Zych, Courtney
(2011)
Development of a high throughput cell-based assay to screen for inhibitors of HIV-1 Vpr oligomerization.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Highly active anti-retroviral treatment (HAART) targets less than a third of the proteinsproduced during HIV-1 infection. Testing the effectiveness of an anti-retroviral drug requiresassays specific for the individual target that take into account its mechanism of action. MostHIV-1 proteins need to undergo dimerization in order to become functional in the viral life cycle. Historically, it has been difficult to visualize and quantify changes in a protein-protein interaction, which has left this characteristic of proteins unexplored as potential antiviral targets. In this study, a bimolecular fluorescence complementation based screening assay is developed that can quantify a change in dimerization, using the HIV-1 accessory protein Vpr as a "proof of concept".Results demonstrated that bimolecular fluorescence complementation of Vpr could be competed off in a dose-dependent manner using untagged, full length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of peptides and a library of small molecules for an effect on Vpr dimerization. None of the Vpr peptides were shown to have an effect; however, one of the small molecules was shown to interfere with Vpr dimerization in a dose-dependent manner.Statement of Public Health relevance: Dimerization is a unique property of many HIV-1 viral proteins and is necessary to complete the viral life cycle, thus it has been identified as a potential drug target. By developing an assay that screens for inhibition of HIV-1 protein dimerization, high throughput screening can be performed to detect inhibitors of a new target in HIV-1 replication. Small molecules identified using this screening method could be developed into a novel anti-retroviral drug.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
29 June 2011 |
| Date Type: |
Completion |
| Defense Date: |
13 April 2011 |
| Approval Date: |
29 June 2011 |
| Submission Date: |
4 April 2011 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Public Health > Infectious Diseases and Microbiology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
drug screening; high content screening; HIV; Vpr; assay development; dimerization |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04042011-225511/, etd-04042011-225511 |
| Date Deposited: |
10 Nov 2011 19:34 |
| Last Modified: |
15 Nov 2016 13:38 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6753 |
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