Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors
Figure 6
RNA interference screen of candidate cellular receptors for EBOV and MBL.
(A–D) We targeted 24 candidate lectin, scavenger and other putative receptors using pLKO.1 lentiviral vectors that expressed 4 or 5 unique short hairpin RNA (shRNA) constructs per gene. We transduced HEK293F cells in quadruplicate using 4.6×108 viral particles (shRNA-expressing vectors or empty control vectors) with hexadimethrine bromide (6 µg/ml) at 37°C for 18 hours. We selected transduced cells with 5 µg/ml puromycin over 48 hours and determined cell viability with alamarBlue reagent (resazurin reduction assay). We then infected cells in quadruplicate with HIV-EBOV GP virion-like particles (1000 pg p24/100 µl) with or without rhMBL. After 48 hours we measured rates of single-round infection (luciferase assay) and adjusted results for cell viability. Percentage change in infection was normalized to the empty pLK0.1 control vector (CTRL). Shown are positive hits (A, C) which were defined as ≥66% reduction in infection by at least two shRNA constructs for any particular gene. Reductions in protein expression produced by shRNAs (western blots; B, D) relative to that produced by the empty pLK0.1 control vector are shown. Relative densitometry was performed with ImageJ (NIH) by adjusting for variations in the actin loading controls (adjusted relative densities for CLEC6A: lane 1, 0.60; lane 2, 0.56; lane 3, 0.51; lane 4, 0.35; control, 1.0. C1QBP: lane 1, 0.32; lane 2, 0.52; lane 3, 0.47; control 1.0).
