Bessong, Pascal O.Shonhai, AddmoreTambani, Tshifhiwa2019-10-072019-10-072019-09-20Tambani, Tshifhiwa (2019) Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease, University of Venda, South Africa.<http://hdl.handle.net/11602/1423>.http://hdl.handle.net/11602/1423PhD (Microbiology)Department of MicrobiologyHigh genetic diversity is a major contributory factor in the development of drug resistance, in addition to challenges in diagnosis and treatment monitoring in the therapeutics of human immunodeficiency virus (HIV) .Within the wide HIV-1 diversity, differences in mutational frequency, disease progression, drug response and transmission amongst HIV-1 subtypes have been shown. In spite HIV-1 subtype C (HIV-1C) being the most prevalent variant globally, none of the available drugs nor screening assays for inhibitory molecules have been developed targeting the genetics of this important subtype. This study therefore aimed to overexpress and biophysically characterize HIV-1C reverse transcriptase and protease to serve as reagents in the development of assays for routine screening of molecules inhibitory to HIV-1C. Heterologous expression of HIV-1C reverse transcriptase and protease isolates that are prevalent in South Africa was carried out in Escherichia coli (E. coli (BL21-DE3). The secondary and tertiary structures of the proteins were determined using, circular dichroism (CD) and fluorescence spectroscopy respectively. Thereafter, interaction studies to delineate interaction properties of natural products for possible inhibition of protease were conducted. Furthermore, in silico studies to determine binding interactions, further confirmed by in vitro binding assays of a pepsin inhibitor homolog (Bm-33) from Brugia malayi , against protease were also conducted. Expressed reverse transcriptase and protease from the globally prevalent HIV-1C were shown to be structurally and functionally intact for application in downstream HIV-1 inhibition assays. Interaction studies on the other hand revealed successful inhibition of the expressed HIV-1C PR with gallotanin. Furthermore, binding interactions of Bm-33 and HIV-1 PR revealed the first intermolecular interactions of the two molecules displaying possible inhibition of HIV-1 PR1 online resource (88 leaves : color illustrations, color maps)enUniversity of VendaHIV-1 Subtype C.UCTDReverse transcriptaseProtease expressionBiophysical characterizationInhibition assaysMolecular docking616.979201HIV (Viruses)HIV infectionsAIDS (Disease) -- PatientsHIV-positive personsOverexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and proteaseThesisTambani T. Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease. []. , 2019 [cited yyyy month dd]. Available from: http://hdl.handle.net/11602/1423Tambani, T. (2019). <i>Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease</i>. (). . Retrieved from http://hdl.handle.net/11602/1423Tambani, Tshifhiwa. <i>"Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease."</i> ., , 2019. http://hdl.handle.net/11602/1423TY - Thesis AU - Tambani, Tshifhiwa AB - High genetic diversity is a major contributory factor in the development of drug resistance, in addition to challenges in diagnosis and treatment monitoring in the therapeutics of human immunodeficiency virus (HIV) .Within the wide HIV-1 diversity, differences in mutational frequency, disease progression, drug response and transmission amongst HIV-1 subtypes have been shown. In spite HIV-1 subtype C (HIV-1C) being the most prevalent variant globally, none of the available drugs nor screening assays for inhibitory molecules have been developed targeting the genetics of this important subtype. This study therefore aimed to overexpress and biophysically characterize HIV-1C reverse transcriptase and protease to serve as reagents in the development of assays for routine screening of molecules inhibitory to HIV-1C. Heterologous expression of HIV-1C reverse transcriptase and protease isolates that are prevalent in South Africa was carried out in Escherichia coli (E. coli (BL21-DE3). The secondary and tertiary structures of the proteins were determined using, circular dichroism (CD) and fluorescence spectroscopy respectively. Thereafter, interaction studies to delineate interaction properties of natural products for possible inhibition of protease were conducted. Furthermore, in silico studies to determine binding interactions, further confirmed by in vitro binding assays of a pepsin inhibitor homolog (Bm-33) from Brugia malayi , against protease were also conducted. Expressed reverse transcriptase and protease from the globally prevalent HIV-1C were shown to be structurally and functionally intact for application in downstream HIV-1 inhibition assays. Interaction studies on the other hand revealed successful inhibition of the expressed HIV-1C PR with gallotanin. Furthermore, binding interactions of Bm-33 and HIV-1 PR revealed the first intermolecular interactions of the two molecules displaying possible inhibition of HIV-1 PR DA - 2019-09-20 DB - ResearchSpace DP - Univen KW - HIV-1 Subtype C. KW - Reverse transcriptase KW - Protease expression KW - Biophysical characterization KW - Inhibition assays KW - Molecular docking LK - https://univendspace.univen.ac.za PY - 2019 T1 - Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease TI - Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease UR - http://hdl.handle.net/11602/1423 ER -