Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain.
Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain.
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来自 PR 结构域 N 末端具有阻断突变的前体的新型人类免疫缺陷病毒 1 型蛋白酶蛋白的蛋白水解活性。
DOI：
10.1128/jvi.68.1.240-250.1994
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发表时间：
1994
影响因子：
5.4
通讯作者：
Carter,C
中科院分区：
文献类型：
--
作者：
Zybarth,G;Krausslich,HG;Partin,K;Carter,C
成熟的人类免疫缺陷病毒 1 型蛋白酶（PR；11 kDa）可以切割 Gag 和 Gag-Pol 多蛋白前体中的所有域间连接。为了确定该酶前体形式的活性，我们通过在 PR 结构域的 N 末端 Phe-Pro 切割位点引入突变，阻止了截短的 Gag-Pol 多蛋白释放成熟 PR。突变体前体在大肠杆菌中表达后可以有效地自动加工。未发布可检测到的成熟 PR；然而，一些与 PR 相关的产品的大小范围从大约 14 到 18 kDa 累积。当用野生型 PR 消化突变体前体时，会产生相同大小的产物。因此，PR 可以利用 PR 结构域上游区域的切割位点，导致形成延伸的 PR 物种。根据活性位点滴定，由突变前体产生的 PR 物种在肽底物上表现出野生型活性。然而，当比较等摩尔量的延伸蛋白和野生型PR蛋白时，这些延伸酶对外源提供的多蛋白底物的蛋白水解活性较低。表达突变前体的哺乳动物细胞主要产生前体，并显着减少成熟产物的数量。释放的颗粒主要由未切割或部分切割的多蛋白组成。我们的结果表明，PR 的前体形式可以自动加工，但在加工 Gag 前体形成成熟病毒颗粒时效率较低。
The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors. To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain. The mutant precursor autoprocessed efficiently upon expression in Escherichia coli. No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated. Products of the same size were generated when mutant precursors were digested with wild-type PR. Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species. On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates. However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared. Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products. Released particles consisted mostly of uncleaved or partially cleaved polyproteins. Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles.