The Involvement of Ubiquitin in Parainfluenza Virus 5 Budding
Open Access
- Author:
- Harrison, Megan Stasik
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 27, 2011
- Committee Members:
- Anthony Paul Schmitt, Dissertation Advisor/Co-Advisor
Anthony Paul Schmitt, Committee Chair/Co-Chair
Eric Thomas Harvill, Committee Member
Edward C Holmes, Committee Member
Richard John Frisque, Committee Member - Keywords:
- virus budding
paramyxovirus
ubiquitin - Abstract:
- Ubiquitin has been implicated in the process of enveloped virus budding based primarily on findings with retroviruses. Ubiquitination of retroviral Gag proteins appears to be important for efficient virus budding, presumably by contributing to interactions with host proteins that are hijacked by viruses to assist in virion release. However, the role of ubiquitin and viral protein ubiquitination in enveloped virus budding remains controversial and poorly understood. Precious little is known about the role of ubiquitin in the assembly and budding of single-stranded, negative-sense RNA viruses, such as paramyxoviruses. Parainfluenza virus 5 (PIV5) is a nonpathogenic prototypic paramyxovirus ideal for the investigation of processes it shares with other pathogenic paramyxoviruses. Paramyxovirus matrix (M) proteins orchestrate virus assembly by accumulating at sites of budding and recruiting viral and cellular components to these sites. It has been found that proteasome inhibitor treatment, which leads to the depletion of free pools of ubiquitin, potently inhibits PIV5 particle and virus-like particle (VLP) production, implicating the ubiquitin-proteasome pathway in PIV5 assembly and budding. We sought to investigate the involvement of ubiquitin in PIV5 budding. We find that PIV5 M protein can be ubiquitinated and the most abundant form of ubiquitinated M protein is a monoubiquitinated form. Free ubiquitin is detected in released PIV5 virions. Ubiquitinated M protein is detected in released virions and VLPs. The primary lysine residues modified by ubiquitin attachment were identified by mass spectrometry (MS) analysis. Cumulative mutagenesis of MS-identified and predicted ubiquitination sites from lysine to arginine that resulted in reduced M protein monoubiquitination also led to reduced VLP production, reduced virion production and infectivity, and reduced incorporation of free ubiquitin into virions. Recombinant PIV5 encoding cumulative lysine-mutant M protein exhibits an early assembly defect as observed by immunofluorescence microscopy. The level of monoubiquitinated PIV5 M protein is also reduced by proteasome inhibitor treatment and treatment of PIV5 infected cells with proteasome inhibitors results in a similar early assembly defect. Our results suggest that the monoubiquitination of PIV5 M protein is important for efficient virus budding. These data suggest that proteasome inhibitors – including the drug bortezomib (Velcade), approved in the United States by the FDA for use in multiple myeloma – have potential as antiviral agents for the treatment of paramyxoviral infections. Additionally, several lysine residues in PIV5 M protein that are crucial for budding, but dispensable for ubiquitination, were identified. These lysine residues may be part of sequence motifs that bind to cellular components for efficient virus budding.