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.

Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

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Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD and NTD of subunit S1 and against subunit S2. The antibody responses of different infected individuals exhibit different efficacies to inactivate (neutralize) the virus.

Phosphatidylserine (PS) receptors enhance infection of a wide range of enveloped RNA 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. Under ACE2lo conditions, expression of members of the TIM and TAM families of PS receptors synergized with ACE2 to enhance SARS-CoV-2 infection; however, PS receptors alone did not mediate infection. PS receptors enhanced SARS-CoV-2 binding to the surface of cells. While PS receptors did not interact directly with purified SARS-CoV-2 spike, addition of PS liposomes reduced entry of VSV-SARS-CoV-2 spike pseudovirions, providing evidence that PS/PS receptor interactions are mediating the effect. AXL being abundant on airway cell 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 some lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. RNAseq studies in Vero E6 and A549-hACE2 cells demonstrated that bemcentinib reduced SARS-CoV-2 transcripts dramatically. The ability of bemcentinib to decrease coronavirus infection in vivo was assessed using the betacoronavirus mouse hepatitis virus (MHV). Liver titers and virus load of MHV were significantly inhibited by bemcentinib at day 5 of infection. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and that inhibition of signaling of the PS receptor AXL has therapeutic potential against SARS-CoV-2.