dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35

Abstract

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions.

Fig. 1

rVP35 shifts the electrophoretic migration of heterologous IVT dsRNAs. (A) Coomassie blue-stained 12% SDS–PAGE showing IMAC-purified, full length ZEBOV rVP35. Lane 1, molecular weight marker; lane 2, E. coli crude extract cell lysate; lane 3, IMAC unbound flow through proteins; lane 4, unspecific washed proteins; lane 5, eluted rVP35. (B) 1.7% non-denaturing agarose EMSA shows that rVP35 is able to bind dsRNA of 500 bp in length, retarding its migration on a gel. Lane 1, molecular weight marker; lane 2, rVP35 alone; lane 3, IVT 500 bp dsRNA; lane 4, IVT 500 bp dsRNA + rVP35; lane 5, IVT 500 bp dsRNA + tRNA + rVP35; lane 6, rVP35 alone (heat-denatured); lane 7, IVT 500 bp + rVP35 (heat-denatured).

Fig. 2

Characterization of optimal binding conditions in the magnetic pull down assay. The formation of a stable complex between VP35 and IVT dsRNA is dependent to pH, ionic strength and concentration of the divalent cation Mg²⁺. Optimal binding was observed at pH 7.5 (A), in the presence of 100 mM NaCl (B) and 20 mM MgCl₂ (C). rVP35 binding to IVT dsRNA was shown to be correlated to the amount of conjugated protein to TALON Dynabeads: a rVP35 dose–response binding curve shows the linear dependence between VP35–dsRNA binding and rVP35 concentration (D).

Fig. 3

rVP35 binds to IVT dsRNAs of different length with a very high affinity. Competition binding experiments, performed with the magnetic pull down assay at 37 °C at optimal biochemical conditions, show that: (A) rVP35 binds IVT dsRNA molecules of 50–500 bp in a length-independent manner. Unlabeled dsRNA of 500 bp (full circle), 150 bp (full square) and 50 bp (full triangle) in length are titrated to compete against a fixed amount of the 500 bp ³H–dsRNA ligand. Comparable values of the apparent equilibrium dissociation constant were observed: 2.8 ± 0.1 nM (500 bp dsRNA), 2.4 ± 0.3 nM (150 bp dsRNA) and 3.2 ± 0.5 nM (50 bp dsRNA); (B) rVP35 binding to synthetic short dsRNAs with different 5′ ends. Unlabeled 5′-phosphate dsRNA (full circle) and 5′-hydroxyl dsRNA (empty circle) of 8 bp in length are titrated to compete against a fixed amount of the 500 bp ³H–dsRNA ligand. Different equilibrium dissociation constant were observed: of 64 ± 9 nM (5′-phosphate) and 1.1 ± 0.2 μM (5′-hydroxyl). Concentration of dsRNA competitor is plotted versus the bound percentage of the radiolabeled ligand, each experimental point represents the mean ± SD of specific bound dsRNA from at least three independent experiments.

Fig. 4

Effect of temperature on rVP35 binding to IVT 500 bp dsRNA. Analysis of the rVP35-binding affinity to IVT 500 bp dsRNA at different temperatures. (A) Homologous-competition binding curves performed with the magnetic pull down assay at optimal binding conditions at different temperatures (full circle, 37 °C; full triangle, 30 °C; full square, 23 °C). Concentration of IVT 500 bp unlabeled dsRNA is plotted versus the bound percentage of the same IVT 500 bp ³H–dsRNA molecule, each experimental point represents the mean ± SD of specific bound dsRNA from three independent experiments. (B) Van’t Hoff plot describing the effect of temperature change upon rVP35 binding affinity to 500 bp dsRNA. The natural log of the equilibrium dissociation constant is plotted versus the reciprocal value (in Kelvin) of the incubation temperature.

Fig. 5

Inhibition of rVP35–dsRNA interaction by ATA. Dose–response curve of the rVP35 binding to IVT 500 bp dsRNA inhibition by ATA. The reactions were performed at 37 °C at optimal biochemical conditions and titrated with compound concentrations ranging from 0.1 to 300 μg/mL. Results are shown as the percentage of specific bound dsRNA and represent the average of three independent determinations.

Fig. 6

R305A, K309A and R312A rVP35 mutants display diminished dsRNA binding on native gel shift and decreased affinity for dsRNA as compared with wt rVP35. Non-denaturing agarose EMSA shows that R305A, K309A and R312A rVP35 mutants are not able to shift the IVT dsRNA of 500 bp in length migration in the gel as wt rVP35, even though they retain a certain ability to bind IVT dsRNA. (A) Lane 1, molecular weight marker; lane 2, IVT 500 bp dsRNA alone; lane 3, wt rVP35 alone; lane 4, wt rVP35 + IVT 500 bp dsRNA; lane 5, R305A rVP35 alone; lane 6, R305A rVP35 + IVT 500 bp dsRNA; lane 7, K309A rVP35 alone; lane 8, K309A rVP35 + IVT 500 bp dsRNA. (B) Lane 1, R312A rVP35 alone; lane 2, R312A rVP35 + IVT 500 bp dsRNA; lane 3, molecular weight marker. (C) Homologous-competition binding, performed with the magnetic pull down assay at 37 °C at optimal biochemical conditions, showed that R305A rVP35 (full square, K_d = 3.85 ± 0.6 nM), K309A rVP35 (empty square, K_d = 4.99 ± 1.03 nM) and R312A rVP35 (empty circle, K_d = 10.6 ± 1.7 nM) bind to 500 bp IVT dsRNA with lower affinity with respect to wt rVP35 (full circle, K_d = 2.8 ± 0.1 nM). (D) Heterologous competition binding experiment, performed with the magnetic pull down assay at 37 °C at optimal biochemical conditions, showed that R312A rVP35 (full square, K_d value of 1.76 ± 0.2 μM) binds to 8 bp 5′-phosphate dsRNA with significantly lower affinity with respect to wt rVP35. Concentration of unlabeled competitor dsRNA is plotted versus the bound percentage of the radiolabeled dsRNA ligand, each experimental point is the mean ± SD of specific bound dsRNA from three independent experiments.