Staudt, Ryan P
(2020)
Role of HIV-1 Nef dimerization in SERINC5 restriction factor antagonism and Src-family kinase recruitment.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The HIV-1 Nef accessory protein plays a prominent role in viral pathogenesis, immune evasion of HIV-infected cells, and AIDS progression. Nef lacks intrinsic biochemical activity, and instead functions through interactions with multiple host cell effector proteins. Previous studies have shown that Nef homo-dimerizes at a hydrophobic interface between two Nef molecules. As Nef dimerization has been implicated in several Nef functions, it may represent a novel and effective target for antiretroviral drug development. Research that further expands our understanding of Nef dimerization may aid in the development of future therapeutics.
Therefore, for the first part of my dissertation, I explored the role of Nef dimerization in Nef antagonism of the host restriction factor SERINC5. We showed that Nef homodimers are required for the AP-2 dependent downregulation of SERINC5, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Disruption of Nef dimerization impaired antagonism of SERINC5, rescuing SERINC5 inhibition of viral infectivity. In addition, dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2 by fluorescence complementation assay, suggesting that the Nef dimer bridges SERINC5 with AP-2 to drive endocytosis of cell-surface SERINC5.
For the second part of my dissertation, I examined the dynamics and individual contribution of predicted dimer-stabilizing residues towards dimerization and complex formation in solution. Crystal structures have revealed distinct orientations of the Nef dimer interface within complexes of Nef bound to Src family kinase SH3 domain vs. Nef complexes bound to SH3-SH2 domain. I was able to validate the orientation of the dimer interface captured in each structure using biophysical and biochemical studies. As part of a collaborative study, I was able to confirm that Nef Asp123 contributes to Nef dimerization within the SH3-bound but not the SH3-SH2 bound structure of Nef. I also identified a Nef residue (Tyr115) that stabilizes Nef dimerization within both complexes, suggesting that it is a consistent feature of Nef dimerization across two potential stages of kinase domain engagement. Taken together, this dissertation helps to further illuminate the mechanics of Nef dimerization, and implicates Nef dimerization in one of the more recently discovered Nef functions: Nef antagonism of SERINC5.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
24 August 2020 |
| Date Type: |
Publication |
| Defense Date: |
17 July 2020 |
| Approval Date: |
24 August 2020 |
| Submission Date: |
5 August 2020 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
189 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
HIV-1, Accessory protein, Nef, SERINC5, Dimerization, Hck |
| Date Deposited: |
25 Aug 2020 03:48 |
| Last Modified: |
25 Aug 2020 03:48 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/39524 |
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