We have used the vaccinia virus-T7 RNA polymerase-based expression system for studies on the activity of proteases from various retroviruses on homologous and heterologous Gag polyproteins in eukaryotic cells. Proteases from human immunodeficiency virus (HIV) types 1 and 2, equine infectious anaemia virus, human T cell leukaemia virus type 1 and human spumavirus were produced and were shown to cleave their cognate Gag substrates produced . Analysis of cross reactivity revealed that lentivirus proteases cleaved only lentivirus Gag proteins and oncovirus proteases acted primarily on oncovirus Gag proteins. The HIV-2 protease cleaved the HIV-1 Gag precursor almost as efficiently as HIV-1 protease. Expression of the 5′ end of the human spumavirus gene revealed that it encodes a functional protease that acts specifically on the human spumavirus Gag polyprotein. This assay will allow further investigation on the activity and specificity of retrovirus proteases in eukaryotic cells.


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