Development of a blocker of the universal phosphatidylserine- and phosphatidylethanolamine-dependent viral entry pathways

Song, Da-Hoon; García, Gustavo; Situ, Kathy; Chua, Bernadette A.; Hong, Madeline Lauren O.; Elyza, A.; Ramirez, Christina M.; Harui, Airi; Arumugaswami, Vaithilingaraja; Morizono, Kouki

doi:10.1016/j.virol.2021.04.013

Cited by 11 publications

(5 citation statements)

References 84 publications

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“…In our previously studies, other enveloped viruses that utilize PS receptors, such as filoviruses, use TIM-1 preferentially when both proteins are expressed [12,19]. Further, a recent study identified that the TIM-1 IgV domain that contains the PS binding pocket serves as an effective inhibitor of enveloped virus infection regardless of the PS receptor utilized for virus uptake [43], consistent with the good affinity the TIM-1 PS binding pocket has for PS [44]. Nonetheless, when TIM-1 is not present in cells and AXL is the sole PS receptor expressed, AXL is used by filoviruses and flaviviruses [11,13,[45][46][47].…”

Section: Discussionmentioning

confidence: 91%

Phosphatidylserine receptors enhance SARS-CoV-2 infection

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 91%

Phosphatidylserine receptors enhance SARS-CoV-2 infection

et al. 2021

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“…A better understanding of the molecular interaction between amino acid residues in the IgV domain of TIM-1, PtdSer and viral glycoproteins may help in the development of antiviral factors. For instance, Song et al recently developed a reagent that specifically binds PtdSer and/or phosphatidylethanolamine and could inhibit ZIKV infection [ 85 ]. Analysis of the diffusive behavior of CHIKV upon binding to the cell surface not only confirms that the PtdSer binding site promotes virus binding but also influences its diffusive behavior.…”

Section: Discussionmentioning

confidence: 99%

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Kirui

Abidine

Lenman

et al. 2021

Cells

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Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

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“…Cell culture supernatant was collected daily from 5 to 11 days posttransfection (dpt). To confirm the titer and viral particle production efficiency, Vero cells ( 40 , 41 ) were transduced with produced ZVAX particles at a 10-fold dilution. Flow cytometry analysis tracking expression of GFP in the cells at 48 h postransduction revealed that optimal production of vaccine particles occurred between days 7 and 9 posttransfection of plasmids in HEK 293T cells ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The probes for ZIKV and mouse GAPDH were labeled by 6-carboxyfluorescein (FAM) and VIC, respectively, which enables quantitation of both genes from the same RNA samples. The sequences of the primers and probe for reverse transcription and droplet PCR were previously published ( 40 ). The RNA samples were diluted 400-fold with water.…”

Section: Methodsmentioning

confidence: 99%