Construction and Characterization of the NS3 Protein of Bluetongue Virus as A Bait for Application in Yeast Two-hybrid System

Background: Bluetongue is an arthropod transmitted viral disease causing range of clinical manifestations in small ruminants. The causative agent, Bluetongue virus (BTV) possesses several structural (SPs) and non-structural proteins (NSPs) associated with its morphogenesis and virulence. NS3, a multifunctional protein of BTV execute a decisive role in virus pathogenesis and release. In the present study, the NS3 protein of BTV was cloned into a pGBKT7-BD vector as bait and characterized for its expression. The suitability of NS3 as a bait to screen interactions with host proteins was also assessed. Methods: The NS3 gene was cloned in the pGBKT7-BD vector to generate the bait plasmid. The recombinant pGBKT7-NS3 bait was sequence confirmed and characterized for auto-activation, toxicity and expression. Thereafter, the bait was mated with yeast two-hybrid (Y2H) cDNA library and screened for the host-BTV protein-protein interactions (PPI). Conclusion: The constructed bait was found suitable for mating, however, no protein hits appeared on forward library screening. The sequence analysis revealed the presence of the transmembrane domains (TM) within the NS3 sequence that may have resulted in the failure of protein interaction studies in Y2H.