Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses

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Abstract
The innate immune system is a vital part of the body's defences against viral pathogens. RIG-I and MDA5 function as cytoplasmic PRRs that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualization of dsRNA and MDA5, which provides unique evidence between the relationship of viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.

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