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. 2022 Apr 6;10(3):e02212-21. doi: 10.1128/spectrum.02212-21

FIG 1.

FIG 1

Ectopic expression of TIM-1 enhances Ebola virus cell entry. (A) Surface TIM-1 expression at 36 h following transfection. 293T cells were transfected with 2 μg of empty plasmid (solid gray histogram) or increasing amounts of TIM-1 (black dotted line, 0.5 μg; black dashed line, 1 μg; black solid line, 2 μg). (B) Cell entry of the transfected cells by pSG3.Δenv.cmvFluc (left panel) and VSV-ΔG* (right panel) bearing EBOV, BDBV, SUDV, and VSV GPs. Relative luciferase intensity was detected at 48 h following infection. Data are the fold increase of viral entry into TIM-1-expressing cells compared with cells transfected with empty vector. VSV-pseudotyped virus was used to infect 293T cells transfected with 2 μg TIM-1 and empty vector. (C) Polymorphic TIM-1 expression at 36 h following transfection with 2 μg TIM-1-359aa and TIM-1-364aa. (D) Cell entry of transfected cells by pSG3.Δenv.cmvFluc bearing EBOV, BDBV, SUDV, and VSV GPs (left panel) and cell entry of transfected cells by pSG3.Δenv.cmvFluc bearing EBOV GP by the Western blot method, with GP detected by monoclonal antibody mab114 (right panel). (E) TIM-1 enhances EBOV attachment, internalization, and infection. 293T cells were transfected with an empty vector or plasmids encoding polymorphic TIM-1 for 36 h. EBOV pseudovirus was incubated with cells at 4°C for 30 min and washed with PBS three times (attachment, left panel). After being washed with PBS to remove unbound EBOV particles, cells were transferred to 37°C for 2 h to allow virus internalization and treated with proteinase K to remove uninternalized virions (internalization, middle panel), and then cells were directly challenged with EBOV for 48 h (infection, right panel). Total RNA was extracted and used for quantification of EBOV GP by qRT-PCR. (F) Function comparison of TIM-1 and TIM-1 bearing mutations in the PS binding pocket (TIM-1-N114A D115A) in EBOV cell entry. (G) Function of annexin V to compete for cellular entry of pSG3.Δenv.cmvFluc bearing EBOV and VSV GPs. **, P < 0.01; ***, P < 0.001.