Fig. 7

PVRL4 is an anti-SARS-CoV-2 ISG that can inhibit its spike-mediated membrane fusion and viral entry. (A) qRT-PCR analysis of PVRL4 expression in Hela-ACE2 infected with SARS-CoV-2 or uninfected for the indicated times. (B) qRT-PCR analysis of PVRL4 expression in HEK293T-ACE2 infected with SARS-CoV-2 pseudovirus for the indicated time. (C) qRT-PCR analysis of SARS-CoV-2 NP RNA levels in HEK293T-ACE2 cells transfected with PVRL4 or vector plasmids for 30 h, followed by infection for 24 h with SARS-CoV-2 (MOI = 0.25). (D) Western blotting analysis of lysates from HEK293T-ACE2 cells transfected with increasing amounts of PVRL4-HA plasmids (0, 100, 300, 500ng). (E-K) HEK293T-ACE2 cells were transfected with PVRL4 or control vector plasmids. After 24 h transfection, the cells were infected with SARS-CoV-2 pseudovirus (E) or various variants (F-K). Then luciferase activity was performed to determine the pseudovirus quantity. (L) Western blotting analysis of lysates from A549-ACE2 cells that overexpressed PVRL4 or not. (M) A549-ACE2 cells that overexpressed PVRL4 or not were infected SARS-CoV-2 pseudovirus. After 24 h infection, the luciferase activity was performed to determine the pseudovirus quantity. (N) HEK293T-ACE2 cells were co-transfected with TMPRSS2 expression plasmid and PVRL4 or vector control plasmid for 24 h. Cells were treated with 100µM chloroquine or DMSO before SARS-CoV-2 pseudovirus infection. The pseudovirus infection was quantified by luciferase activity at 24 h post-infection. (O and P) HEK293T-ACE2 cells were cotransfected with SARS-CoV-2-spike-mCherry and PVRL4 or vector for 24 h. The white arrows highlight the syncytia formation. Scale bars, 100 μm. Relative membrane fusion was determined by normalizing the number of nuclei per syncytia to the vector cells set to 100% (P). (Q and R) PVRL4 or control vector-transfected HEK293T-ACE2 cells were co-cultured with HEK293T cells expressing spike-mCherry at the ratio of 1:1, and then the syncytium formation was visualized by laser scanning confocal microscope at the 6-8 h post co-culture (Q). Scale bars,100 μm. White arrows indicated syncytia. Relative membrane fusion was determined by normalizing the number of nuclei per syncytia to the vector cells set to 100% (R). Mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test