Identification of host factors involved in borna disease virus cell entry through a small interfering RNA functional genetic screen

Abstract

Borna disease virus (BDV), the prototypic member of the Bornaviridae family, within the order Mononegavirales, is highly neurotropic and constitutes an important model system for the study of viral persistence in the central nervous system (CNS) and associated disorders. The virus surface glycoprotein (G) has been shown to direct BDV cell entry via receptor-mediated endocytosis, but the mechanisms governing cell tropism and propagation of BDV within the CNS are unknown. We developed a small interfering RNA (siRNA)-based screening to identify cellular genes and pathways that specifically contribute to BDV G-mediated cell entry. Our screen relied on silencing-mediated increased survival of cells infected with rVSVDeltaG*/BDVG, a cytolytic recombinant vesicular stomatitis virus expressing BDV G that mimics the cell tropism and entry pathway of bona fide BDV. We identified 24 cellular genes involved in BDV G-mediated cell entry. Identified genes are known to participate in a broad range of distinct cellular functions, revealing a complex process associated with BDV cell entry. The siRNA-based screening strategy we have developed should be applicable to identify cellular genes contributing to cell entry mediated by surface G proteins of other viruses.

FIG. 1.

Ol as cell substrate for siRNA-based HTS to identify cellular genes involved in BDV cell entry. (A) Ol cells exhibited high silencing efficiency. HeLa, known to be highly susceptible to siRNA silencing in the absence of significant toxicity, and Ol cells were subjected to reverse transfection with a specific siRNA to CD29, and 48 h later the cells were fixed and CD29 expression detected by IFA. Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole). (B) Ol cells transfected with control siRNAs remained highly susceptible to rVSVΔG*/BDVG-mediated CPE. Ol cells were reverse transfected with control siRNAs and subsequently infected (MOI = 5) with either rVSVΔG*/BDVG or rVSVΔG*/VSVG or else left uninfected. At 48 h p.i., cell survival was determined by using the ATPlite assay.

FIG. 2.

Flow chart of the siRNA-based HTS and subsequent confirmation and validation steps to identify cellular genes involved in BDV entry. The screen consisted of three distinct steps. In the first step, initial candidates were selected based on the results of siRNA-mediated increased cell survival upon rVSVΔG*/BDVG infection. In the second step, siRNAs selected in the first step were examined with respect to their specific effects on BDV G-mediated cell entry. For this, Ol cells were reverse transfected with siRNAs corresponding to the selected siRNA candidates and subsequently infected with either rVSVΔG*/BDVG or rVSVΔG*/VSVG. At 48 h p.i. the cell survival was determined to identify siRNAs that specifically reduced the rVSVΔG*/BDVG-mediated CPE. In the third step, candidates selected in the second step were examined for their effect on Ol cells susceptibility to infection with bona fide BDV.

FIG. 3.

Frequency distribution of screening results. A survival score was calculated for each target as described in the text. The histogram shows the distribution of binned survival scores obtained for all targets assayed in the screen. Quantile-quantile plot analysis revealed a good fit with a normal distribution (R² = 0.992, not shown). Arrows indicate the area from which hits were selected. From the initial list, we considered hits targets with two or more siRNAs inducing twofold activation or greater. The lower limit (survival score = 2.38) was determined based on TRIM11, the 57th and last ranked selected hit (see Table 3).

FIG. 4.

Identification of candidate genes involved in cell entry-mediated by BDV G. Ol cells were reverse transfected with the indicated siRNAs prespotted in triplicates onto M386 plates. After incubation for 36 h to induce gene silencing, the plates were infected with either rVSVΔG*/BDVG or rVSVΔG*/VSVG, and cell viability was determined at 48 or 24 h p.i. by using the ATPlite assay. Values obtained were normalized and corrected by readouts of cells transfected with each siRNA assayed.

FIG. 5.

Validation of candidate genes identified by the screen as host factors involved in cell entry mediated by BDVG. Ol cells were reverse transfected with the indicated siRNAs and 36 h later were infected with BDV (MOI = 2). At 48 h p.i. the cells were fixed and examined by IFA using a rabbit serum to BDV N to identify BDV-infected cells. To normalize numbers of BDV-infected cells in each sample, a value of 1 was assigned to the number of BDV N-positive cells observed in cells transfected with a nonspecific control siRNA and infected with BDV. *, P ≤ 0.05 by Student's t test.

FIG. 6.

Validation as bona fide BDV cell entry factors of cellular genes whose siRNA induced knockdown conferred increased resistance to both rVSVΔG*/BDVG and rVSVΔG*/VSVG. Ol cells were reverse transfected with the indicated siRNAs and 36 h later infected with BDV (MOI = 2). At 48 h p.i. the cells were fixed and examined by IFA using a rabbit serum to BDV N to identify BDV-infected cells. To normalize numbers of BDV-infected cells in each sample, a value of 1 was assigned to the number of BDV N-positive cells observed in cells transfected with a nonspecific control siRNA and infected with BDV. *, P ≤ 0.05 by Student's t test.

FIG. 7.

(A) Inhibition of BDV infection by additional siRNA targeting cellular genes identified by the screen as host factors involved in BDV G-mediated cell entry. Ol cells were reverse transfected with the indicated siRNAs and 36 h later infected with BDV (MOI = 2). At 48 h p.i. cells were fixed and examined by IFA using a rabbit serum to BDV N to identify BDV-infected cells. To normalize the numbers of BDV-infected cells in each sample, a value of 1 was assigned to the number of BDV N-positive cells observed in cells transfected with a nonspecific (NS) control siRNA and infected with BDV. (B) Ol cells express candidate genes identified by the screen and host cell factors involved in BDV cell entry. RNA from Ol cells was first subjected to RT and then amplified by PCR using specific oligonucleotides to detect gene expression in Ol cells. (C) Gene silencing by transfection with specific siRNAs targeting genes identified by the screen. Ol cells were transfected with siRNAs targeting LDLR, furin, DNCL2B, and nonspecific siRNA. At 36 h posttransfection, cellular RNA was purified, and mRNAs from genes targeted were detected by RT-PCR. As a control, we also detected GAPDH mRNA from every sample analyzed. RT-PCRs were performed as follows. Total cellular RNA (5 μg) was treated with DNase and then subjected to an RT reaction in a final volume of 50 μl. In each case different amounts (0.1, 0.5, 2, and 10 μl) of the RT reaction product (cDNA) were used for PCR (three cycles of 1 min at 55°C in a final volume of 50 μl), and 10 μl of each PCR product was analyzed by agarose gel electrophoresis. Amplified DNA fragments were detected by ethidium bromide staining. The amount of amplified DNA product followed a dose-dependent response with respect to the amount of cDNA used for the PCR. (C) Results of PCR assays with 2 μl of cDNA. *, P ≤ 0.05 by Student's t test.

FIG. 8.

Effect of furin inhibitor on BDV G-mediated cell entry. Ol cells were pretreated with increasing concentrations of the furin inhibitor hexa-d-arginine 1 h prior infection (MOI = 0.1) with rVSVΔG*/BDVG, bona fide BDV, or (as a control) rVSVΔG*/VSVG. (A) At 7 h p.i. cells infected with rVSVΔG*/BDVG or rVSVΔG*/VSVG were trypsin treated and fixed for green fluorescent protein detection by fluorescence-activated cell sorting analysis. (B) At 48 h p.i., BDV-infected cells were subjected to IFA to determine the numbers of cells expressing BDV N.

FIG. 9.

Effect of ADAM17 and cathepsin L inhibitors on BDV G-mediated cell entry. Ol cells were pretreated with the indicated concentrations of each inhibitor at 1 h prior infection (MOI = 0.1) with BDV or (as a control) LCMV. At 48 h p.i. BDV- and LCMV-infected cells were subjected to IFA to determine the numbers of cells expressing BDV or LCMV nucleoprotein (N) antigen. The numbers of infected cells were normalized by considering as 100% the numbers of infected cells detected in the absence of inhibitor.