Fig. 4

PVRL4 inhibits the entry of VSV and IAV. (A-C) WT or PVRL4^−/−HEK293T cells and HEK293T cells overexpressing PVRL4 or vector were infected with IAV (MOI = 5) for 3 h and processed for immunofluorescence staining (A) and western blotting assay (B and C) with anti-NS1 antibody. (D and E) PVRL4 or control vector-transfected HEK293T cells and WT or PVRL4^−/− HEK293T cells were infected with IAV (MOI = 5) for 1 h at 4℃ and then the cells were harvested to assess the viral NP vRNA copy number through qRT-PCR assay. (F and G) PVRL4 or control vector-transfected HEK293T cells and WT or PVRL4^−/− HEK293T cells were infected with IAV (MOI = 5) for 1 h at 4℃ and then incubated at 37℃ for 10 min. After that, the cells were washed with acidic-PBS and the internalized viral particles were analyzed by qRT-PCR. (H-K) For the VSV binding experiment, PVRL4 or control vector-transfected HEK293T cells, and WT or PVRL4^−/− HEK293T cells were infected with VSV (MOI = 5) at 4℃ for 1 h. Cell-surface binding was assessed by determning the viral copy number in the cell lysates through qRT-PCR assay (H and I). For the VSV entry experiment, after infected at 4℃ for 1 h, the cells were incubated at 37℃ for another 30 min to internalize bound virion before an low pH-PBS wash. The internalized virions were determined by qRT-PCR assay (J and K). (L and M) HEK293T cells transfected with PVRL4 or control vector and WT or PVRL4^−/− HEK293T cells were infected with VSV pseudovirus for 24 h. The cell lysates were collected and measured for luciferase activity. Then the results was normalized to the control cells. Mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test