The multifunctional VP35 protein is a major virulence factor by which the highly pathogenic Ebolaviruses (EBOVs) evade host innate immune response. VP35 is essential for EBOVs replication as a component of the viral RNA polymerase complex, it is a key participant to the nucleocapsid assembly, and it also binds to dsRNA antagonizing RIG-I like receptors antiviral signaling and, ultimately, inhibiting the host interferon (IFN) production. Insights in the VP35 dsRNA binding, ascribed to its C-terminal IFN inhibitory domain (IID), have been recently revealed through structural and functional analysis of the C-terminal truncated version of the protein. Here, we report the first biochemical characterization of the dsRNA binding of the full length, His-tagged recombinant VP35 (rVP35) of the Zaire ebolavirus species. For this purpose we established a new in vitro magnetic pull down assay, validating it for compound screening also by assessing the inhibitory ability of the auryntricarboxylic acid which showed an IC50 value of 50 μg/mL. Optimal biochemical parameters for dsRNA binding and Kd values for dsRNA with different length were obtained through competition binding studies. Furthermore, the dsRNA binding properties of the R305A, K309A and R312A rVP35 mutants, known for their defective dsRNA binding-mediated inhibition of the host IFN response in cell culture were assessed. Interestingly, results showed that, as compared to wild type rVP35, all three rVP35 mutants displayed a modified migration pattern in gel mobility shift assays and, when tested in the magnetic pull down assay, they displayed a significantly increase of the Kd values for dsRNA binding.
dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35
ZINZULA, LUCA;ESPOSITO, FRANCESCA;TRAMONTANO, ENZO
2011-01-01
Abstract
The multifunctional VP35 protein is a major virulence factor by which the highly pathogenic Ebolaviruses (EBOVs) evade host innate immune response. VP35 is essential for EBOVs replication as a component of the viral RNA polymerase complex, it is a key participant to the nucleocapsid assembly, and it also binds to dsRNA antagonizing RIG-I like receptors antiviral signaling and, ultimately, inhibiting the host interferon (IFN) production. Insights in the VP35 dsRNA binding, ascribed to its C-terminal IFN inhibitory domain (IID), have been recently revealed through structural and functional analysis of the C-terminal truncated version of the protein. Here, we report the first biochemical characterization of the dsRNA binding of the full length, His-tagged recombinant VP35 (rVP35) of the Zaire ebolavirus species. For this purpose we established a new in vitro magnetic pull down assay, validating it for compound screening also by assessing the inhibitory ability of the auryntricarboxylic acid which showed an IC50 value of 50 μg/mL. Optimal biochemical parameters for dsRNA binding and Kd values for dsRNA with different length were obtained through competition binding studies. Furthermore, the dsRNA binding properties of the R305A, K309A and R312A rVP35 mutants, known for their defective dsRNA binding-mediated inhibition of the host IFN response in cell culture were assessed. Interestingly, results showed that, as compared to wild type rVP35, all three rVP35 mutants displayed a modified migration pattern in gel mobility shift assays and, when tested in the magnetic pull down assay, they displayed a significantly increase of the Kd values for dsRNA binding.
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