Using CRISPR/Cas9 and human haploid genetic screens to identify host factors essential for dengue virus infection
Abstract/Contents
- Abstract
- Dengue virus (DENV) is an emerging pathogen worldwide, causing an estimated 100 million symptomatic infections annually, yet recent large clinical trials for a DENV vaccine candidate have showed only partial protection. Additionally, no licensed antiviral drugs are available, motivating our approach to evaluate host functions to accelerate development of antiviral therapies. We applied tools for unbiased discovery of host functions exploited by DENV to replicate and spread by performing two genome-scale knockout screens in parallel: one using CRISPR/Cas9 mutagenesis and one using gene trap mutagenesis in haploid human cells. We identified distinct ER-associated multi-protein complexes, which are critically required for DENV infection involved in protein translocation, N-linked glycosylation, and ER associated degradation. We observed that DENV replication was almost completely eliminated in cells deficient in the oligosaccharyltransferase (OST) complex. Additionally, other Flaviviruses, particularly Yellow Fever Virus, West Nile Virus and Zika Virus, also showed partial dependence on the OST complex. Our studies of the interaction mechanism between these Flaviviruses and the OST complex revealed that viral RNA replication was the critical step dependent on the OST complex possibly through interactions with viral nonstructural proteins. Entry or translation of the virus did not require the OST complex. We created catalytic dead mutants and found that dengue virus replication was efficiently rescued in knockout cells. Thus, we reveal a function of the OST in viral replication that is independent from its cellular function in N-linked glycosylation. This striking feature of the OST complex establishes it as a host target for antiviral drug development and targeting the OST complex will result in a higher barrier of resistance compared to drugs targeting viral proteins.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Marceau, Caleb Daniel |
|---|---|
| Associated with | Stanford University, Department of Microbiology and Immunology. |
| Primary advisor | Carette, Jan, 1971- |
| Thesis advisor | Carette, Jan, 1971- |
| Thesis advisor | Kirkegaard, Karla |
| Thesis advisor | Schneider, David (David Samuel) |
| Thesis advisor | Stearns, Tim |
| Advisor | Kirkegaard, Karla |
| Advisor | Schneider, David (David Samuel) |
| Advisor | Stearns, Tim |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Caleb Daniel Marceau. |
|---|---|
| Note | Submitted to the Department of Microbiology and Immunology. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Caleb Daniel Marceau
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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