Berkowitz R D, Luban J, Goff S P
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Virol. 1993 Dec;67(12):7190-200. doi: 10.1128/JVI.67.12.7190-7200.1993.
Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.
在病毒粒子组装过程中，逆转录病毒基因组RNA的包装被认为是由gag多聚蛋白与基因组5'端附近的RNA序列（通常称为ψ或E区）之间的特异性相互作用介导的。对于许多逆转录病毒，包括1型人类免疫缺陷病毒（HIV-1），这种相互作用所需的gag蛋白和RNA部分仍不清楚。我们使用RNA凝胶迁移率变动分析来测量纯化的谷胱甘肽S-转移酶-HIV-1 gag融合蛋白与RNA核糖探针的体外结合。发现完整的gag多聚蛋白和单独的核衣壳（NC）蛋白都能特异性结合HIV-1核糖探针。NC的任何一个半胱氨酸-组氨酸框被去除后，都不会消除与ψ核糖探针的特异性结合，但仅包含MA和CA蛋白而没有NC的gag部分不与RNA结合。HIV-1基因组RNA中至少有两个与gag多聚蛋白结合的位点：一个完全在gag的5'端，另一个完全在gag内部。HIV-1 NC蛋白与含有其他逆转录病毒ψ序列的核糖探针的结合几乎与HIV-1 ψ核糖探针一样好。
