Luban J, Lee C, Goff S P
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032.
J Virol. 1993 Jun;67(6):3630-4. doi: 10.1128/JVI.67.6.3630-3634.1993.
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 Pr55gag 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.
我们已在细菌中表达了1型人类免疫缺陷病毒（HIV-1）蛋白酶（PR），其形式为Gag-PR多聚蛋白（J. Luban和S.P. Goff，《病毒学杂志》65:3203 - 3212，1991年）。该蛋白具有酶活性，可将Gag多聚蛋白前体Pr55gag切割成预期产物。PR酶仅作为二聚体具有活性，我们推测PR激活可能用作多聚蛋白多聚化的指标。我们在gag基因上构建了25个连接子插入突变，并通过细菌裂解物中Gag切割产物的出现来评估突变型Gag-PR多聚蛋白的PR活性。所有突变体构建体在细菌中都产生了稳定的蛋白质。大多数Gag-PR突变体的PR活性与野生型无差异。六个突变体，一个在基质（MA）中有插入，四个在衣壳（CA）中有插入，一个在核衣壳（NC）中有插入，全面破坏了多聚蛋白的加工过程。当在单独的质粒上反式提供PR时，可以以野生型效率切割Gag蛋白。这些结果表明，被鉴定为破坏多聚蛋白加工的gag突变并未掩盖多聚蛋白的可裂解键。相反，这些突变可能通过阻止多聚蛋白二聚化，从而在Gag-PR多聚蛋白的背景下阻止了PR激活。
