Determining the HIV Vif Gene Interactome by Screening a Human Protein Library using a Yeast Two Hybrid System

• Main Author: Katta, Suma
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Biochemistry; Biology; Genetics
• ISBN: 9798837518799

HIV Vif gene encodes the Vif protein expressed by lentivirus HIV-1 which plays an important role in replication and pathogenesis of the virus. Vif stands for viral infectivity factor. In the genome, its gene is located in the central region overlapping the 3' end of the pol ORF. This study helps to determine the protein-protein interactions between the HIV Vif gene product and the protein product from genes expressed in human genome library using a yeast two hybrid system (Y2H). It is important to understand interactions between viral and host proteins because interfering with these interactions may have therapeutic value. HIV Vifc DNA was sub-cloned into the pGBKT7 (pBD) vector such that it was fused to the Cterminus of the GAL4 DNA binding domain. The presence of HIV Vif gene in its correct orientation was verified by PCR. The Vif/pGBKT7 recombinant plasmid was transformed into yeast strain Y2H gold. Verifying the presence and the expression of Vif in yeast cells was performed using PCR and SDS gel electrophoresis. Once the expression of Vif was confirmed, the yeast two hybrid system screening was undertaken. To accomplish this, a human cDNA library was obtained from CloneTech. In this library, human cDNAs were fused with the activation domain of the GAL4 transcription factor within the pGADT7 (pAD) expression plasmid. This library was pre-transformed in yeast strain AH109. The two yeast strains were mated to produce diploid cells expressing both plasmids. Plating on DDO plates enables selection for diploid cells with possible protein-protein interaction. Colonies were further analyzed using selective plates (QDO--X-gal) that allowed growth and turned the yeast colonies blue whenever there was a protein-protein interaction between Vif (bait) and a cellular protein expressed from the cDNA (prey). From these blue interactive colonies, the pAD-cDNA plasmid was obtained, and the cDNA insert was identified through DNA sequencing.