Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins
Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins
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DOI：
10.1006/jmbi.1998.1968
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发表时间：
1998-11-20
影响因子：
5.6
通讯作者：
Wainberg, MA
中科院分区：
文献类型：
--
作者：
Cherry, E;Liang, C;Wainberg, MA
我们选择性诱变 1 型人类免疫缺陷病毒 (HIV-1) 的蛋白酶 (PR) 和逆转录酶 (RT) 基因之间连接处的特定残基，以研究 PR-RT 融合蛋白在全长、感染性原病毒构建体中的作用。分析源自用该构建体转染的COS-7细胞的突变病毒的病毒复制、成熟；和传染性。免疫印迹分析表明，该突变阻止了 PR 和 RT 蛋白之间的裂解，并且两者都以 PR-RT 融合蛋白的形式存在于每种细胞和病毒裂解物中。有趣的是，存在于 PR-RT 融合蛋白内的细胞内 PR 仍保持功能活性，从而有效地加工 HIV-1 前体蛋白。此外，如 RT 测定所示，融合蛋白的 RT 成分也保留了其酶活性。电镜显示含有PR-RT融合蛋白的突变病毒具有野生型形态。这些病毒还对 HIV-1 PR 和 RT 活性的抑制剂表现出野生型敏感性。然而，含有PR-RT融合蛋白的病毒的传染性比野生型病毒低20倍。当突变的 Gag-Pol 蛋白由于干扰移码的额外突变而过度表达时，这种缺陷更加明显。因此，与 PR N 末端的裂解位点突变不同，PR 和 RT 之间的裂解位点突变不会影响 PR 或 RT 的酶活性，并且含有 PR-RT 融合蛋白的病毒是可行的。 (C) 1998 年学术出版社。
We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral con struct. Mutant viruses derived from COS-7 cells transfected with this con struct were analyzed in regard to each of viral replication, maturation; and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable. (C) 1998 Academic Press.