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. 2014 Feb 6;10(2):e1003911. doi: 10.1371/journal.ppat.1003911

Figure 2. The sterol regulatory complex promotes infection of ANDV.

Figure 2

(A) Infection of Chinese Hamster Ovary (CHO) cells null for S1P, S2P, or SCAP (Figure S2). CHO cells were infected with VSV-(G), rVSV-ANDV or rVSV-HTNV for 10 hours. Infections were quantified via flow cytometry and normalized relative to wild-type (CHO-K1) cells. Mean±SEM shown for three independent experiments; *** p<0.001. (Raw infection percentage (CHO-K1): VSV-(G) = 21%, rVSV-ANDV = 15%, VSV-(HTNV) = 8%) (B) siRNAs directed against SREBF2 efficiently knock down protein expression as measured by immunoblot to SREBP-2 with GAPDH as a loading control. (C) HEK293T cells depleted of SREBF2 were infected with non-replicating VSV pseudotypes bearing the indicated glycoproteins encoding red fluorescent protein (RFP) and infection was quantified 10 h.p.i.. Infectivity was normalized relative to control cells. Mean±SEM shown for three independent experiments; * p<0.05. (Raw infection percentage (control): VSV-(G) = 4%, VSV-(ANDV) = 19%, VSV-(HTNV) = 3%) (D) TALENs were used to genetically disrupt SCAP in HEK293T cells. These insertions were enriched upon challenge with rVSV-ANDV and were all killed by VSV-G. Enrichment was measured by a non-homologous end joining (NHEJ) endonuclease assay where a single band (top asterisk) indicates the presence of wild-type sequence and the lower molecular weight doublet (lower 2 asterisks) represents the disrupted alleles. Densitometry was used to quantify the levels of the undisrupted and disrupted alleles, which is shown below.