Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which consists of viruses with genomes 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 those of other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and the 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 advance, 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 test sensitively 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 we 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 such as those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Although cofactor phosphorylation is present in most of these interactions, its effect, if any, on this proteinprotein 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. Since phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggests that the structural picture of L during infection might be more complex than previously appreciated.

Automated summary

VSV, a member of Mononegavirales, plays a vital role in molecular biology research with its RNA polymerase composed of L and P proteins. Phosphorylation of P-NTD modulates its interaction with L and affects the conformation of L during infection; this study is the first to detail the effects of phosphorylation on protein-protein interactions, providing important insights into mononegavirus RNA synthesis.