Fig. 4: VAMP5 overexpression in tissue cells persists its effect on viral restriction. | Nature Communications

Fig. 4: VAMP5 overexpression in tissue cells persists its effect on viral restriction.

From: Vesicle-associated membrane protein 5 is an intrinsic defense factor for embryonic stem cells against coronaviruses

Fig. 4

a Representative images of the infection of SARS-CoV-2 S pseudotyped VSV-ΔG (MOI = 0.1, 24 hpi) with GFP as reporter in VAMP5 KO Caco-2-N cells with VAMP5 and CD74 overexprssion via transiently transfection system. Scale bars, 100 μm. The results are representative of three independent experiments. b Representative images of SARS-CoV-2 trVLP replication with GFP expression in VAMP5 KO Caco-2-N cells with VAMP5 overexpression via transiently transfection system (MOI = 0.1, 24 hpi). Scale bars, 50 μm. The results are representative of three independent experiments. c Representative images of western blot analysis of SARS-CoV-2 spike protein (S) expression when different concentration of SARS-CoV-2 trVLP infecting VAMP5 KO Caco-2-N cells for 24 h with VAMP5 and negative control plasmids transfection. The results are representative of three independent experiments. d TCID50 analysis of extracellular SARS-CoV-2 titers at different time points after viral infection in Caco-2-N cells with or without VAMP5 knockdown (MOI = 0.05, 24 hpi). e–i RT-qPCR analysis of viral RNA levels from SARS-CoV-2 different variants (MOI = 0.05, 24 hpi) like Alpha (e), Delta (f), Gamma (g), Kappa (h) and Omicron (i) infecting in VAMP5 KO Caco-2-N cells with VAMP5 or negative control (Ctrl) transfection. j-n RT-qPCR analysis of viral RNA levels of other coronaviruses like MERS-CoV (j) in Huh7 cells, OC43 (MOI = 1, 24 hpi) (k) and NL63 (MOI = 1, 24 hpi) (l) in Caco-2-N cells, MHV (MOI = 0.1, 24 hpi) (m) in 17CI-1 cells and PEDV (MOI = 0.1, 24 hpi) (n) in the Vero E6 cells with VAMP5 or negative control (Ctrl) transfection. o, p Luciferase activity analysis of AAV2 (MOI = 0.1, 24 hpi) (o) and VSV-G-pseudotyped HIV-1 lentivirus (LV) (MOI = 0.1, 24 hpi) (p) replication in HEK293T cells with VAMP5 or negative control (Ctrl) transfection. q–t RT-qPCR analysis of viral RNA levels from ZIKV (q), DENV2 (r), VSV (s) and IAV (t) infecting in Vero E6 or HEK293T cells with VAMP5 or negative control (Ctrl) transfection (MOI = 0.1, 24 hpi). Data are represented as mean ± SEM, n = 3 biological replicates in each group. Student’s two tailed t test was used. *p < 0.05, **p < 0.01, ***p < 0.001. MOI, multiplicity of infection; Ctrl, control; NC, negative control.

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