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Muhammad Khalil Thesis

dc.contributor.advisorChelico , Linda
dc.contributor.committeeMemberUppalapati , Maruti
dc.contributor.committeeMemberBull, Harold
dc.contributor.committeeMembervan der Hurk, Sylvia
dc.contributor.committeeMemberVizeacoumar, Franco
dc.creatorKhalil, Muhammad 1993- 2018
dc.description.abstractAccording to recent data, an additional one million people have died of HIV-1/AIDS related illness in 2016 bringing the death toll to 35.0 million people worldwide. Currently, to inhibit HIV-1 replication a combination of various drugs is used that targets different steps of its life cycle. This Highly Active Anti-Retroviral Therapy (HAART) involves the use of a combination of inhibitors to stop viral replication. Although HAART is predominantly used in clinical settings, it is facing various challenges that encourages the scientific community to pursue other avenues of therapy. One of the options is to promote the enzymatic function of host APOBEC3 restriction factors. APOBEC3 is an acronym for Apolipoprotein B mRNA Editing Enzyme-catalytic polypeptide 3. The APOBEC3 (A3) are cytidine deaminases that possess the ability to deaminate deoxycytidine in single stranded DNA and can become encapsidated into HIV-1 particles to induce mutagenesis of the antisense DNA strand of HIV-1 after synthesis of first DNA strand and prior to the synthesis of second DNA strand. But, this activity is hindered by the HIV-1 Viral Infectivity Factor (Vif). Vif enables the ubiquitination of APOBEC3 enzymes by a Cullin RING E3 Ligase complex, resulting in their subsequent degradation through the 26S proteasome. Vif exists in infected cells as a heterotetramer where Vif obligately binds the co-transcription cofactor CBFβ and Elongin C for stability. Elongin C exists as a dimer with Elongin B. Both Elongin C and Vif need to independently interact with Cullin 5 to form the Cullin RING E3 Ligase complex. Thus, the physiologically relevant form of Vif is in the following complex, Vif/CBFβ/EloC/EloB. Vif acts as the substrate receptor of the ubiquitination complex, by directly binding an A3 to bring it into the complex. Vif has a unique interaction site for each A3 and each A3 has a unique interaction site for Vif. The inhibition of the interaction site between Vif and A3, specifically APOBEC3G and APOBEC3F, proteins are a tempting site for drug development. Organic compounds have been discovered that can inhibit HIV-1 Vif function. However, these compounds have high toxicity issues, they are too small to cover the interface and are prone to resistance. We propose that a protein-protein interaction based binder will work more efficiently to inhibit the interaction between Vif and APOBEC3 proteins. To achieve this goal, we proposed to screen for stable scaffold proteins specific against Vif through a high throughput M13 phage display system. We also proposed to design an engineered chimeric A3-Vif binding domain that contains the Vif binding domains of both A3G and A3F which will act as a “super binder”. Instead of Vif binding to A3G or A3F during viral infection, the super binder (chimeric A3) can interfere with this process by having a high affinity for Vif. Various scaffold libraries were tested against Vif/CBFβ/EloC/EloB heterotetramer and GST-Vif targets. We successfully isolated binders specific to the entire Vif/CBFβ/EloC/EloB heterotetramer. In addition, we obtained similar results for a GST-Vif target. The GST-Vif isolated binders showed low level of specificity for the targets compared to our controls and were not studied further. Three binders were isolated that had high specificity for the Vif/CBFβ/EloC/EloB heterotetramer and warrant further investigation. Additionally, we were able to display a chimeric A3G on the M13 phage, demonstrating feasibility of this approach for the purposes of determining its specificity for Vif binding site. All together this thesis work is a proof of principle that protein binders that disrupt Vif function can be obtained by screening protein-based M13 phage display libraries. Since the use of A3 enzymes as an HIV-1 therapy has the potential to have less side effects than conventional HIV-1 antiretroviral treatment, it is important to continue the study of inhibitors that disrupt Vif function
dc.subjectHIV, VIF, Inhibitors
dc.titleMuhammad Khalil Thesis
local.embargo.terms2020-07-29 and Immunology and Immunology of Saskatchewan of Science (M.Sc.)


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