7] Identification, purification, and cell culture assays of retroviral proteases

Abstract

This chapter focuses on identification, purification, and cell culture assays of retroviral proteases. The retroviral protease is activated by autocatalytic processing from the zymogen-type gag-pol precursor. The mature protease then processes the remaining precursor to active reverse transcriptase, RNase H, and integrase. The gag precursor is cleaved to yield mature viral capsid. Only mature virus is infectious and in the case of HIV, only infectious virus has a cytotoxic effect to the host cell. Virus protease inhibitors impair all of these functions including the autocatalytic protease activation. In vitro assays of the activity and kinetics of purified protease and the effect of inhibitors are discussed in the chapter. Assays are described for testing the activity of HIV protease in infected cells, screening simultaneously for function on maturation, infectivity, and cytotoxicity of the progeny viruses.