Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly
Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly
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DOI：
10.1128/jvi.76.11.5452-5462.2002
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发表时间：
2002-06-01
影响因子：
5.4
通讯作者：
Vogt, VM
中科院分区：
文献类型：
--
作者：
Ma, YM;Vogt, VM
结构蛋白 Gag 是逆转录病毒组装所需的唯一病毒产物。纯化的 Gag 蛋白或 Gag 片段能够在体外自发形成类似于未成熟病毒粒子的颗粒，但此过程需要核酸以及 Gag 的核衣壳结构域。为了检查核酸在体外组装中的作用，我们使用了劳斯肉瘤病毒 Gag 蛋白的纯化、稍微截短的版本、DeltaMBDDeltaPR 和由简单重复序列 GT 组成的 DNA 寡核苷酸。确定不同长度寡核苷酸的表观结合常数，并根据这些值计算 DNA 上蛋白质的结合位点大小。通过基于电子显微镜的定量测定来评估寡核苷酸促进体外组装的能力。我们发现过量的锌或镁离子抑制病毒样颗粒的形成，但不干扰蛋白质-DNA 结合，这意味着与核酸的相互作用是必要的，但对于体外组装来说还不够。在 EDTA 存在下纯化以去除两个半胱氨酸-组氨酸基序上的锌离子后，DeltaMBDDeltaPR 蛋白的结合位点大小估计为 11 个核苷酸 (nt)。当蛋白质在低浓度锌离子存在下纯化时，该值降低至 8 nt。对于无锌形式的蛋白质，促进体外有效组装的 DNA 寡核苷酸的最小长度为 22 nt，对于锌结合形式，DNA 寡核苷酸的最小长度为 16 nt。为了解释结合位点大小和寡核苷酸长度要求之间惊人的 1:2 比例，我们提出了一个模型，其中核酸在组装中的作用是促进一种 Gag 二聚体的形成，该二聚体本身是 Gag 聚合形成未成熟病毒颗粒蛋白壳的关键中间体。
The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, DeltaMBDDeltaPR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the DeltaMBDDeltaPR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.