Characterization of Ebola virus VP35-dsRNA binding for drug development against Ebola virus disease.

The Ebola virus (EBOV) VP35 protein plays an important role in the inhibition of the initial innate immune responses to EBOV infection leading to Ebola virus disease development. In fact, VP35 interaction with the RIG-I like receptors (RLR) cascade components inhibits the interferon (IFN) production, impeding proper host immune response. Hence, it has been shown that VP35 is a validate drug target. Full-length His-tagged recombinant VP35 (rVP35) has been previously expressed in prokaryotic system and used to validate a biochemical pull-down assay for the screening of small molecules targeted to the VP35-double strand (ds)-RNA interactions. However, low rVP35 amount of purified protein and the use of radioactive substrate for binding evaluation, strongly limited the screening system. In the present study, a new method for high-yield rVP35 expression and purification, based on denaturation and subsequent protein refolding was established. Subsequently, a novel assay based on the use of Nickel-coated plates using a fluorescent labeled 30mer dsRNA was validated, showing a VP35 Kd value for dsRNA binding around 4 nM, comparable to the one previously reported, and a Z'-factor equal to 0.69, that indicate a good assay. The use of this biochemical assay to screen the ability of plant extracts and derived compounds to inhibit the rVP35 binding to dsRNA allowed to identified a few small molecules able to inhibit the VP35-dsRNA binding with IC50 values in the low micromolar range. Active plant extracts and derived compounds were also tested in a cellular assay to evaluate their ability to counteract the inhibitory activity of VP35 on the IFN response. We identified a number of compounds able to inhibit the VP35 function in biochemical assay but ineffective in the cellular system. Conversely, some other compound, unable to inhibit rVP35 binding to dsRNA in biochemical assay, subverted VP35 inhibition of IFN production in cellular assay. These results suggested that the VP35 binding to dsRNA may not be the driving force of the VP35 inhibition of the IFN cascade and that an alternative mechanisms of action could be hypothesized for those compounds not able to inhibit VP35-dsRNA binding but effective against the VP35 IFN inhibitory effect. An interesting compound showed activity in both biochemical and cellular assays, suggesting the possibility that some small molecules interact with VP35 in such way to disrupt its interaction with multiple targets. In order to understand the relative role of VP35-dsRNA binding in inhibiting the RLR cascade, we studied the effects of the lack of VP35 dimerization, considered essential for VP35 binding to dsRNA. For this purpose, three single point mutations, proposed to be essential for VP35 dimerization, were introduced into the coiled-coil VP35 domain. We confirmed by in silico studies that introduction of these three mutations disrupted coiled-coil dimerization. The importance of VP35 dimerization for VP35-dsRNA binding was confirmed by biochemical assay studies, where the mutant rVP35 showed a reduce ability to bind dsRNA. However, the mutant VP35 expressed in a cellular system showed a limited reduction of the ability of IFN production inhibition compared to the wild-type VP35. These results seem to confirm the hypothesis that VP35 binding to dsRNA is not the main interaction needed for VP35 inhibition of the IFN production and suggested that the VP35 interactions with cellular components do not require VP35 dimerization. In conclusion, we demonstrated that small molecules interacting with VP35 can subvert its inhibition of the IFN production, possibly inhibiting its interactions with cellular components of RLR pathway and suggested that VP35 binding to dsRNA is not the driving for VP35 inhibition RLR cascade activation.