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Cell Surface Env Transformation and Membrane Disruption by HIV-1 Entry Inhibitors

Thursday, June 4, 2020

10:00 AM-12:00 PM

BIOMED PhD Thesis Defense

Cell Surface Env Transformation and Membrane Disruption by HIV-1 Entry Inhibitors
Charles G. Ang, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Irwin Chaiken, PhD
Professor of Biochemistry and Molecular Biology
College of Medicine
Drexel University

Ken Barbee, PhD
Professor, Senior Associate Dean, and Associate Dean for Research
School of Biomedical Engineering, Science and Health Systems
Drexel University

This work examined transformations of the HIV-1 fusion protein Env and its anchoring membrane that can be subverted by lytic entry inhibitors to inactivate Env on viruses and cells. Two entry inhibitors were investigated: KR13 and M*DAVEI. KR13 is a chemically synthesized peptide triazole thiol (PTT) comprised of a CD4 binding site pharmacophore linked to a disulfide-targeting thiol, while M*DAVEI is a chimeric recombinant protein containing a gp120-glycan-binding lectin linked to Trp3, a gp41-binding fragment of Env’s membrane proximal external region (MPER). Both inhibitors have previously been shown to cause virus lysis, indicating that they can trigger membrane disruption. Env-presenting cells were selected for these experiments to facilitate mechanistic studies and provide preliminary data for further cell-based studies.

To investigate KR13, we developed a flow cytometry protocol to examine the nature of its Env rearrangements by measuring changes in epitope availability for key regions of Env upon treatment with serial dilutions of KR13 or its thiol-blocked analogue KR13b. HEK293T cells were transiently transfected for surface presentation of JRFL Env and pre-loaded with fluorescent dye. Epitopes examined were: intact gp120-gp41 association (Ab 35O22), formation of the 6HB (Ab NC-1), MPER exposure (Ab 10E8), the immunodominant loop (Ab 50-69), fusion peptide (Ab VRC34.01), and membrane disruption (loss of intracellular dye). Env transformations caused by KR13 resembled several steps of native fusion, including loss of attached gp120, increase in 6HB detection, increase in MPER exposure, and membrane disruption, suggesting that the mechanism by which KR13 induces transformation may incorporate and subvert the conformational “program” Env uses to enter target cells. In turn, KR13 and PTT compounds in general may be useful not only as antagonist leads but also as probes to interrogate HIV entry and the exposure of key post-fusion epitopes from the unique approach of transforming Env and membrane without target cells. Further experiments with KR13 and KR13b showed that the membrane disruption and leakage effects of KR13 could be recapitulated by the combination of KR13b and the redox protein Thioredoxin-1 (Trx1). Virions were treated with either KR13 alone, or a combination of fixed concentration KR13b and serial dilutions of Trx1, showing comparable p24 release between the two. Along with literature showing Trx1’s presence in and necessity to native fusion, we established that the KR13’s free terminal thiol may be mimicking the reducing effects of Trx1, likely targeting Env’s C296-C331 disulfide.  

The second part of this research examined the engineered protein M*DAVEI, which causes severe membrane disruptions in virions, speculated to result from the dual engagement of Env by the gp120-binding lectin and the MPER/membrane-interacting Trp3. We investigated whether the destructive effect of lectin-DAVEI on viruses could be used to target cells presenting Env on their surfaces. HEK293T cells were transiently transfected with DNA encoding JRFL Env, pre-loaded with calcein dye, treated with serial dilutions of M*DAVEI, and stained for intact gp120-gp41 association (Ab 35O22). The observed effects of M*DAVEI on cells were proportional to individual levels of Env expression: "Low-Env" cells experienced transient poration and calcein leakage, while "High-Env" cells died. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and its associated cytotoxicity. Controls with unlinked M*DAVEI components showed no effect on calcein leakage or cell viability, confirming covalently linked DAVEI components as necessary to induce transformation.  

Cumulatively, this work has demonstrated Env metastability as an intrinsic property of the transmembrane protein complex and not limited to fusogenic or fusion-like transformations of Env alone. As such, the Env-membrane complex can be perturbed to trigger membrane disruption in virus and cell contexts, an extension of our understanding of these entry inhibitors’ effects and their mechanisms of action.

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Natalia Broz

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