| Autor: |
Boggs KB; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Edmonds K; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Cifuentes-Munoz N; Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago, Chile., El Najjar F; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Ossandón C; Facultad de Ciencias del Mar y Recursos Biológicos, Universidad de Antofagasta, Antofagasta, Chile., Roe M; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Wu C; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA., Moncman CL; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Creamer TP; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA., Amarasinghe GK; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA., Leung DW; Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA., Dutch RE; Department of Molecular and Cellular Biochemistry, University of Kentuckygrid.266539.d, College of Medicine, Lexington, Kentucky, USA. |
| Abstrakt: |
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 (N 0 ) 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 (N 0 -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 (N 0 ) and oligomeric (N-RNA) HMPV N for IB formation. |
