Figure 4. Naturally occurring TMEM41B SNPs negatively impact flavivirus infection but are able to maintain normal lipid distribution in cells.

(A) Table shows the frequency of a SNP (rs78813996) in TMEM41B in several human populations. (B) WT, TMEM41B KO, and TMEM41B KO HAP1 cells expressing WT or TMEM41B SNP variants infected with YFV 17D, MOI = 0.005 PFU/cell for 48 h. Left, cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Right, supernatants were collected and titrated by tissue culture infectious dose (TCID₅₀/ml) assay on Huh-7.5 cells. (C) Same as panel (B, left) with ZIKV at MOI = 0.25 PFU/cell for 48 h; DENV-GFP at MOI = 0.1 PFU/cell for 96 h; WNV-GFP at MOI = 1 PFU/cell for 72 h; and hPIV-3-GFP at MOI = 0.02 IU/cell for 48 h. (D) WT and TMEM41B KO Huh-7.5 cell clones stained with Nile red to visualize lipid droplets. (E) Cumulative frequency of droplets plotted vs. droplet area (μm²) for six independent single cell clones generated with two independent sgRNAs. Inset shows the mean lipid droplet area (μm²) for the six TMEM41B KO clones compared to WT Huh-7.5 cells. (F) Mean lipid droplet area (μm²) for WT, TMEM41B KO, and TMEM41B KO Huh-7.5 cells (clone 1–1) expressing the indicated TMEM41B variants. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Error bars depict SD for three replicates.