Figure 1.
A forward genetic screen in human haploid cells identifies MBTPS1, MBTPS2, SCAP, and SREBF2 as required for ANDV infection.
(A) pLentiET gene-trap vector schematic. pLentiET is diagramed 3′ to 5′, self-inactivating (SIN) 3′LTR, splice acceptor site (SA), internal ribosome entry site (IRES), eGFP, polyadenylation signal (pA), tk promoter (tk), G418 resistance gene (Neo), splice donor site (SD), mRNA destabilization element, 5′ LTR. Upon a successful gene-trapping event, eGFP is spiced into the coding transcript downstream of the native promoter. A second transcript encoding G418 is simultaneously produced. (B) Genetic screen strategy. 75×106 human haploid (HAP1) cells were mutagenized using a gene-trap vector (pLentiET) delivered through transduction. Cells were expanded for several passages and infected with either rVSV-ANDV or rVSV-G virus to select for cells resistant to infection. Resistant cells were used for gene-trap integration site analysis (total chromosomal DNA from all resistant cells), integrations aligned to the human genome, and integration frequency ranked. (C) MBTPS1 (Site 1 Protease, S1P), MBTPS2 (Site 2 Protease, S2P), SCAP (Sterol Regulatory Element Binding Protein Cleavage Activating Protein), and SREBF2 (Sterol Regulatory Element Binding Protein 2, SREBP-2) were found to have the highest total number of independent integration events. Genes are depicted 5′ to 3′ with horizontal bars denoting exons. Sequenced integration sites are demarked based on the orientation of the gene-trap vector relative to the human genome (red dots: sense strand, blue dots: antisense stand). Significance values were calculated using the 1-sided Fisher's Exact test based on the unselected control library are indicated above each gene.
Table 1.
List of candidate genes identified during siRNA screen.
Figure 2.
The sterol regulatory complex promotes infection of ANDV.
(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.
Figure 3.
PF-429242 and mevastatin prevent efficient infection by rVSV-ANDV.
(A) A549 cells were pretreated with the indicated concentrations of PF-429242 24 hours prior to infection with VSV-(G) (solid line), rVSV-ANDV (dashed line) or VSV-(HTNV) (dotted line). Cells were harvested 16 hours later and viral infection levels were quantified via flow cytometry. Values are normalized to untreated infection levels for each virus. Mean±SEM shown for three independent experiments; p<0.001 for 20 µM drug concentration for both rVSV-ANDV and VSV-(HTNV) compared to untreated control. (Average raw infection percentage (untreated): VSV-(G) = 45%, rVSV-ANDV = 35%, VSV-(HTNV) = 14%). (B) Treatment of A549 cells with the HMG-CoA Reductase inhibitor, mevastatin, blocks infections with Sindbis (SINV) and ANDV pseudoviruses. VSV-derived pseudoviruses (VSV-(G) and VSV-(SINV)), rVSV-ANDV or vaccinia virus were used to infect cells pretreated with the indicated concentrations of mevastatin. Infections were quantified using flow cytometry either by fluorescent protein expression or indirect antibody staining (rVSV-ANDV). Infectivity is normalized relative to untreated control cells. Mean±SEM shown for three independent experiments; p<0.001 for 10 µM drug concentration for rVSV-ANDV compared to untreated control. (Average raw infection percentage (untreated): VSV-(G) = 33%, VSV-(SINV) = 16%, rVSV-ANDV = 52%, vaccinia = 30%). (C) Viral infectivity in the presence of 5 µM mevastatin can be rescued with the addition of normal FBS and mevalonate. A549 cells were treated as described above with DMSO or 5 µM mevastatin with delipidated FBS or normal FBS (not subjected to delipidation) with 25 µM mevalonate. Cells were infected with pseudoviruses (VSV-(G)or VSV-(SINV)) or rVSV-ANDV encoding RFP and harvested 10 h.p.i. Infections were quantified using flow cytometry. Infectivity has been normalized relative to untreated control cells. Mean±SEM is shown for three independent experiments; ** p<0.01, ***p<0.001. (Average raw infection percentage (FBS+DMSO): VSV-(G) = 49%, VSV-(SINV) = 14%, rVSV-ANDV = 45%).
Figure 4.
Cholesterol is required for ANDV infection.
Infection of cells pretreated with (A) PF-429242 or (B and C) mevastatin blocks infection with wild-type ANDV (strain 9717869). Vero E6 cells were pretreated with DMSO, PF-429242, or mevastatin at the indicated concentrations and 24 hours later, cells were infected with virus at an MOI of 3 for 2 hours and subsequently overlaid with fresh media. All infections and incubations were carried out in the presence of indicated concentrations of PF-429242 or mevastatin. 72 h.p.i (ANDV) or 16 h.p.i. (VSV-(G)) cells were harvested for flow cytometry, and the percentage of infected cells was determined by expression of a fluorescent reporter gene (VSV) or indirect fluorescent staining of ANDV N (ANDV). Values are normalized to infection rate of DMSO treated cells. Mean±SEM shown for three independent experiments; ** p<0.01, ***p<0.001. (Average raw infection percentage of PF-429242 experiments (No Drug): VSV-(G) = 53%, ANDV = 9% and mevastatin experiments (No Drug): ANDV = 16%).
Figure 5.
Quantitative PCR analysis of viral RNA during infection of human cells.
(A) HAP1WT or HAP1S1P cells were infected with VSV-(G) or rVSV-ANDV for 12 hours and cells collected for flow cytometric analysis. Infection was normalized to infection levels in HAP1WT cells. Mean±SEM is shown for five independent experiments; ** p<0.01. (Average raw infection percentages (HAP1 WT): VSV-(G) = 55%, rVSV-ANDV = 71%) (B) Binding and internalization of rVSV-ANDV. rVSV-ANDV was bound to three sets of HAP1WT and HAP1S1P cells for one hour on ice. One set of cells were scraped into PBS and washed to measure bound virions (bound). A second set of cells were treated with trypsin for 10 minutes to remove externally bound virions (background). A third set of cells were warmed to 37°C for one hour to permit endocytosis before being treated with trypsin to remove any remaining external virions (internal). Cells were washed extensively, cell pellets and associated virions were lysed for RNA extraction and viral RNA (vRNA) was quantified by qRT-PCR. Viral RNA values were normalized to GAPDH to control for input RNA levels and plotted relative to virus bound to HAP1WT cells. Mean±SEM is shown for three independent experiments; ** p<0.01, ***p<0.001.
Figure 6.
Detection of viral particles during cellular entry.
Purified and DiO-labeled rVSV-ANDV (left) or VSV-(G) (right) where bound to Vero E6 cells at 4°C for 90 minutes pretreated for 24 hours with DMSO (A) or 40 µM PF-429242 (B). Cells were subsequently washed with cold PBS and fixed immediately (‘0 min’), or following a 20 minute incubation at 37°C, with paraformaldehyde. Cellular membranes where counterstained Wheat Germ Agglutinan-647 and imaged using confocal microscopy. Internalized virus is indicated with an arrowhead. Representative images from 2 independent experiments are shown.