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.

AUTHOR SUMMARY: 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.

Orthoebolavirus zairense is the species name for Zaire Ebola virus (EBOV) within Filoviridae. This group of viruses can cause severe disease in humans, characterized by hemorrhagic shock, coagulation abnormalities, and severe inflammation. While tissue macrophages are critical targets early during EBOV infection, other cell types support viral replication as disease progresses. At late stages of infection, infectious EBOV is found on the surface of the skin, which may be a critical source of infectious virus transmitted between individuals during outbreaks. Human skin contains a number of cellular targets of EBOV, including keratinocytes. Here, we demonstrate EBOV infection of telomerase-immortalized normal human skin keratinocytes (NHSK-1), as well as EBOVΔVP30 infection of NHSK-1 cells that were stably complemented with EBOV transcription factor VP30. Infection with EBOVΔVP30 did not elicit detectable endogenous interferon responses; however, exogenous pre-treatment of NHSK-1 cells with type I, II, and III interferon (IFN) inhibited EBOVΔVP30 infection and infection of an additional low-containment model of EBOV, rVSV/EBOV GP, in a dose-dependent manner. Analysis of the transcriptome of IFN-treated keratinocytes identified multiple genes unique to each IFN and a subset of ISGs upregulated by all three IFNs. Our results indicate that ISGs induced by IFN pre-treatment of keratinocytes can reduce infection, underlining that ISGs may serve as EBOV-targeting therapeutics.

Orthoebolavirus zairense, or Zaire Ebola virus (EBOV) is a species within the virus family Filoviridae, a group of viruses that can cause severe disease in humans characterized by hemorrhagic shock, coagulation abnormalities, and severe inflammation. While tissue macrophages are critical targets early during EBOV infection, other cell types support viral replication as disease progresses. At late stages of infection, infectious EBOV is found on the surface of the skin, which may be a critical source of infectious virus transmitted be-tween individuals during outbreaks. Human skin contains a number of cellular targets of EBOV, including keratinocytes. Here, we demonstrate EBOV infection of telomerase-immortalized normal human skin keratinocytes (NHSK-1) as well as EBOVΔVP30 infection of NHSK-1 cells that were stably complemented with the EBOV transcription factor VP30. Infection with EBOVΔVP30 did not elicit detectable endogenous interferon responses, however, exogenous pre-treatment of NHSK-1 cells with type I, II, and III interferon (IFN) inhibited EBOVΔVP30 infection and infection of an additional low containment model of EBOV, rVSV/EBOV GP, in a dose-dependent manner. Analysis of the transcriptome of IFN-treated keratinocytes identified multiple genes unique to each IFN and a subset of ISGs upregulated by each IFN. Our results indicate that ISGs induced by IFN pre-treatment of keratinocytes can reduce infection, underlining possible anti-EBOV ISGs for development of EBOV-targeting therapeutics.

Ebola virus (EBOV) disease (EVD) is a hemorrhagic disease caused by EBOV infection. EVD outcomes in pregnant women are similar to non-pregnant women however, EVD is associated with negative fetal outcomes in ∼99% of cases. There is a critical need for a tractable small animal model to study maternal/fetal transmission of EBOV. We utilized interferon α/β receptor knock out mice infected and authentic EBOV or the model virus, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (rVSV/EBOV). Infection with either virus during late pregnancy resulted in placental infection and vertical transmission to the fetus within 2-3 days. Robust levels of maternal and fetal proinflammatory cytokines were evident by day 5 after EBOV infection. Within the placenta, trophoblasts and endothelial cells were viral antigen positive. Elimination of the endosomal receptor NPC1 in junctional zone trophoblasts reduced placental infection and virus transmission to the fetus. These studies establish an infectious model that provides EBOV trafficking and pathogenesis insights during pregnancy. Teaser: This model provides key insights into how viral trafficking and maternal immune responses drive adverse fetal outcomes during gestational Ebola virus infection.