OVEREXPRESSION OF THE HIV-1 GAG-POL POLYPROTEIN RESULTS IN INTRACELLULAR ACTIVATION OF HIV-1 PROTEASE AND INHIBITION OF ASSEMBLY AND BUDDING OF VIRUS-LIKE PARTICLES
OVEREXPRESSION OF THE HIV-1 GAG-POL POLYPROTEIN RESULTS IN INTRACELLULAR ACTIVATION OF HIV-1 PROTEASE AND INHIBITION OF ASSEMBLY AND BUDDING OF VIRUS-LIKE PARTICLES
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DOI：
10.1006/viro.1993.1174
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发表时间：
1993-04-01
期刊：
影响因子：
3.7
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
KARACOSTAS, V;WOLFFE, EJ;MOSS, B
一些逆转录病毒，包括HIV-1，通过翻译移码机制调节gagandpolgene产物的相对量。使用牛痘病毒表达载体测试了改变Gag和Pol蛋白比例的结果，其中Gag和Pol基因通过将它们置于相同的开放阅读框中而融合。细胞裂解物的免疫印迹表明，约160 kDa的蛋白质，预期的翻译产物的fusedgag-polgene，是主要的物种检测与HIV特异性抗血清在感染这种重组病毒的前几个小时。随后，全长多聚蛋白的数量减少，并出现了一系列的GAG相关的中间大小的蛋白质。在感染后期，p24和豆蔻酰化p17 Gag蛋白占主导地位，并且与野生型gag-polgene形成的量相比，检测到更大量的细胞内加工逆转录酶、整合酶和蛋白酶。当野生型gag-polgene表达时，通过电子显微镜观察到大量出芽、未成熟和成熟的逆转录病毒样颗粒，而在表达融合型gag-polgene的细胞中未检测到颗粒。通过（i）用拟肽抑制剂抑制HIV-1蛋白酶，（ii）诱变蛋白酶的活性位点，或（iii）通过缺失大部分逆转录酶基因缩短Gag-Pol多蛋白，部分克服了对病毒组装的阻断。然而，出芽是低效的，结构似乎不成熟，经常异常。这些结果表明，全长Gag-Pol多聚蛋白的过量生产和细胞内蛋白酶活性的增加都不利于病毒组装。进一步的实验表明，当豆蔻酰化被阻止时，Gag和Gag-Pol多聚蛋白的细胞内加工在不存在颗粒形成的情况下发生。
Some retroviruses, including HIV-1, regulate the relative amounts ofgagandpolgene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which thegagandpolgenes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fusedgag-polgene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-typegag-polgene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-typegag-polgene was expressed, whereas no particles were detected in cells that expressed the fusedgag-polgene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.