Processing of retroviral gag polyproteins: An in vitro approach

This chapter focuses on work using in vitro systems to examine HIV-1 protease activation and specificity. By comparisons with results obtained in intact cell systems to illustrate some of the unique advantages of cell-free expression systems for the study of dimeric protein interactions. Overexpression systems utilizing Escherichia coli or baculovirus are essential for production of large quantities of protease and gag-related substrates and facilitate purification and biochemical analysis of these viral components. Transient precursor forms of HIV protease are particularly elusive because the high concentration of these enzymes in such expression systems facilitates their dimerization and autoprocessing even if they are intrinsically poor enzymes. The chapter describes that gag substrates can be produced efficiently for analysis of substrate determinants. The ability to directly control the system by manipulation of the input messenger RNA uniquely permits precise comparison of the intrinsic functionality of the encoded substrate or enzyme.