Phosphatidylserine receptors enhance SARS-CoV-2 infection

Abstract

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

Fig 1. PS receptors synergize with ACE2, enhancing SARS-CoV-2 infection of HEK 293T cells.

A) Cells transfected with PS receptor expression plasmids, AXL or TIM-1, with or without 50 ng of ACE2 and infected 48 hours later with SARS-CoV-2 (MOI = 0.5). Resulting cellular infection was determined by viral loads 24 hours after initial infection using RT-qPCR. B-C) PS receptors, TIM-1 (B) and AXL (C), enhance rVSV/Spike infection at low concentrations of transfected ACE2. D) Virus binding of cells transfected with PS receptor plasmids with or without 50 ng of ACE2. rVSV/Spike was bound to transfected cells at 48 h following transfection and bound virus was measured via RT-qPCR. E) PS receptors mediate internalization of rVSV/Spike. Virion internalization was measured at 24 h after transfection with 1 μg of the indicated plasmids. FITC-labeled rVSV/Spike was bound for 1-hour, unbound virions washed away, and cells shifted to 37°C for 30 minutes. Non-internalized virus was then cleaved from cell surface by trypsin. Cells were washed, and FITC retention quantified by flow cytometry. F) HEK 293T cells transfected with PS receptor plasmids, TYRO3 or TIM-4, with or without 250 ng of ACE2 and infected 48 hours later with VSV/Spike. Viral loads were determined 24 hours following infection. Data shown are pooled from at least 3 independent experiments (A, B, C, D, E, F). Data represented as means ± SEM. Student’s t-test (A, E) and multiple t-test (B, C), One-Way ANOVA with multiple comparisons (D, F); asterisks represent p < 0.05.

Fig 2. PS receptors interact with SARS-CoV-2 by binding to virion PS.

A) PS is readily detectable on UV irradiated SARS-CoV-2 virions and rVSV/Spike. Indicated quantities of viral particles (determined by protein content) were coated in ELISA plates, and PS was detected using bavituximab followed by secondary antisera. B-C) PS liposomes interfere with rVSV/Spike infection. HEK 293T cells transfected with 50 ng of ACE2 plasmid and 1 μg of TIM-1 (B) or AXL (C) plasmid. Cells were infected with rVSV/Spike in the presence of increasing concentrations of PS or PC liposomes and assessed for nanoluciferase activity 24 hours later. D) HEK 293T cells were transfected with 1 μg of plasmid expressing WT or PS binding pocket mutant TIM-1 (ND115DN) with or without 250 ng of ACE2 plasmid and infected 48 hours later with rVSV/Spike. Luminescence fold change were compared to Mock transfected lysates that were set to a value of 1. E) Surface expressed AXL is unable to directly interact with purified SARS-CoV-2 spike/Fc proteins. HEK 293T cells transfected with AXL or ACE2 were incubated with soluble Spike protein-Fc, S1 RBD-Fc or S1 NTD-Fc and subsequently incubated with an Alexa 647 secondary against Fc. Transfected cells bound to spike constructs were detected by flow cytometry. F) Purified AXL does not bind to the NTD of SARS-CoV-2 spike. Biolayer interferometry association curves show that immobilized AXL-Fc fails to interact with purified NTD of spike. Data are pooled from at least 3 independent experiments (B, C) or are representative of at least 3 experiments (A, D, E, F). Data represented as means (or individual datapoints) ± SEM. Multiple t-test (B, C), One-way ANOVA with multiple comparisons (A, D); asterisks represent p < 0.05.

Fig 3. The route of SARS-CoV-2 entry is altered by TMPRSS2 expression.

A) HEK 293T cells were transfected with 50 ng ACE2 plasmid and TMPRSS2 plasmid as noted and infected at 48 h with VSV/Spike. At 24 hpi, luminescence activity was determined. Findings are shown relative to empty vector (Mock) transfected cells. B) TMPRSS2 expression enhances rVSV/Spike infection at low levels of ACE2 expression. HEK 293T cells were transfected as indicated and rVSV/Spike infection assessed by measuring luminescence activity at 24 hpi. C) Transfected HEK 293T cells were transfected and infected with VSV/Spike at 48 h in the presence or absence of E-64 (300 μM). Luciferase activity was determined 24 hpi. Data are pooled from at least 3 independent experiments (B, C) or are representative of at least 3 experiments (A). Data represented as means ± SEM. Student’s t-tests (A) Multiple t-tests (B), Two-way ANOVA with row-wise multiple comparisons (C); asterisks represent p < 0.05.

Fig 4. AXL has a prominent role in SARS-CoV-2 entry into Vero E6 cells.

A) PS liposomes interfere with SARS-CoV-2 pseudovirion entry. Vero E6 cells were treated with increasing concentrations of PS or PC liposomes and infected with VSV/Spike pseudovirions for 24 hours. Infection was detected by GFP fluorescence expressed from the VSV genome. B) PS liposomes disrupt SARS-CoV-2 binding. Vero E6 cells were incubated with SARS-CoV-2 (MOI = 5) at 10°C for 1 hour in the presence of indicated liposomes, washed extensively, and viral load assessed by RT-qPCR. C) AXL signaling inhibitor bemcentinib inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with bemcentinib and infected with SARS-CoV-2 (MOI = 0.01). Viral loads were measured 24 hpi by RT-qPCR. E) RNAseq studies in Vero E6 cells demonstrate bemcentinib inhibition. Cells were treated with 1 μM bemcentinib, infected with SARS-CoV-2 (MOI = 0.01) and mRNA harvested 18 hpi. mRNA was deep sequenced on an Illumina platform, and ‘Percent Viral Reads’ were calculated by alignment to the SARS-CoV-2 genome. E) Broad spectrum TAM inhibitor BMS-777607 inhibits SARS-CoV-2 infection in Vero E6 cells. Cells were treated with inhibitor at indicated concentrations prior to challenged (MOI = 0.01), and viral loads measured 24 hpi by RT-qPCR. F-G) Enhanced colocalization of AXL and ACE2 during SARS-CoV-2 infection. STED micrographs shows staining for ACE2 (red) and AXL (green) and merged in Vero E6 cells (F). Insets are enlarged images from regions highlighted by yellow rectangles. White arrows indicate shared vesicular structures between the two channels. Yellow arrowheads indicate objects that are only seen in one channel. Plot profiles are shown in S4F, representing signal intensity along the yellow lines in the merged panels. Pearson’s correlation coefficients of ACE2 and AXL were calculated for n = 20 mock and infected cells (ROI determined by cell borders) (G). Data are pooled from at least 3 independent experiments (B, E) or are representative of at least 3 experiments (A, C, F, G). Data are represented as means ± SEM. Multiple t-tests (A) Student’s t-test (B, C, D, G); asterisks represent p < 0.05.

Fig 5. AXL inhibition reduces SARS-CoV-2 infection in human lung cells.

A-E) SARS-CoV-2 infection (MOI = 0.5) is reduced by AXL inhibition by bemcentinib or E64 in multiple human lung cell lines, including A549^ACE2(A), H1650 (B), HCC1944 (C), HCC2302 (D). Inhibitors were added to cells 1 h prior to infection and maintained on the cells for the entire infection. At 24 hpi, viral load was determined. E) Infectious virus produced by HCC2302 cells was inhibited by bemcentinib. HCC2302 cells were treated with bemcentinib at the indicated concentrations and infected with SARS-CoV-2 (MOI = 0.5). Input virus was removed 6 hpi and media containing the appropriate bemcentinib concentration was added. Supernatant was collected at 24 and 48 hpi and titered by TCID₅₀ assays on Vero E6-TMPRSS2 cells. TCID₅₀/mL was calculated by the Spearmann-Karber method. F) Timing of effect bemcentinib inhibition of SARS-CoV-2 infection. In time-of-addition studies, SARS-CoV-2 (MOI = 0.01) was added to infected H1650 cells (human lung cells) to initiate the experiment and 1 μM bemcentinib was added at the times noted. Cells were harvested at 24 hpi for viral load determinations. G) Calu-3 are insensitive to bemcentinib and E64. Studies were performed as described for panels A-E. H) A549^ACE2 were treated with bemcentinib, infected with SARS-CoV-2 (MOI = 0.5) and mRNA harvested 24 hpi. mRNA was deep sequenced and viral loads calculated by alignment to the SARS-CoV-2 genome. Data are representative of at least 3 experiments (A, B, C, D, E, F, G). Data represented as means ± SEM. Student’s t-test; asterisks represent p < 0.05.

Fig 6. AXL knockout reduces viral loads and ablates inhibition by bemcentinib.

A) Flow cytometry histograms depicting AXL surface staining (black) and secondary only background (gray) on parental, Cas9 expressing H1650 cells or Cas9/guide RNA treated and cloned cells, demonstrating loss of AXL expression. H1650 AXL knockout cells were generated by lentiviral transduction of Cas9 and gRNA targeting AXL, followed by selection, enrichment, and biological cloning. B) H1650 AXL^neg and H1650 Cas9 (parental) lines were challenged with SARS-CoV-2 at indicated MOIs. At 24hpi, viral loads were assessed by RT-qPCR. C) H1650 parental and AXL^neg lines were treated with the indicated concentration of bemcentinib and challenged with SARS-CoV-2 (MOI = 0.5) and viral loads determine by RT-qPCR 24 hpi. Data are pooled from 3 independent experiments (B, C) or are representative of at least 3 experiments (A). Data represented as means ± SEM. Multiple t-tests; asterisks represent p < 0.05 (B). Student’s t-tests comparing Mock and 1μM; p values shown (C).