Membrane-associated RNA-dependent RNA replication of viruses
Abstract/Contents
- Abstract
- Positive strand RNA viruses are major human pathogens that result in significant morbidities and mortalities worldwide. All positive strand RNA viruses induce rearrangement of intracellular membranes to establish a membranous platform where genome replication occurs. This replication factory, or replication complex, contains a collection of host and viral proteins as well as RNAs in organelle-like structures with distinct membrane composition. In this thesis, I aim to dissect the contribution of different components of the replication complex of one important human RNA virus, the hepatitis C virus (HCV). To characterize the importance of RNA secondary structure for viral replication, I utilized and improved a novel chemical mapping method termed 2' selective hydroxyl acylation analyzed by primer extension (SHAPE). This method allowed me to rapidly determine the RNA secondary structure of the HCV 5' untranslated region (UTR) at high resolution and its interaction with the liver-specific microRNA (miR), miR-122. I also utilized a supported lipid-bilayer (SLB) platform coupled with quartz-crystal microbalance with dissipation (QCM-D) to reconstitute and monitor how the interactions of key components of the viral replication complex including the viral proteins, the RNA template, and the membrane phosphoinositides composition affect in vitro RNA-dependent RNA transcription and viral RNA replication inside the cell. The above studies employed a combination of chemical, biophysical, and genetics tools and reveal that 1) the RNA secondary structure of the HCV 5' UTR and the structural integrity of the HCV 5' UTR -- miR-122 complex mediate viral replication; 2) SLB coupled with QCM-D can monitor the reconstitution of RNA synthesis activity of the HCV membrane-anchored RNA-dependent RNA polymerase NS5B and its interaction with other replicase complex proteins; and 3) the replication complex of HCV is enriched with phosphatidylinositol 4,5-bisphosphate (PIP2), PIP2 is a ligand for another replicase component protein NS5A, and NS5A binds PIP2 the basic amino acids PIP2 pincer (BAPPP) domain, and this interaction modulates HCV RNA replication.
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 | Pham, Edward A |
|---|---|
| Associated with | Stanford University, Department of Microbiology and Immunology. |
| Primary advisor | Glenn, Jeffrey S, 1962- |
| Thesis advisor | Glenn, Jeffrey S, 1962- |
| Thesis advisor | Greenberg, Harry B |
| Thesis advisor | Peltz, Gary, 1956- |
| Thesis advisor | Sarnow, P. (Peter) |
| Advisor | Greenberg, Harry B |
| Advisor | Peltz, Gary, 1956- |
| Advisor | Sarnow, P. (Peter) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Edward A. Pham. |
|---|---|
| Note | Submitted to the Department of Microbiology and Immunology. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Edward Anhoa Pham
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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