Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein.
|Journal||Journal of Virology|
|State||Published - May 2023|
Bibliographical noteFunding Information:
Funding for this work was provided by NIH/NIAID grant RO1AI40758.
We thank Makoto Takeda (National Institute of Infectious Diseases, Tokyo, Japan) for providing the HMPV rescue system, Trevor Creamer (University of Kentucky) for providing purified HMPV P, and the University of Kentucky Center for Molecular Medicine Protein Core (funded by NIH/NIGMS COBRE grant P30GM110787) for generating nanobody.
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- inclusion bodies
- protein phosphorylation
- replication complex
ASJC Scopus subject areas
- Insect Science