Fig. 7: PIP5K1C and PIKfyve are key regulators of SARS-CoV-2 spike protein-mediated viral entry.

a Pseudotyped virus entry assay in the presence of the indicated concentrations of UNI418. Expression plasmids for VSV-G and the SARS-CoV-2 spike protein from WuHan-Hu-1, the Delta variant (B.1.617.2) and the Omicron variant (BA.1) were used to produce pseudotyped virus particles with a luciferase reporter. The 293T-ACE2 cells were infected with each pseudotyped virus under the indicated concentrations of UNI418. The relative viral entry efficiency was determined by the luciferase activity 24 h postinfection and is presented as the mean ± SEM, n = 3. Statistical significance was determined by two-way ANOVA, with ***p < 0.001. b, c Entry of Wuhan-Hu-1 spike-pseudotyped virus was assessed after PIKfyve and PIP5K1C knockdown in 293T-ACE2 cells. HEK293T-ACE2 cells were transfected with the indicated siRNAs and subjected to a luciferase-based virus entry assay (top panel) and Western blot analysis (bottom panel). The data are presented as the means ± SEMs, n = 3. Statistical significance was determined by Student’s t-test, with *p < 0.05 and **p < 0.01. d Entry of Wuhan-Hu-1 spike-pseudotyped virus was measured by luciferase activity after treatment with apilimod or UNC3230 and their cotreatment in 293T-ACE2 cells. The data are presented as the means ± S.D.s, n = 3. Statistical significance was determined by Student’s t test, with *p < 0.05, **p < 0.01 and ***p < 0.001. e, f Synergistic effect of UNC3230 and apilimod against SARS-CoV-2 infection. Vero cells infected with SARS-CoV-2 at an MOI of 0.01 were treated with 3-fold serial dilutions of different ratios of UNC3230 and apilimod: 5:0 (300 µM UNC3230 only), 4:1 (240 µM UNC3230 and 60 nM apilimod), 3:2 (180 µM UNC3230 and 120 nM apilimod), 2:3 (120 µM UNC3230 and 180 nM apilimod), 1:4 (60 µM UNC3230 and 240 nM apilimod), and 0:5 (300 nM apilimod only). e On Day 1 after infection, the antiviral efficacy of UNC3230 (red) and apilimod (blue) was individually determined in each combination. f Isobologram graph showing the sums of fractional EC₅₀ values (ΣFEC₅₀) for each combination. The mean value from the fixed dose ratios of 4:1, 3:2, 2:3 and 1:4 was 0.68. g The graphical model shows how UNI418 inhibits SARS-CoV-2 entry into cells by targeting PIP5K1C and PIKfyve.