Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV‐specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pol gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two‐step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27‐kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy‐terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27‐kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence‐ but also conformation‐dependent. This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.

SummaryThe integration protein of the human immunodeficiency virus type 1 was purified from recombinant bacteria overproducing this enzyme. The final step of purification, namely chromatography on polyUsepharose, yielded a homogeneous protein preparation showing specific DNA cutting and joining activities. For a convenient assay of the endonuclease reaction, a 21-mer duplex oligonucleotide corresponding to the U5-LTR end of the viral DNA was radiolabelled at the dinucleotide that is removed by the enzyme. After the reaction, assay mixtures were passed through DEAEcellulose filters which bind the substrate, but not the short radiolabelled product. Thus, an enzyme-dependent decrease of bound radioactivity was observed with time. Reaction rate was linearly dependent on enzyme concentration and the amount of substrate used was far below saturating concentrations. The reaction showed a pH-optimum~t 7.5 and was strictly dependent on the presence of Mn 2 +. The presence of reducing agents like 2-mercaptoethanol was not essential for enzymatic activity. The assay was used to test selected compounds for their inhibitory potential against integrase. Typical inhibitors of DNA-topoisornerases did not inhibit the endonuclease reaction, with the exception of the intercalative agent actinomycin D which blocked the reaction with an ICso-value of 3jLM. Dextran sulphate inhibited the enzyme with an IC oo = 1.6jLg mr".

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