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Dissertation
Proteasome-Depedent Entry of Alphaherpesviruses and the Role of Host Cell Neddylation in Alphaherpesvirus Entry and HSV Internalization
Washington State University
Doctor of Philosophy (PhD), Washington State University
2022
DOI:
https://doi.org/10.7273/000005256
Abstract
HSV-1 commandeers the host cell proteasome at several steps of its replication cycle including entry. Incoming capsids rely on 20S proteasome activity for trafficking to the nuclear periphery, and this transport is facilitated by ICP0 present in the inner tegument layer. Here we demonstrate that PRV, BoHV-1, and HSV-2 entry are blocked by proteasome inhibitors MG132 and Bortezomib. Proteasome-dependent entry of HSV-1 is thought to be ubiquitin-independent. To interrogate further the proteasome-dependent mechanism of entry, we determined the involvement of the ubiquitin-like molecule NEDD8 and the neddylation cascade in alphaherpesvirus entry and infection. MLN4924 is a small-molecule inhibitor of neddylation that binds directly to the NEDD8-activating enzyme, and it has antiviral activity against HSV-1 and HCMV. Cell treatment with MLN4924 inhibited plaque formation and infectivity by HSV-1, HSV-2, PRV and BoHV-1 at non-cytotoxic concentrations. Thus, the neddylation pathway is broadly important for alphaherpesvirus infection. However, the neddylation inhibitor had less of an effect on the entry of the veterinary viruses but had a more significant inhibitory effect on entry of HSV-1 and HSV-2 into seven different cell types that are both experimentally and biologically relevant. Time-of-addition assay suggested that the drug was acting on an early step in the entry process. Wash-out experiments indicated that MLN4924’s effect on entry was reversible. siRNA knockdown of NEDD8 significantly inhibited HSV entry, confirming the critical role of host cell neddylation. In probing the precise neddylation-dependent step in entry, we found that MLN4924 dramatically blocked endocytic uptake of HSV from the cell surface by >90%. In contrast, the rate of HSV entry into cells that support direct fusion of HSV with the cell surface was unaffected by MLN4924. Interestingly, proteasome activity was not critical for the endocytic internalization of HSV from the cell surface. Our results identify the NEDD8 cascade as critical for the internalization step of HSV entry.
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Details
- Title
- Proteasome-Depedent Entry of Alphaherpesviruses and the Role of Host Cell Neddylation in Alphaherpesvirus Entry and HSV Internalization
- Creators
- Becky Lee
- Contributors
- Anthony Nicola (Advisor)Dana Shaw (Committee Member)Alan Goodman (Committee Member)Kyle Taylor (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Veterinary Medicine, College of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 75
- Identifiers
- 99901019940901842
- Language
- English
- Resource Type
- Dissertation