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. 2023 Apr 18;6(7):e202302005. doi: 10.26508/lsa.202302005

Figure 4. Mapping the LRP1-promoted infection steps.

Figure 4.

(A) Influence of cellular cholesterol levels. LRP1 siRNA-transfected A549 cells and their controls (see Fig 3A) were pretreated for 1 h with methyl-β-cyclodextrin to deplete cholesterol, or enriched with additional cholesterol (CH), before synchronized infection with RVFV MP-12 at an MOI of 1 for 5 h. (B) Role of endocytosis. Cells were pretreated for 1 h with bafilomycin A1 to block endosomal acidification, or incubated for 3 min with an acidic medium (pH 5.0) to force the fusion of viral particles at the cell surface (bypass). (C) RVFV ZH548 RNA levels in LRP1 knockout cells over the course of infection. HuH-7 LRP1 knockout cells and HuH-7 NTC (no template control) cells were infected in a synchronized manner at an MOI of 1, washed three times, and further incubated in a medium. Samples were collected after the three washes post-infection (attachment step), or at 2, 5, or 24 h post-infection. Two-step RT–qPCR was done to detect viral RNA and the GAPDH reference gene. The RNA levels in the infected NTC cells were set to 100%. Statistics were done on three independent experiments, using a paired one-tailed t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and n.s., non-significant.