Arrigo S J, Haines J K, Huffman K M
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston 29425-2230.
DNA Cell Biol. 1995 Jan;14(1):15-23. doi: 10.1089/dna.1995.14.15.
We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein.
我们构建了多种哺乳动物表达载体，用于生产人类免疫缺陷病毒1型（HIV-1）蛋白酶（PR）与HIV-1 Nef蛋白的融合蛋白。这些蛋白的表达可被HIV-1 Tat蛋白诱导。嵌入Nef编码序列且两侧带有PR切割位点的PR能产生高水平的蛋白水解活性PR表达。融合蛋白几乎完全被切割，且未表现出病毒编码PR所具有的调控加工过程。未检测到PR表达的细胞毒性作用。HIV-1 PR的特异性抑制剂（U75875）可抑制PR的自我切割。去除氨基末端PR切割位点对前体融合蛋白的切割没有可测量的影响。切割后的融合蛋白在转染细胞中似乎极其不稳定。这些发现证明了在异源融合蛋白背景下，HIV-1 PR在哺乳动物细胞中的内在活性。
