The Molecular Characterization of NiV C protein and the Identification of Henipavirus-Host Protein Interactions
Open Access
- Author:
- Mccrory, Thomas Sean
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 19, 2012
- Committee Members:
- Anthony Paul Schmitt, Dissertation Advisor/Co-Advisor
Anthony Paul Schmitt, Committee Chair/Co-Chair
Kumble Sandeep Prabhu, Committee Member
Pamela Hankey Giblin, Committee Member
Craig Eugene Cameron, Special Member - Keywords:
- Henipavirus
Yeast two-hybrid
Nipah virus
Virus-like particles
Ubiquitination
Protein Purification - Abstract:
- Since the emergence of the highly pathogenic Henipaviruses in 1990’s, hundreds of human fatalities have been reported throughout Asia and Australia. Like other paramyxoviruses, Henipaviruses are enveloped viruses; successful replication requires the cooperation of numerous virus and host factors. One of these factors, Nipah virus (NiV) C protein, was investigated in the current research to characterize some of the molecular characteristics C protein may require for its role in viral pathogenesis and immune evasion. Many paramyxoviruses express multifunctional C proteins; potential roles of C proteins during virus infection include: immune evasion, regulation of viral polymerase function, viral pathogenesis, and recruitment of host factors for virus particle assembly. Here, it was observed that NiV C protein is a membrane-bound protein that is also highly unstable when expressed in mammalian cells. NiV C protein turnover was also found to be ubiquitin-proteasome dependent. It was confirmed that NiV C protein is ubiquitinated and the elimination of all the sites for ubiquitin attachment was required to prevent ubiquitination and partially restore protein stability. These findings have defined new characteristics of NiV C protein, which are likely important during viral infections. In an effort to identify proteins important for Henipavirus assembly and budding, several screens were performed to identify host protein interactions with Henipavirus matrix (M) proteins. Several novel host factors that interact with Henipavirus M proteins were identified. In addition, minimal binding fragments were constructed that, when overexpressed, inhibited the production of Nipah virus-like particles. A more comprehensive analysis of the identified host proteins revealed that they were frequently found in known mammalian cellular complexes with each other. These complexes are involved in a variety of cellular functions such as transcriptional regulation, protein translation, protein sorting and transport. Any number of these may be critical for Henipavirus M protein function. Additionally, more than half of the confirmed Henipavirus M-interacting proteins contained large serine-rich acidic regions. These regions were critical for binding and likely allow for electrostatic interactions to occur with Henipavirus M proteins. Together, these data provide a model where Henipavirus M protein uses electrostatic interactions with host factors for a variety of functions.