oa The magnetic pull down: A biochemical assay for the screening of antiviral candidates

Type I interferon α/β (IFN-α/β) cytokines are produced by the host cell upon sensing viral proteins and nucleic acids as pathogen associated molecular patterns (PAMPs) and thereby elicit an antiviral response that represents the frontline of innate immunity against a virus infection. Among the signatures of the "non self" presence, double-stranded RNA (dsRNA) is the most potent PAMP in triggering the IFN-α/β-based antiviral response. dsRNA is synthesized during the course of infection by RNA viruses as a byproduct of replication and transcription, or it may be originated through the panhandle self-pairing occurring at viral genome ends. dsRNA is targeted by specific pathogen recognition receptors (PRRs), of which the class of cellular helicases termed as RIG-I-like receptors (RLRs) is the subset deputed to its prompt detection in the cytosol. This is the reason why avoiding dsRNA recognition by RLRs is a strategy adopted by RNA viruses to inhibit the IFN-α/β induction. Moreover, in most cases, such circumvention of the innate immune antiviral response relies on the function of virally-encoded proteins that act binding viral dsRNA to impede its interaction with RLRs. To date, proteins displaying dsRNA binding-mediated IFN-antagonism have been described for several RNA viruses that are highly lethal to humans. Among these there are emerging pathogens that recently affected Middle East and the Arabian Peninsula, such as influenza A viruses and - putatively - also the newly identified SARS-related coronavirus. Therefore, given the role of IFN-inhibiting proteins as key determinants of virulence and pathogenesis, characterization of their dsRNA binding function is of urgent need, and disruption of their interaction with viral dsRNA is an attractive and promising target for the development of effective antiviral agents. Within this picture, we have recently developed a new in vitro assay, namely the Magnetic Pull Down (MPD), as a method for measuring dsRNA binding activity of viral proteins. Relying on the properties of the paramagnetic TALON Dynabeads, an His-tagged recombinant protein can be stably coated to beads in this assay, and subsequently incubated with a labeled dsRNA substrate. Next, application of a magnetic field allows the precipitation and further isolation of a quantifiable dsRNA:protein complex. By using as a paradigm model a full length recombinant version of the Ebola virus VP35 (EBOVrVP35) protein - a potent dsRNA binding-dependent IFN-α/β suppressor - we characterized its dsRNA binding function and validated the MPD assay for antiviral screening. Results showed that EBOV rVP35 binds to 3H-radiolabeled in vitro transcribed dsRNA molecules of different length (500-50bp) with very high affinity and KD values that are within the low nanomolar range. Moreover, the formation of EBOV rVP35:dsRNA complex was inhibited in this assay by using the tester compound auryntricarboxylic acid, which showed an IC50 value of 50μg/mL. In summary, the MPD assay herein described provides a straightforward tool i) to identify viral IFN-antagonists displaying dsRNA binding activity; ii) to quantitatively characterize dsRNA binding function by measuring the dsRNA:protein complex formation and iii) to screen for antiviral agents inhibiting such interaction, either they are synthetic compounds or natural herbal extracts.