Mizuno A, Ido E, Goto T, Kuwata T, Nakai M, Hayami M
Laboratory of Pathogenic Virus, Institute for Virus Research, Kyoto University, Japan.
AIDS Res Hum Retroviruses. 1996 Jun 10;12(9):793-800. doi: 10.1089/aid.1996.12.793.
To clarify the physiological function of two zinc finger motifs in the nucleocapsid (NC) domain of the Gag protein of human immunodeficiency virus type 1 (HIV-1), we changed cysteine to serine in either of the two motifs or both by site-directed mutagenesis. Viral infectivity was lost by any of the mutations, but their effects appeared differently in the respective mutants. Northern blot analysis showed that the first finger mutant was far less efficient (approximately 10% of the wild type) in genomic RNA encapsidation and that the dual mutant of both fingers completely failed to encapsidate the RNA. In contrast, the second finger mutant retained its ability for RNA encapsidation with an efficiency similar to that of the wild type. Immunoblot analysis of the lysates of CD4-positive M8166 cells transfected with the mutant proviral DNAs showed that the processing of Gag precursors was delayed in two mutant viruses having alterations in the first finger sequence, whereas the processing of the second finger mutant appeared to be normal. On the other hand, immunoblot analysis of the virus particles showed that the second finger mutant particles contained some proteins that were thought to be degradation products of p24CA. Electron microscopic observation showed that all particles of these mutant viruses were morphologically alike except that they had a slightly larger diameter than that of the wild type. These results indicate that these finger motifs of HIV-1 NC protein do not function equivalently. Namely, the first finger is primarily responsible for RNA encapsidation and the second is required for stabilization of virus particles.
为阐明人类免疫缺陷病毒1型（HIV-1）Gag蛋白核衣壳（NC）结构域中两个锌指基序的生理功能，我们通过定点诱变将两个基序中的任意一个或两个中的半胱氨酸突变为丝氨酸。任何一种突变都会导致病毒感染力丧失，但它们在各自突变体中的作用表现不同。Northern印迹分析表明，第一个锌指突变体在基因组RNA包装方面效率极低（约为野生型的10%），而两个锌指的双突变体完全无法包装RNA。相比之下，第二个锌指突变体保留了RNA包装能力，效率与野生型相似。对用突变前病毒DNA转染的CD4阳性M8166细胞裂解物进行免疫印迹分析表明，在第一个锌指序列发生改变的两种突变病毒中，Gag前体的加工过程延迟，而第二个锌指突变体的加工过程似乎正常。另一方面，对病毒颗粒的免疫印迹分析表明，第二个锌指突变体颗粒含有一些被认为是p24CA降解产物的蛋白质。电子显微镜观察表明，这些突变病毒的所有颗粒在形态上相似，只是直径比野生型略大。这些结果表明，HIV-1 NC蛋白的这些锌指基序功能并不等同。也就是说，第一个锌指主要负责RNA包装，第二个锌指是病毒颗粒稳定所必需的。
