title: Nuclear magnetic resonance structure revealed that the human polyomavirus JC virus agnoprotein contains an α-helix encompassing the Leu/Ile/Phe-rich domain.
abstract: Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic α-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the α-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A+L32A+L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection.
  
  
  OBJECTIVE
  Agnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic α-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the α-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.
authors.affiliation: Université Paris Descartes, Sorbonne Paris Cité, Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Paris, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Coric
authors.affiliation: Department of Neuroscience, Laboratory of Molecular Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: A Sami
authors.initials: AS
authors.lastname: Saribas
authors.affiliation: Temple University School of Pharmacy, Moulder Center for Drug Discovery Research, Philadelphia, Pennsylvania, USA.
authors.forename: Magid
authors.initials: M
authors.lastname: Abou-Gharbia
authors.affiliation: Temple University School of Pharmacy, Moulder Center for Drug Discovery Research, Philadelphia, Pennsylvania, USA.
authors.forename: Wayne
authors.initials: W
authors.lastname: Childers
authors.affiliation: Department of Neuroscience, Laboratory of Molecular Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Martyn K
authors.initials: MK
authors.lastname: White
authors.affiliation: Université Paris Descartes, Sorbonne Paris Cité, Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Paris, France serge.bouaziz@parisdescartes.fr msafak@temple.edu.
authors.forename: Serge
authors.initials: S
authors.lastname: Bouaziz
authors.affiliation: Department of Neuroscience, Laboratory of Molecular Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA serge.bouaziz@parisdescartes.fr msafak@temple.edu.
authors.forename: Mahmut
authors.initials: M
authors.lastname: Safak
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D007577:JC Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D027601:Polyomavirus Infections / Q000821:virology*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D000086082:Viroporin Proteins
chemical_list: D054334:Viral Regulatory and Accessory Proteins; C042559:agnoprotein, polyomavirus; D000086082:Viroporin Proteins