THE BACTERIAL EXPRESSION AND CHARACTERIZATION OF BIV REV EXON 2 AND THE EXTRACELLULAR DOMAIN OF BIV TRANSMEMBRANE PROTEIN

Abstract

To determine if BIV rev exon 2 is post-translationally removed from the Env polypeptide and if the extracellular domain of BIV TM protein is the region responsible for virus-cell fusion, PCR generated fragments of these two regions were cloned and bacterially expressed as maltose binding protein (MBP) fusion proteins in the pMAL expression system. The expressed fusion proteins were isolated and purified by affinity chromatography using amylose resin. The fusion proteins were immunologically characterized by immunoblotting using BIV specific sera, as well as the rabbit anti-MBP serum. The purified proteins were used to immunize mice and guinea pigs for the production of antisera. The antisera were immunologically characterized by western blotting and radioimmunoprecipitation of BIV 127-infected cell lysates. The experimental findings suggest that rev exon 2 translation appears in both the Env precursor and the mature Rev protein. Furthermore, rev exon 2 is posttranslationally cleaved from the gp100 Env surface glycoprotein as evidenced by the recognition of the mouse antisera to MBP-rev exon 2 to p102 Env precursor and the p23rev but not gp100. The presence of the putative BIV primary fusion domain within the extracellular domain of the BIV transmembrane protein (TM-ecd) was not determined due to the lack of reagents.