Author ORCID Identifier

https://orcid.org/0009-0008-5584-3845

Date Available

5-10-2023

Year of Publication

2023

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Rebecca Dutch

Abstract

Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus (NNSV) that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. Despite the initial identification of HMPV in 2001, there are currently no FDA approved antivirals or vaccines available. Therefore, understanding the mechanism of HMPV replication is critical for the identification of novel therapeutic targets. A key feature in the replication cycle of HMPV and other NNSVs is the formation of membrane-less, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). Recent work on NNSV IBs suggests they display characteristics of biomolecular condensates formed through a liquid-to-liquid phase transition. Intrinsically disordered proteins are common drivers of biomolecular condensate formation, and post-translational modifications have been shown to regulate condensate properties. The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures and are both required for the viral polymerase to synthesize RNA during infection. HMPV P is a necessary co-factor for the viral polymerase, has regions of intrinsic disorder, and has several known and predicted phosphorylation sites. I hypothesized that 1) the HMPV P intrinsically disordered domains facilitate IB formation and 2) changes to P phosphorylation state regulates the properties of IBs and the function of P as a polymerase co-factor.

Upon deletion of regions of HMPV P, we found that the 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 were dispensable. Alanine substitution at a single tyrosine residue in the CTD was a minimal mutation which abrogated IB formation and reduced co-immunoprecipitation with HMPV N, identifying this residue as a key interaction site for N and as a facilitator of IB formation. Phospho-dead and phosphomimetic 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.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.195

Funding Information

This study was funded by the National Institutes of Health and the National Institute of Allergy and Infectious Disease grant RO1AI40758 from 2018-2023.

Download

Share

COinS