A New Protein Delivery Technology Based On Manipulation Of Paramyxovirus Genome Packaging Interactions
Restricted (Penn State Only)
- Author:
- Panthi, Santosh
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 02, 2022
- Committee Members:
- Anthony Schmitt, Chair & Dissertation Advisor
Troy Sutton, Major Field Member
Kumble Prabhu, Major Field Member
Anthony Paul Schmitt, Professor in Charge/Director of Graduate Studies
Jason Rasgon, Outside Unit & Field Member - Keywords:
- VLP
nucleocapsid
matrix
packaging
paramyxovirus
protein delivery - Abstract:
- Enveloped virus particles are formed by budding from infected cell membrane. This process involves the collection of viral proteins and genomes at the assembly site, acquisition of envelopes, and release of viral particles. Viruses assemble in a way that is meant to maximize the packaging of viral components and prevent the packaging of most non-viral components. For paramyxoviruses and many other RNA viruses, genome packaging into budding particles is mediated through matrix (M)-nucleocapsid (NP) proteins interaction. In addition to generation of genome containing infectious particles, M-NP interaction also stimulates efficient production of virus-like particles (VLPs). Previous studies have shown that paramyxovirus NP proteins harbor short DxD-containing sequences near their C-terminal ends that mediate M protein interaction and direct the virus assembly function. Interestingly, fusions of such residues onto the C-terminal ends of foreign proteins allow them to participate in viral assembly and package efficiently into paramyxovirus-like particles. Building upon these observations, we have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using multiple paramyxoviruses based VLP production platforms including parainfluenza virus 5 (PIV5), mumps virus (MuV), and Nipah virus (NiV), and in each case efficient cargo packaging and delivery could be achieved. In addition, we tested a series of second-site M mutants derived from PIV5 for improved Cre recombinase packaging and delivery. When compared to wild type M, VLPs generated using second–site M mutant significantly improved VLP production, Cre packaging, and delivery of functional Cre into target cell nuclei. Together, these findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. Paramyxovirus M-NP interactions could be a potential target for a rational design of antivirals as this interaction is critical for both viral genome packaging and viral particle formation. However, the molecular mechanisms governing this interaction are yet to be fully elucidated. Our lab previously identified a DxD-containing region of NP protein that mediates interaction with M protein. Alterations to DxD-sequences near the C-terminal ends of NP proteins inhibit M-NP interaction and VLP production function. This study expanded upon such discoveries and established an in vitro binding assay using purified viral components. PIV5 M protein was expressed and purified using E. coli, and it was tested binding with DxD-containing synthetic peptides corresponding to the C-terminal region of PIV5 NP protein. E. coli expressed M protein was retained efficiently by wild type peptide, whereas the peptide lacking DxD sequence failed to retain M protein. This work demonstrates for the first time that a paramyxovirus matrix-nucleocapsid interaction could be established by utilizing purified components in an experimental system that is not affected by the host cell components. This assay could serve as the foundation for identifying the structural regions of M protein that interact directly with NP protein.