Bipartite interface of the measles virus phosphoprotein X domain with the large polymerase protein regulates viral polymerase dynamics

PLoS Pathog. 2019 Aug 5;15(8):e1007995. doi: 10.1371/journal.ppat.1007995. eCollection 2019 Aug.

Abstract

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Humans
  • Measles virus / enzymology*
  • Models, Molecular
  • Models, Theoretical
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Protein Interaction Domains and Motifs*
  • RNA Replicase / chemistry
  • RNA Replicase / metabolism*
  • Sequence Homology
  • Virus Replication*

Substances

  • Phosphoproteins
  • RNA Replicase