Expression and Characterization of Human Immunodeficiency Virus Type 1 Envelope Mutant Glycoproteins by Using Baculovirus Expression System

Considerable effort has been directed at understanding the structure and function of HIV-1 envelope glycoproteins. It has been difficult to characterize HIV-1 envelope glycoproteins due to the limited availability of these proteins from virus particles or infected cells. To facilitate the structural and functional analysis of HIV-1 envelope glycoproteins, recombinant baculoviruses were generated to express wild type or mutant HIV-1 envelope glycoproteins. The gp160 precursor protein as well as the gp120 glycoprotein were detected in the cell infected with recombinant BacENVw.t containing wild type HIV-1 envelope gene. In the insect cells infected with recombinant BacENVc with mutations at the cleavage site of gp160, a precursor form of envelope glycoprotein was produced, but not secreted into the culture medium. However, the insect cells infected with recombinant BacENVc/t containing both mutations at the cleavage site and membrane spanning region produced mutant envelope glycoproteins that were efficiently secreted into the culture medium in the form of precursor. Therefore the recombinant HIV-1 envelope glycoproteins produced in this system would be useful as immunogens in the development of a vaccine against AIDS.

Expression and Characterization of the Human Immunodeficiency Virus Type 1 Mutant Envelope Glycoproteins in Mammalian Cells

Human immunodeficiency virus type 1(HIV-1) envelope glycoprotein is synthesized as a 160KDa precursor, gp160, that is cleaved by a cellular protease to form the gp120 and gp41 subunits. Mammalian expression vectors were designed that are capabae of efficient expression of various mutant envelope glycoproteins derived from a molecular clone of HIV-1. To construct these vectors, one type of mutation was made at the gp120-gp41 cleavage site by oligonucleotide directed mutagenesis. And another mutation was made to change amino acids in the membrane spanning region of HIV-1 gp41 important for membrane anchorage. Next, these two mutations were combined to generate a vector to have double mutations in cleavage site and membrane spanning region. These mutants were transiently expressed in mammalian cells. The effect of these mutations on envelope glycoprotein synthesis, proteolytic processing and secretion was determined. In addition, cell surface expression and ability of the glycoprotein to induce syncytium formation were examined. This study provides a mammalian expression system that is capable of efficient expression and secretion of soluble gp160.