Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System

Abstract

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, 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_NTD) coordinates the C-terminal accessory domains to produce a "compacted" L conformation. Despite this advancement, the role of the three phosphorylation sites in P_NTD has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between P_NTD and the L protein C-terminal domain (L_CTD), 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_NTD on L_CTD is longer than was previously appreciated. We demonstrated that phosphorylation of P_NTD modulates its interaction with L_CTD and used a minigenome reporter system to validate the functional significance of the P_NTD-L_CTD interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated P_NTD displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of P_NTD modulates its cofactor and conformational regulatory activities with L.IMPORTANCE 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.