Arrigo S J, Huffman K
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston 29425-2230, USA.
J Virol. 1995 Oct;69(10):5988-94. doi: 10.1128/JVI.69.10.5988-5994.1995.
Constructs were generated in which expression of human immunodeficiency virus type 1 (HIV-1) protease (PR) was placed under control of the HIV-1 long terminal repeat, thus requiring the HIV-1 Tat protein for expression of PR. The activity of PR was assessed by cotransfection with a construct producing a Gag substrate. Expression of PR as an intramolecular multimer resulted in a large increase in PR activity in comparison with the level obtained with the expression of PR as a monomer. A cytotoxic effect of PR expression was also exhibited by the constructs expressing PR multimers. CD4+ T-cell lines were generated with a construct producing PR as a linked tetramer and screened for PR activity and inducibility. The replication of HIV-1 in these cell lines was several orders of magnitude reduced in comparison with that in cell lines not expressing PR. Infection in these cell lines could be detected early after infection but disappeared over time. Infection of the PR-expressing cell lines could be increased several orders of magnitude by the addition of a specific inhibitor of PR, U75875 (Upjohn), after infection of the cells, demonstrating that the potent inhibition of HIV-1 replication in these cells was directly due to the expression of PR.
构建了一些载体，其中人类免疫缺陷病毒1型（HIV-1）蛋白酶（PR）的表达置于HIV-1长末端重复序列的控制之下，因此PR的表达需要HIV-1反式激活因子（Tat）蛋白。通过与产生Gag底物的载体共转染来评估PR的活性。与以单体形式表达PR相比，以分子内多聚体形式表达PR导致PR活性大幅增加。表达PR多聚体的载体也表现出PR表达的细胞毒性作用。用产生作为连接四聚体的PR的载体生成CD4 + T细胞系，并筛选PR活性和诱导性。与未表达PR的细胞系相比，HIV-1在这些细胞系中的复制降低了几个数量级。在这些细胞系中感染可在感染后早期检测到，但随时间消失。在细胞感染后添加PR的特异性抑制剂U75875（Upjohn）可使表达PR的细胞系中的感染增加几个数量级，这表明这些细胞中HIV-1复制的有效抑制直接归因于PR的表达。
