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Molecular Mechanism of Bruton’s Tyrosine Kinase Activation by the HIV-1 Nef Virulence Factor

Aryal, Manish (2022) Molecular Mechanism of Bruton’s Tyrosine Kinase Activation by the HIV-1 Nef Virulence Factor. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Aryal, Manishmanish.aryal@pitt.edumaa287
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairConway, Jamesjames.conway@pitt.edu
Thesis AdvisorSmithgall, Thomastsmithga@pitt.edu
Committee MemberIshima, Riekoishima@pitt.edu
Committee MemberAlvarado, John Jeffjja24@pitt.edu
Committee MemberAndreotti, Amyamyand@iastate.edu
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: 03 Jan 2022 17:22
URI: http://d-scholarship.pitt.edu/id/eprint/42132

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