Dissertation
Generation of human immunodeficiency virus Type-1 escape mutants to peptide triazole entry-inhibiting virus inactivators
Doctor of Philosophy (Ph.D.), Drexel University
Aug 2016
DOI:
https://doi.org/10.17918/etd-7147
Abstract
A study was performed to evolve resistant HIV-1 variants to multiple classes of peptide triazole entry inhibitors in order to understand the mechanisms of virus escape and inactivation. The macrocyclic and sulfhydryl-containing peptides used in this study are both capable of inactivating the virus through gp120 shedding, but the latter class has the additional ability to induce cell-free virolysis. Generation of HIV-1 escape mutants was accomplished through virus passaging on an immortalized T-cell line with dose escalation of inhibitor. Over the course of approximately four months, resistant virus cultures were established that grow under peptide concentrations that are two orders of magnitude greater than the starting concentration. Traditional Sanger sequencing of the escape mutant HIV-1 genome revealed similar escape profiles for both classes despite the difference between their inactivating effects. Mutations within the binding site and around the HIV-1 Env protein were discovered, including at the gp120-gp41 interface and the trimer apex, supporting the hypothesis that peptide triazoles actuate inactivating pressure throughout the protein complex. From this pool of mutations, individual and combinational mutations were generated in a pseudoviral system by site-directed mutagenesis to investigate the mechanism of escape. Small changes in the side chain at a pivotal location between the peptide binding cavities on gp120, V255I/T, were found to have a profound effect on inhibitor function. Flexible molecular docking of peptide triazoles onto HIV-1 Env trimers in the presence and absence of the V255 mutations suggests that the mutation fills an important binding cavity and prevents inhibitor penetration into the protein. The results of this work demonstrate that a major pathway of HIV-1 escape from peptide triazole virus inactivators occurs through binding site disruption, with other escape potential observed by alterations in Env gp120 shedding and infectivity.
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Details
- Title
- Generation of human immunodeficiency virus Type-1 escape mutants to peptide triazole entry-inhibiting virus inactivators
- Creators
- Andrew Patrick Holmes - DU
- Contributors
- Irwin Chaiken (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 7147; 991014632151104721