A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

Author summary Many positive-strand RNA viruses replicate without transcribing subgenomic RNAs otherwise often used to temporally coordinate the expression of proteins involved either in genome replication (early) or virion formation (late). Instead, the RNA genomes of the Flaviviridae are translated into a single polyprotein. Their nonstructural proteins (NS), while not present in the virions, are known to be crucially involved in RNA replication and virion formation. The important question how the same proteins form specific complexes required for fundamentally different aspects of the viral replication cycle is not solved yet. For pestiviruses the mature NS3/4A complex is an essential component of the viral RNA-replicase but is incapable of participating in virion morphogenesis which in turn depends on uncleaved NS2-3 in complex with NS4A. However, a gain of function mutation in NS3 enabled the NS3/4A complex to function in virion assembly. Using structure guided mutagenesis in combination with functional studies we identified the interface between NS3 and the C-terminal NS4A region as a module critical for the decision whether a NS3/4A complex serves in RNA replication or as a packaging component. Thus, we propose that subtle changes in local protein interactions represent decisive switches in viral complex formation pathways.