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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 bind PS and 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. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. This clever use of this uptake mechanism by enveloped viruses is termed apoptotic mimicry. 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 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. PS is readily detectable on the surface of SARS-CoV-2 virions, and contrary to prior reports we were unable to identify any interaction between AXL and SARS-CoV-2 spike. Pharmacological inhibition of AXL activity and knockout of AXL expression suggest it is the preferred PS receptor during SARS-CoV-2 entry. We propose that AXL is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry. [ABSTRACT FROM AUTHOR]

Enveloped virus utilization of PS receptors: Advantages and disadvantages of this functionall... One common viral lifestyle strategy that is benefited by the use of PS receptors is virus targeting of a wide variety of host species. As PS, PS receptor structure, and PS receptor function are highly conserved attributes among mammalian hosts and functionally conserved in insect hosts, the potential of PS receptors to facilitate zoonotic transmission under specific circumstances should not be ignored. Enveloped RNA virus entry is a conceptually simple stepwise process: A virus attaches to host cells, which leads to viral membrane/cellular membrane fusion and viral genome injection into the cytoplasm. While the inherent low-affinity interactions between PS and PS receptors is likely a disadvantage for a broad range of viruses to use this uptake mechanism, specific viruses are directly antagonized by PS receptors. [Extracted from the article]

Background: Ebolavirus (EBOV) outbreaks, while sporadic, cause tremendous morbidity and mortality. No therapeutics or vaccines are currently licensed; however, a vaccine has shown promise in clinical trials. A critical step towards development of effective therapeutics is a better understanding of factors that govern host susceptibility to this pathogen. As macrophages are an important cell population targeted during virus replication, we explore the effect of cytokine polarization on macrophage infection. Methods/Main findings: We utilized a BSL2 EBOV model virus, infectious, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (GP) (rVSV/EBOV GP) in place of its native glycoprotein. Macrophages polarized towards a M2-like anti-inflammatory state by combined IL-4 and IL-13 treatment were more susceptible to rVSV/EBOV GP, but not to wild-type VSV (rVSV/G), suggesting that EBOV GP-dependent entry events were enhanced by these cytokines. Examination of RNA expression of known surface receptors that bind and internalize filoviruses demonstrated that IL-4/IL-13 stimulated expression of the C-type lectin receptor DC-SIGN in human macrophages and addition of the competitive inhibitor mannan abrogated IL-4/IL-13 enhanced infection. Two murine DC-SIGN-like family members, SIGNR3 and SIGNR5, were upregulated by IL-4/IL-13 in murine macrophages, but only SIGNR3 enhanced virus infection in a mannan-inhibited manner, suggesting that murine SIGNR3 plays a similar role to human DC-SIGN. In vivo IL-4/IL-13 administration significantly increased virus-mediated mortality in a mouse model and transfer of ex vivo IL-4/IL-13-treated murine peritoneal macrophages into the peritoneal cavity of mice enhanced pathogenesis. Significance: These studies highlight the ability of macrophage polarization to influence EBOV GP-dependent virus replication in vivo and ex vivo, with M2a polarization upregulating cell surface receptor expression and thereby enhancing virus replication. Our findings provide an increased understanding of the host factors in macrophages governing susceptibility to filoviruses and identify novel murine receptors mediating EBOV entry. Author summary: Ebola virus causes outbreaks in Central and West Africa, often resulting in high mortality rates. Macrophages are important cell targets for the virus, yet infection of these cells remains poorly understood. Here, we show that macrophages stimulated with the immunomodulatory cytokines IL-4 and IL-13 are significantly more susceptible than unstimulated cells to a model virus that expresses the Ebola virus glycoprotein. These cytokines increase virus entry by enhancing the expression of the cell surface receptors DC-SIGN in humans and SIGNR3 in mice. Blocking availability of those receptors reduced virus load. Consistent with an important role for macrophages during EBOV infection, reconstitution of mice with macrophages treated with IL-4 and IL-13 exacerbated virus pathogenesis. Our studies argue for the critical importance of the macrophages and their response to immunomodulatory cytokines in controlling the pathological consequences of Ebola virus glycoprotein-dependent infections and highlight an important aspect of filovirus/macrophage interaction that if controlled could decrease virus pathogenesis. [ABSTRACT FROM AUTHOR]

Background: T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein to demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection. Methodology/Principal findings: Infectious, GFP-expressing recombinant vesicular stomatitis virus encoding either full length EBOV glycoprotein (EBOV GP/rVSV) or mucin domain deleted EBOV glycoprotein (EBOV GPΔO/rVSV) was used to assess the role of TIM-1 during in vivo infection. GFP-expressing rVSV encoding its native glycoprotein G (G/rVSV) served as a control. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/β receptor-/- mice were challenged with these viruses. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV or EBOV GPΔO/rVSV challenge. EBOV GP/rVSV or EBOV GPΔO/rVSV in spleen of infected animals was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection. Conclusions: Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection. [ABSTRACT FROM AUTHOR]

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks. [ABSTRACT FROM AUTHOR]

The article discusses the significance of the series of events which lead up to the entry of the Ebola virus (EBOV) into the human body. Topics include the outbreak of EBOV in new geographic regions and the need for a vaccine, how the virions initiate the Infection by entering macrophages and dendritic cells and the structure of the EBOV particle which includes viral glycoprotein and and viral ribonucleoprotein complex.