Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation




Molecular and Cellular Biochemistry

First Advisor

Dr. Rebecca Dutch


Hendra virus (HeV) and human metapneumovirus (HMPV) are negative-sense, singled-stranded RNA viruses. The paramyxovirus HeV is classified as a biosafety level 4 pathogen due to its high fatality rate and the lack of a human vaccine or antiviral treatment. HMPV is a widespread pneumovirus that causes respiratory tract infections which are particularly dangerous for young children, immunocompromised individuals, and the elderly. Like HeV, no vaccines or therapies are available to combat HMPV infections. These viruses fuse their lipid envelopes with a cell to initiate infection. Blocking cell entry is a promising approach for antiviral development, and many vaccines are designed based on the envelope protein responsible for fusion. Following fusion, the coated genome and its associated proteins are released into the cytoplasm for replication and transcription. HMPV and other negative-strand viruses form membrane-less inclusion bodies (IBs) which act as viral factories to promote these processes. HMPV IBs represent another promising target for developing new antivirals since they house the replication machinery. Viral fusion and cytoplasmic replication are ubiquitous to most negative-strand viruses and are addressed in this work through analysis of HeV and HMPV.

HeV utilizes a trimeric fusion protein (F) within its lipid bilayer to mediate membrane merger with a cell for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the prefusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, and N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events, likely due to the limited flexibility in the TMD. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a prefusion conformation-specific antibody prior to cell disruption, indicating that the introduced disulfide bonds did not significantly affect protein folding. This study strengthens the current model for HeV fusion and provides important insight for understanding the basic mechanisms of membrane fusion for negative-strand RNA viruses.

HMPV IBs are dynamic structures required for efficient replication and transcription. The minimum components needed to form IB-like structures in cells are the nucleoprotein (N), which coats the RNA genome, and the tetrameric phosphoprotein (P), which acts as a cofactor to the polymerase. HMPV P binds to two versions of N protein in infected cells: C-terminal P residues interact with oligomeric, RNA-bound N (N-RNA), and N-terminal P residues interact with monomeric N (N0) to maintain a pool of protein to encapsidate new RNA. Recent work on other negative-strand viruses has shown that IBs are liquid-like organelles formed via liquid-liquid phase separation (LLPS). Recombinant versions of HMPV N and P proteins were purified to analyze the interactions required to drive LLPS in vitro. Purified HMPV P was shown to form liquid droplets in the absence of other protein binding partners, suggesting that it functions as a scaffold to recruit client proteins to IBs. HMPV P recruited a monomeric N protein construct, N0-P, to liquid droplets. In addition, HMPV P incorporated N-RNA into liquid droplets, though N-RNA formed aggregates independently. These findings support that HMPV P acts as a scaffold protein to mediate multivalent interactions with monomeric and oligomeric HMPV N to promote phase separation of IBs. Collectively, the work presented here provides important insight into the processes of viral entry, replication, and IB formation for negative-strand RNA viruses.

Digital Object Identifier (DOI)

Funding Information

This study was supported by the University of Kentucky College of Medicine Fellowship for Excellence in Graduate Research in 2018. It was also supported by the Max Steckler Fellowship from the University of Kentucky in 2020.