In vivo processing of Pr160gag-pol from human immunodeficiency virus type 1 (HIV) in acutely infected, cultured human T-lymphocytes.

The processing of the HIV-1 Pr160gag-pol precursor polyprotein was analyzed in freshly HIV-1-infected MT-4 cultured cells. Single intermediates of the processing cascade were characterized by immunoblotting using distinct antisera. A potent inhibitor of the HIV protease (PR), Ro 31-8959, was employed to block cleavage by the mature PR, thus allowing insights into initial stages of the gag-pol (auto)-catalytical processing. While most known gag-pol cleavages were blocked in the presence of the inhibitor, the cleavage site between the gag-NC and the pol-p6 domains was still cleaved even in presence of high amounts (1 microM) of inhibitor, leading to the accumulation of a novel 114-kDa polyprotein comprising p6-PR-RT-IN. In the absence of inhibitor no accumulation of p114 was observed. In inhibitor-treated, HIV-1-infected cells a p6-PR intermediate was also detected, indicating subsequent cleavage of the PR/RT scissile bond. These results demonstrate initial cleavage(s) of the gag-pol precursor hydrolyzed by a proteolytic activity different from the mature PR and indicate that p114 (p6-PR-RT-IN) and p6-PR intermediates could play an essential role in the PR activation process.