Fig 1.
Identification of bacterial effectors that promote arbovirus replication in bat and human cells. A-B.
Fold-change in viral GFP signals (normalized to CellTracker Blue signal) relative to cells transduced with lentivirus expressing firefly luciferase (LUC) (negative control) for RRV-GFP (A) and VSVM51R-GFP (B) screens 20 hpi. The cutoff for effectors to be scored as hits was a >10-fold change in over negative controls (cutoffs represented by dotted lines). Data points are means. C-E. Representative immunoblots of Flag-tagged bacterial effector expression in bat R06E cells 48 h post-transfection with pcDNA3.1 vectors. Additionally, fold-change in normalized viral GFP signal relative to cells transfected with empty vector (EV) is shown. Cells were stained 20 hpi with CellTracker Orange dye and imaged to calculate fold-change in normalized GFP signal over signals in EV treatments. Wild-type (WT) effectors are compared to their mutants (SopBC420S, SidMA435E, IpaH4C339S). F-H. Representative immunoblots and fold-change in normalized viral GFP signal for effectors experiments conducted as in C-E but in human U2OS cells. Quantitative data in C-H are means ± SEM; n=3. Statistical significance in graphs in C-H was determined with One-way ANOVA and Dunnett’s post-test; ns (not significant), *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.
Fig 2.
The central domain of the conserved host protein, RNF214, is sufficient for IpaH4-mediated ubiquitination and degradation.
A. Schematic of human RNF214 with mapping of prey clones collectively identified in two yeast two-hybrid screens using IpaH4 bait [5]. Numbers indicate RNF214 a.a. encoded by each prey clone. B. Schematic displaying the relative conservation and length of RNF214 across indicated organisms. C. PhyloTv2-generated phylogenetic tree of RNF214 orthologs. D. Table of RNF214 orthologs in various vertebrate and invertebrate species generated via reciprocal BLAST analysis. E. Representative immunoblot of degradation assay using Flag-tagged RNF214 co-transfected into U2OS cells with GFP-IpaH4 (WT) or GFP-IpaH4C339S (C339S) catalytic mutant expression vectors. F-G. Representative immunoblot of degradation assays using Flag-tagged RNF214 truncations, RNF2141-275 (F) or RNF214276-504 (G), co-transfected into U2OS cells with GFP-IpaH4 (WT) or GFP-IpaH4C339S (C339S) vectors. H. Representative immunoblot of recombinant human Flag-RNF214 following GST pull-down after incubating with either GST, GST-IpaH4, or GST-IpaH4C339S. I-J. Representative immunoblot of in vitro ubiquitination assays showing direct IpaH4-mediated ubiquitination of purified, recombinant human Flag-RNF214 (I) and Flag-RNF214276-504 (J) proteins.
Fig 3.
Overexpression of RNF214 suppresses arbovirus replication in bat and human cells
. A. Cartoon protein maps and a.a. alignment of human RNF214 with other selected human RNF proteins. Residues in the characteristic RING domain “C4-H-C3” motif are highlighted in red, and the C658 residue targeted for substitution mutation is boxed. AAA+ ATPase domain in RNF213 is indicated [38]. B. a.a. alignment of human RNF214 with R. aegyptiacus RNF214. Residues are highlighted as described in A. C. Representative immunoblot of Flag-tagged RNF214 and RNF214C658S (C/S) overexpression in R06E cells for 48 h. D. Percent of GFP signal relative to empty vector (EV) control after R06E cells overexpressing RNF214 constructs (WT or RNF214C658S) were infected with RRV-GFP (MOI=0.05) or VSVM51R-GFP (MOI=0.005) for 20 h. E. Representative immunoblot of Flag-tagged RNF214 constructs overexpressed in U2OS cells for 48 h. F. Percent of GFP signal relative to empty vector (EV) control in R06E cells overexpressing Flag-RNF214 constructs (WT or RNF214C658S) and infected with RRV-GFP (MOI=0.05) or VSVM51R-GFP (MOI=0.005) for 20 h. Data are means ± SEM; n=3. Statistical significance was determined with One-way ANOVA and Dunnett’s post-test; ns (not significant), *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.
Fig 4.
Depletion of RNF214 enhances arbovirus replication in bat and human cells.
A. Representative fluorescence microscopy images (GFP channel) of R06E cells transfected with firefly luciferase (Luc)- or RNF214-targeting siRNAs. 48 h post-knockdown, cells were infected with RRV-GFP (MOI=0.001) for 20 h. Cells were then stained with CellTracker Orange, imaged, and fold-change in GFP signal compared to the Luc control was calculated. Supernatants were also collected to assess viral titers by plaque assay. B-C. Similar experiments were performed as in A but for VSVM51R-GFP (MOI=0.0001) (B) and ONNV-GFP (MOI=0.001) (C). D. Representative immunoblot of endogenous bat RNF214 levels following siRNA knockdown in R06E cells for 48 h. E. Representative immunoblot of endogenous human RNF214 levels in U2OS cells transduced with control lentivirus (C) or lentivirus expressing guide RNAs targeting RNF214 (KO). F. Representative fluorescence microscopy images (GFP channel) in control or U2OSΔRNF214 cells infected with RRV-GFP (MOI=0.001) for 20 h. Cells were stained with CellTracker Orange, imaged, and fold-change in GFP signal compared to control U2OS cell infections was calculated. Supernatants were also collected to assess viral titers by plaque assay. G-I. Similar experiments were performed as in F but for VSVM51R-GFP (MOI=0.0001) (G), ONNV-GFP (MOI=0.001) (H) and SINV-GFP (MOI=0.001) (I). Data are means ± SEM; n=3. Statistical significance for A-C was determined with One-way ANOVA and Dunnett’s post-test, and with unpaired Student’s t-test for F-I; ns (not significant), *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. Scalebars indicate 200 μm on all microscopy images.