We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr559a`to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.

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