Fig 2: siRNA profiling of selected genes required for recombinant ANDV pseudovirion infection.Gene-specific siRNA against integrin ß3 (ITB3), DNM2, CLTC, AP2M1, CDC42, or ARF6, or the non-targeting siRNA transfection control (NT), were transfected into HMVEC-L at the concentration of 100 nM for 48 h. Cells were infected with pseudovirions (MOI = 1) for 48 h. All pseudovirions consisted of an HIV backbone with firefly luciferase, and were pseudotyped with ANDV GPC (ANDV) or vesicular stomatitis virus G protein (VSV). Effects of gene knockdown were measured by quantifying luciferase activity in the host genome. Solid bars represent ANDV, and open bars represent VSV. Luciferase activity is expressed as units relative to non-targeting siRNA control (% infection). The effect of ITB3-specific siRNA knockdown is shown in the inset. Results presented are the averages (± SD) of 10 experiments (n = 10), and significant decreases in pseudovirion infection (p < 0.05) after siRNA knockdown are shown with an asterisk (*).

Fig 3: N-WASP is a key plaque regulator.(a) Characterization of control (CTR) knockdown (KD) and N-WASP KD HeLa cells. Total cell lysates were compared using the indicated antibodies. One of three experiments that were performed with similar results is shown. (b) N-WASP regulates the association between CCSs and F-actin. Representative two-colour SR images of control (CTR) KD and N-WASP KD cells stained for CHC (green in merge) and F-actin (red in merge). Dashed white boxes mark the position of the zoom-in. Scale bar, 1 µm. (c) Knockdown of N-WASP increases plaque presence on the basal membrane. Representative TIRF and SR images of CHC on the basal membrane of control (CTR) KD and N-WASP KD cells. Scale bar TIRF, 10 µm. Scale bar SR, 1 µm. (d) Bar graphs show the number of pits per µm2 and percentage of total area of the ROI covered by plaques (plaque covered area). ROIs were defined as three µm-wide regions on the periphery of the cells and segmented for quantification (mean±s.e.m., **P<0.01, t-test, ns=not significant). (e) Representative TIRF and SR images of CHC in N-WASP KD cells transfected with full-length shRNA-resistant N-WASP tagged with GFP (N-WASP-GFP). Scale bar TIRF, 10 µm; SR, 1 µm. (f) Number of pits and plaque-covered area were obtained as above (mean±s.e.m., ****P<0.0001, t-test, ns, not significant).

Fig 4: PICALM associates with LRP1 during Aß transcytosis across endothelial monolayera, PICALM (red), ZO–1 (green) and F–actin (magenta) in a monolayer. b, Colocalization of PICALM (red) with LRP1 (green) and FAM–Aß40 (magenta) 1 min after LRP1–Aß internalization at the basolateral membrane. c–d, Proximity ligation assay (PLA) depicting PICALM–LRP1 association (c, bar=10 µm) and kinetics of association between PICALM and LRP1 and clathrin and LRP1 in endothelium after Aß40 (1 nM) addition to the basolateral membrane studied by PLA (d). e, Internalization of Aß40 (1 nM) at the basolateral membrane of the monolayer transfected with si.LRP1, si.PICALM and si.CHC compared si.Scramble or untransfected control. f–g, Basolateral–to–apical transcytosis of Aß40 (1 nM) across endothelial monolayer transfected with si.PICALM and si.CHC compared to si.Scramble (100%) over 60 min (f) and quantification of unidirectional trans–endothelial Aß40 transport within 30 min (g). Mean ± s.e.m. from 3 primary isolates in triplicates. p<0.05 by ANOVA followed by Tukey’s posthoc test.

Fig 5: Knockdown of selected genes required for ANDV infection.Western blots of indicated proteins after knockdown with gene-specific siRNA against (A) dynamin 2 (DNM2); (B) clathrin heavy chain (CLTC); (C) AP2M1; (D) caveolin 1 (CAV1); (E) CDC42; (F) ARF6; (G) NSF; (H) RAB5C; or (I) TSG101; or non-targeting siRNA transfection control (NT). siRNAs were transfected into HMVEC-L at the concentration of 100 nM for 48 h (see Table 1 for more information regarding these genes). The cells were then infected with ANDV (MOI = 0.5) for 24 h or 48 h. Western blots were performed post infection to ensure knockdown of the specific protein expression. Molecular weight of each specific protein is indicated on the right side of each panel. The blots were also probed with ß-actin specific antibody as the gel-loading control. (A) and (B) show only the results collected at 48 h.