Viral evolution identifies a regulatory interface between paramyxovirus polymerase complex and nucleocapsid that controls replication dynamics

Paramyxoviruses are negative-polarity RNA viruses of major clinical importance. The dynamic interaction of the RNA-dependent RNA polymerase (RdRP) complex with the encapsidated RNA genome is mechanistically and structurally poorly understood. Having generated recombinant measles (MeV) and canine distemper (CDV) viruses with truncated nucleocapsid (N) protein showing defects in replication kinetics, we have applied a viral evolution approach to the problem. Passaging of recombinants resulted in long-range compensatory mutations that restored RdRP bioactivity in minigenome assays and efficient replication of engineered viruses. Compensatory mutations clustered at an electronically compatible acidic loop in N-core and a basic face of the phosphoprotein X domain (P-XD). Co-affinity precipitations, biolayer interferometry, and molecular docking revealed an electrostatic-driven transiently forming interface between these domains. The compensatory mutations reduced electrostatic compatibility of these microdomains and lowered coprecipitation efficiency, consistent with a molecular checkpoint function that regulates paramyxovirus polymerase mobility through modulation of conformational stability of the P-XD assembly.