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Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus.

ABSTRACT: Human metapneumovirus (HMPV) of the family Paramyxoviridae is a major cause of respiratory illness worldwide. Phosphoproteins (P) from Paramyxoviridae are essential co-factors of the viral RNA polymerase that form tetramers and possess long intrinsically disordered regions (IDRs). We located the central region of HMPV P (P(ced)) which is involved in tetramerization using disorder analysis and modeled its 3D structure ab initio using Rosetta fold-and-dock. We characterized the solution-structure of P(ced) using small angle X-ray scattering (SAXS) and carried out direct fitting to the scattering data to filter out incorrect models. Molecular dynamics simulations (MDS) and ensemble optimization were employed to select correct models and capture the dynamic character of P(ced). Our analysis revealed that oligomerization involves a compact central core located between residues 169-194 (P(core)), that is surrounded by flexible regions with ?-helical propensity. We crystallized this fragment and solved its structure at 3.1 Å resolution by molecular replacement, using the folded core from our SAXS-validated ab initio model. The RMSD between modeled and experimental tetramers is as low as 0.9 Å, demonstrating the accuracy of the approach. A comparison of the structure of HMPV P to existing mononegavirales P(ced) structures suggests that P(ced) evolved under weak selective pressure. Finally, we discuss the advantages of using SAXS in combination with ab initio modeling and MDS to solve the structure of small, homo-oligomeric protein complexes.

SUBMITTER: Leyrat C

PROVIDER: S-EPMC3817118 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus.

Leyrat Cedric C Renner Max M Harlos Karl K Grimes Jonathan M JM

PloS one 20131104 11

Human metapneumovirus (HMPV) of the family Paramyxoviridae is a major cause of respiratory illness worldwide. Phosphoproteins (P) from Paramyxoviridae are essential co-factors of the viral RNA polymerase that form tetramers and possess long intrinsically disordered regions (IDRs). We located the central region of HMPV P (P(ced)) which is involved in tetramerization using disorder analysis and modeled its 3D structure ab initio using Rosetta fold-and-dock. We characterized the solution-structure ...[more]

PMID: 24224051

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Project description:Human metapneumovirus (HMPV) inclusion bodies (IBs) are dynamic structures required for efficient viral replication and transcription. The minimum components needed to form IB-like structures in cells are the nucleoprotein (N) and the tetrameric phosphoprotein (P). HMPV P binds to the following two versions of the N protein in infected cells: N-terminal P residues interact with monomeric N (N0) to maintain a pool of protein to encapsidate new RNA and C-terminal P residues interact with oligomeric, RNA-bound N (N-RNA). Recent work on other negative-strand viruses has suggested that IBs are, at least in part, liquid-like phase-separated membraneless organelles. Here, HMPV IBs in infected or transfected cells were shown to possess liquid organelle properties, such as fusion and fission. Recombinant versions of HMPV N and P proteins were purified to analyze the interactions required to drive phase separation in vitro. Purified HMPV P was shown to form liquid droplets in isolation. This observation is distinct from other viral systems that also form IBs. Partial removal of nucleic acid from purified P altered phase-separation dynamics, suggesting that nucleic acid interactions play a role in IB formation. HMPV P also recruits monomeric N (N0-P) and N-RNA to droplets in vitro. These findings suggest that HMPV P may also act as a scaffold protein to mediate multivalent interactions with monomeric and oligomeric N, as well as RNA, to promote phase separation of IBs. Together, these findings highlight an additional layer of regulation in HMPV replication by the viral P and N proteins. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of respiratory disease among children, immunocompromised individuals, and the elderly. Currently, no vaccines or antivirals are available for the treatment of HMPV infections. Cytoplasmic inclusion bodies (IBs), where HMPV replication and transcription occur, represent a promising target for the development of novel antivirals. The HMPV nucleoprotein (N) and phosphoprotein (P) are the minimal components needed for IB formation in eukaryotic cells. However, interactions that regulate the formation of these dynamic structures are poorly understood. Here, we showed that HMPV IBs possess the properties of liquid organelles and that purified HMPV P phase separates independently in vitro. Our work suggests that HMPV P phase-separation dynamics are altered by nucleic acid. We provide strong evidence that, unlike results reported from other viral systems, HMPV P alone can serve as a scaffold for multivalent interactions with monomeric (N0) and oligomeric (N-RNA) HMPV N for IB formation.

Project description:Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (NNuc), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the NNuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and NNuc, which has not been demonstrated yet. Here, we finely characterized the binding P-NNuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc and that P binds to the N-terminal domain of N (NNTD), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-NNuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

Dataset Information

Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus.

Publications

Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus.

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