Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System.

Vesicular stomatitis virus (VSV) is a member of the order , which consists of viruses with a genome of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (P) coordinates the C-terminal accessory domains to produce a "compacted" L conformation. Despite this advancement, the role of the three phosphorylation sites in P has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between P and the L protein C-terminal domain (L), we demonstrated our ability to sensitively test for changes in the interface between the two proteins. This method showed that the binding site for P on L is longer than was previously appreciated. We demonstrated that phosphorylation of P modulates its interaction with L and used a minigenome reporter system to validate the functional significance of the P-L interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated P displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of P modulates its cofactor and conformational regulatory activities with L. Polymerase-cofactor interactions like those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Despite cofactor phosphorylation being present in most of these interactions, what effect if any it has on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. As phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggest that the structural picture of L during infection might be more complex than previously appreciated.