Aryal, Manish
(2022)
Molecular Mechanism of Bruton’s Tyrosine Kinase Activation by the HIV-1 Nef Virulence Factor.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Antiretroviral therapy has significantly boosted the effort in the fight against HIV. However, a cure has still not been found. The shortcomings associated with antiretroviral therapy underscore the need for alternative approaches to eliminate the latent viral reservoirs. HIV-1 Nef represents an attractive drug target in this regard because of its role in immune escape of HIV- infected cells.
I explored the mechanism of interaction between HIV-1 Nef and Bruton’s tyrosine kinase (BTK), a member of the TEC tyrosine kinase family previously shown to enhance the HIV-1 life cycle in cells of myeloid lineage. I discovered that HIV-1 Nef activates BTK by a unique mechanism dependent upon SH3-SH2-mediated dimerization. In a solution-based assay, complex formation with HIV-1 Nef increased the stability of the BTK SH3-SH2 dimer. A cell-based bimolecular fluorescence complementation assay also showed increased recruitment of the BTK dimer to the cell membrane in presence of Nef. Alanine substitution of Pro327 in BTK SH2 domain CD-loop reduced the stability of the BTK dimer in solution, and also reduced BTK homodimerization in cells. Introduction of the P327A mutation completely uncoupled BTK from Nef-mediated activation in a kinetic kinase assay, and also prevented BTK dimerization and activation in response to Nef in cells. The effect of the P327A mutation is consistent with molecular modeling studies which identified a novel interface between the SH2 CD loop and the SH3 domain, where Pro327 is predicted to stabilize a specific CD loop conformation for SH3 engagement. An analogous interaction was demonstrated previously by NMR for the related kinase ITK, which also contributes to the HIV life cycle in CD4 T cells.
Remarkably, this mechanism of BTK activation through stabilization of SH3•SH2 interaction is distinct from the SH3 domain displacement mechanism previously described for Nef- mediated activation of SRC-family kinases, indicating that this viral protein has evolved two separate mechanisms for host cell tyrosine kinase activation. Molecular details of the Nef-driven BTK activation process may guide future drug targeting of this unique interaction. The requirement of HIV-1 Nef homodimerization in this model suggests that HIV-1 Nef dimerization can be targeted for potential therapeutic intervention.
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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: |
3 January 2022 |
| Date Type: |
Publication |
| Defense Date: |
15 December 2021 |
| Approval Date: |
3 January 2022 |
| Submission Date: |
20 December 2021 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
246 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Bruton’s Tyrosine Kinase, HIV-1 Nef, Btk dimerization, HIV-1 Nef dimerization |
| Date Deposited: |
03 Jan 2022 17:22 |
| Last Modified: |
18 May 2023 22:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/42132 |
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