used to determine significance. *, P ≤ 0.02; **, P ≤ 0.007; B) VIAs were performed with SARS-2-S pseudoviruses and 293T-ACE2 cells transfected with the indicated siRNAs. RT-qPCR for the luciferase gene was used to measure internalized pseudovirus. 293T cells and no reverse transcriptase (no RT) served as controls. Shown is the average ± SD of 3 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.02; ***, P ≤ 0.001. C) VIAs were performed with BMDMs isolated from mice of the indicated genotypes with JUNV-C1, LCMV or VSV. Shown is the average ± SD of 3 independent experiments. One-way ANOVA was used to determined significance. *, P ≤ 0.03; **, P ≤ 0.007. D) MnCl2 treatment increases virus internalization in ear fibroblasts. Shown is the average ± SD of 3 experiments. Student T test was used to determine significance. ***, P ≤ 0.0002.

Fig 5: SIRPA’s cytoplasmic tail is required for its antiviral activity.A) Top: Diagram of WT and truncated (ΔCyto) human SIRPA constructs. Y1 through Y4 represent the tyrosines needed for SIRPA’s anti-phagocytic activity. B) U2OS cells were transfected with the indicated plasmids and 24 hr later, infected with the indicated viruses. At 24 hpi, RNA was isolated and examined by RT-qPCR for infection levels. Shown is the average ± SD of 3–10 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.015; **, P ≤ 0.003; ***, P ≤ 0.0002. C) 293T-ACE2 cells were transfected with the indicated plasmids and 24 hours later, infected with the indicated pseudoviruses. At 48 hpi, luciferase assays were performed. Shown is the average ± SD of 3–4 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.01; ****, P ≤ 0.0001. D) The four Y residues in SIRPAs cytoplasmic tail were mutated to A residues. Experiments were carried out as in panel B. Shown is the average ± SD of 3–4 independent experiments. One-way ANOVA was used to determine significance. **, P ≤ 0.002; ****, P ≤ 0.0001.