Consequences of phosphorylation in a mononegavirales polymerase-cofactor system

Academic Article

Abstract

  • 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 ) coordinates the C-terminal accessory domains to produce a “compacted” L conformation. Despite this advance, 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 test sensitively 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 we 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. 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 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. 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. NTD NTD NTD CTD NTD CTD NTD CTD NTD CTD NTD NTD
  • Authors

    Published In

    Digital Object Identifier (doi)

    Author List

  • Gould JR; Qiu S; Shang Q; Dokland T; Ogino T; Petit CM; Green TJ
  • Volume

  • 95
  • Issue

  • 7