Confirmation of interaction of a novel HIV NS2 Protein with host proteins in living mammalian cells

Abstract

NS2 is a novel HIV protein confirmed to be expressed from an open reading frame alongside the Pol open reading frame. Furthermore, NS2 has been shown to be critical to viral replication and to localize in the nucleolus of the nucleus. The mechanism of NS2 function is currently unknown. A yeast-2-hybrid assay (Y2H) yielded a number of possible mammalian protein interactions with NS2. Bimolecular fluorescence complementation (BiFC) is utilized to confirm these positive interactions. The first step to performing a BiFC experiment is inserting the coding sequence for the protein of interest into a vector plasmid. From the list of Y2H positive interactions, we chose to analyze programmed cell death 5 (PDCD5), heat shock protein-40 (DNAJB1), and lantibiotic synthetase C-like protein-2 (LANCL2). Our results presented here are the successful plasmid construction of pBiFC-PDCD5-YN155 and pBiFC-DNAJB1-YN155. Both of these proteins are now ready for BiFC experiments, however, LANCL2 will require a site-directed mutagenesis to remove a restriction site within the coding sequence. The next step in this project is constructing the NS2 plasmid, pBiFC-NS2-YC155. Afterwards, BiFC experiments can be conducted and finally, the mechanism of NS2 determined, which may potentially lead to HIV-1 drug therapy.Keywords: PDCD5, DNAJB1, HIV, LANCL