Fig 2: Cytosolic RNA sensing by RIG-I and MDA-5 is required for SARS-CoV-2 induced innate immune responses in macrophages(A-B) CD169-expressing THP1 monocytes were transduced with lentivectors expressing shRNA against non-specific control (scramble), or specific sequences against STING, UNC93B1, MAVS, RIG-I, and MDA-5. Knockdown of host proteins targeted by shRNAs compared to scramble analyzed by RT-qPCR (A), and immune blotting (B). (C-H) CD169+ THP1 cells with stable knockdown of target genes were PMA-differentiated and infected with SARS-CoV-2 (MOI=1, 24 hpi), and total RNA was analyzed by RT-qPCR for total nucleocapsid (gRNA) (C), sgRNA (Envelope) (D), and pro-inflammatory cytokines/ISGs, IL6, TNFa, IL-1ß and IFN?1 (E-F), normalized to mock-infected controls for each group. Each knockdown is represented by different colors as in A. The means ± SEM from 3 independent experiments are shown, and significant differences were determined by one-way ANOVA followed by the Dunnett’s post-test comparing to scramble THP1 (C-D), or by two-way ANOVA followed by Bonferroni post-test comparing Mock to CoV-2+ in each group (E-H). P-values: *<0.1; **<0.01; ***<0.001; ****<0.0001), ns: not significant.

Fig 3: CD169 is a SARS-CoV-2 attachment and entry factor in macrophages.(A) Binding of SARS-CoV-2 S protein (Wuhan isolate) to THP1 monocytes expressing wt CD169, mutant CD169 (R116A), ACE2, or both wt CD169 and ACE2 (CD169/ACE2). (B) THP1/PMA macrophages were infected with S-pseudotyped lentivirus (20 ng p24Gag), and infection was quantified by measuring luciferase activity at 3 dpi. Relative light units (RLUs) from each cell line were normalized to no virus control (mock). The means ± SEM are shown from at least five independent experiments. (C) Primary MDMs from three donors overexpressing either CD169 or ACE2, or control were infected with S-pseudotyped lentivirus (20 ng p24Gag) for 3 days, followed by analysis of luciferase activity in whole cell lysates. (D) Untransduced primary MDMs (representative of 3 donors) were pre-treated with anti-CD169 mAb (20 µg/ml, 7D2), IgG1, or empty media for 30 min at 4°C prior to infection with S-pseudotyped lentivirus (20 ng p24Gag) for 3 days, followed by analysis of luciferase activity. RLUs from each donor in each group were normalized to no virus control (mock). The means ± SEM from at least 3 independent experiments are shown. P-values: paired t-test, two-tailed (A), one-way ANOVA followed by the Dunnett’s post-test (B) or Tukey’s post-test comparing to parental (THP1) cells (C) or each pre-treatment condition (D). ***: p < 0.001, ****: p < 0.0001, ns: not significant.

Fig 4: Restricted expression of SARS-CoV-2 gRNA and sgRNA induces pro-inflammatory responses in non-productively infected macrophages(A-B) PMA-differentiated THP1 cells from parental (THP1) or those expressing CD169, ACE2, or CD169/ACE2 were infected with SARS-CoV-2 (MOI=1, 24 hpi) in the absence or presence of RDV (1 µM, 30 mins), and total RNA was quantified by RT-qPCR for pro-inflammatory cytokines and ISGs. Values were normalized to mock-infected control for each group. Fold-induction for indicated pro-inflammatory cytokines (A, top panel) and ISGs (A, bottom panel) in the absence of RDV, or in the presence of RDV (B). (C-F) Kinetics of pro-inflammatory cytokine/ISG mRNA expression in the absence of RDV in parental (C), CD169-expressing (D), ACE2-expressing (E), and CD169/ACE2-expressing (F) PMA-differentiated THP1 cells. (G) MDMs from multiple donors were infected with SARS-CoV-2 (MOI=1, 24 hpi) and total RNA analyzed by RT-qPCR. MDMs were pre-treated with DMSO (control) or RDV (1 µM, 30 mins) prior to infection, and fold-induction of indicated cytokine mRNAs normalized to mock-infected controls from each donor. RDV: Remdesivir, ISGs: interferon-stimulated genes. Minus sign represents DMSO, and plus sign represents RDV pre-treatment. The means ± SEM from at least 3 independent experiments are shown. Significant differences between groups were determined by one-way ANOVA followed by the Dunnett’s post-test between groups (A) or Tukey’s multiple comparisons test within groups (B and G), comparing to parental THP1 (A) or DMSO-treated (B, G) in each group. P-values: *<0.1; **<0.01; ***<0.001; ****<0.0001, ns: not significant.

Fig 5: MDM immune activation is initiated via a MAVS-dependent pathway. a MDMs were transduced with lentivectors expressing shRNA against scrambled sequence (Sc), MAVS or STING, and the expression of host proteins targeted by shRNAs was analyzed by immune blotting. b–e shRNA-transduced MDMs were infected with HIV-1 (Lai?envGFP/G) and viral expression (GFP) (b, c) and CD169 expression (d, e) were analyzed 6 days post infection. f MDMs were transduced with lentivectors expressing shRNA against scrambled sequence (Sc), RIG-I or MDA5, and the expression host proteins targeted by shRNAs analyzed by immune blotting. g–j shRNA-transduced MDMs were infected with Lai?envGFP/G and viral expression (GFP) (g, h) and CD169 expression (i, j) were analyzed 6 days post infection. k–m Effect of inhibitors on HIV-1 infection (k), CD169 expression (l), and IP-10 production (m) in MDMs. MDMs were infected with HIV-1 (MOI of 2) and cultured in the presence of indicated inhibitors for 6 days. mock1/DMSO1: 0.1% DMSO, mock2/DMSO2: 0.01% DMSO. The means ± SEM are shown and each symbol represents an independent experiment. Two-tailed p-values: one-way ANOVA followed by the Tukey-Kramer post-test (d, e, i, j, l, and m) or paired t-test (b, c, g, and h). *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant