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. 2021 Jun 18;4(8):e202000910. doi: 10.26508/lsa.202000910

Figure 1. RNAi screen targeting membrane-trafficking proteins reveals 42 EV egress factor candidates.

Figure 1.

(A, B, C) A siRNA screen targeting 224 cellular membrane-trafficking factors was used to identify host proteins required for EV related viral egress. siRNA-transfected HeLa cells were infected with a VACV (E− EGFP/L-mCh) and fractions of early and late infected cells were quantified. (A) Workflow and screening strategy are shown in (A). (B) Gene knockdowns that decreased late/early infection ratio ≥25% were selected as hits. The 42 hit candidate genes are shown grouped into functional modules. n = 2. See also Table S1. (C) Representative images of endosomal sorting complexes required for transport machinery hits. Blue = DAPI, magenta = late infection, green = early infection. Scale bar 200 µm. (D) Western Blot validations of TSG101 and ALIX siRNA knockdown. Mean ± SEM, n = 3. Statistical analysis was performed using unpaired two-tailed t test (****P < 0.0001). (E, F) 24-h intracellular mature virion and extracellular enveloped virion yields from control (siSCR), TSG101, and ALIX depleted cells. Data are mean ± SEM, n = 4, normalized to control (siSCR). Statistical analysis was performed using unpaired two-tailed t tests (*P < 0.05; **P < 0.01).