Fig 2: SIRPA KO renders cells and mice more susceptible to virus infection.A) SIRPA expression in BMDMs isolated from WT and SIRPA KO mice was analyzed in total cell extracts by western blot, using antibodies to the ectodomain or cytoplasmic tail and by FACS, using antibody to the ectodomain. The molecular weight (MW) of mouse SIRPA is 110kD. B) BMDMs isolated from mice of the indicated genotype were infected with the specified viruses and RNA was isolated at 14 hpi (VSV, mNoV), 24 hpi (JUNV-C1, TCRV, LCMV) or 48 hpi (ZIKV). HSV-1 DNA was isolated at 24 hpi. Viral nucleic acids were examined by qPCR for infection levels. Shown is the average ± SD of 4–6 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.02; **, P ≤ 0.002; ***, P ≤ 0.0005. C) Adult mice of the indicated genotype were infected with JUNV-C1 by IP injection, and at 7 or 14 dpi, virus and RNA was isolated from spleens and analyzed for titers and by qPCR. Each point represents an individual mouse. Shown is the average ± SD. Student’s T test with Welch’s correction was used to determine significance. *, P ≤ 0.01; **, P ≤ 0.004. D) Newborn WT and SIRPA KO mice were infected with TCRV by IP injection. Viral titers and RNA levels in spleen were analyzed at 7 dpi. Student’s T test with Welch’s correction was used to determine significance. *, P ≤ 0.01; ***, P ≤ 0.001. E) Newborn mice were infected with 2000 ICs of MLV and virus titers in spleen were measured at 16 dpi. F) WT and SIRPA KO mice were orally infected with mNoV strain CW3 and 3 dpi viral RNA levels in spleens were examined. Unpaired t test with Welch’s correction was used to determine significance.

Fig 3: Integrin activation, rather than interaction with CD47, modulates SIRPA antiviral activity.A) Viruses (JUNV-C1, LCMV) and pseudoviruses (VSV-G, SARS-2-S) were incubated with the indicated antibodies for 1 hr on ice before being added to U2OS and 293T-ACE2 cells, respectively. RT-qPCR for viral RNA or luciferase assays were carried out at 24 and 48 hpi, respectively. The data shown represent the average ± SD of 2 (convalescent serum) - 4 independent experiments. One-way ANOVA was used to determine significance. *, P ≤ 0.02; **, P ≤ 0.002. Panel on the right shows western blot analysis of JUNV-C1-infected U2OS cell extracts and virions (5E5 ICs) probed with the same anti-CD47 antibody, anti-Junín virus NP monoclonal antibody or anti-GAPDH. B) U2OS and 293T-mCAT cells were transfected with the indicated siRNAs and 48 hours later were treated with 1mM MnCl2 for 2 hr. After MnCl2 treatment, U2OS cells were infected with JUNV-C1, LCMV, VSV, MLV and HSV-1 for 24 hr and 293T-mCAT cells with MLV for 48 hr. Viral RNA and DNA levels were determined by qPCR. The data shown represent the average ± SD of 3–4 independent experiments. Two-way ANOVA was used to determine significance. *, P ≤ 0.05; **, P ≤ 0.005: ***, P ≤ 0.0004; ***, P ≤ 0.0001. C) Primary fibroblasts isolated from WT and SIRPA KO mice were treated with 1mM MnCl2 for 2 hr and infected with JUNV-C1 for 24 hr. Viral RNA was examined by RT-qPCR. Shown is the average ± SD of 3 independent experiments. Two-way ANOVA used to determine significance. *, P ≤ 0.02; ****, P ≤ 0.0001.

Fig 4: 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 5: 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.