CIS-ACTING LESIONS TARGETED TO THE HYDROPHOBIC DOMAIN OF A POLIOVIRUS MEMBRANE-PROTEIN INVOLVED IN RNA REPLICATION
CIS-ACTING LESIONS TARGETED TO THE HYDROPHOBIC DOMAIN OF A POLIOVIRUS MEMBRANE-PROTEIN INVOLVED IN RNA REPLICATION
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DOI：
10.1128/jvi.66.10.6045-6057.1992
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发表时间：
1992-10-01
影响因子：
5.4
通讯作者：
SEMLER, BL
中科院分区：
文献类型：
--
作者：
GIACHETTI, C;HWANG, SS;SEMLER, BL
采用分子遗传学方法结合体外生化分析研究了脊髓灰质炎病毒多肽3AB中疏水结构域的结构要求。我们在这里报道了旨在降低该结构域疏水特性的缺失、插入和氨基酸替换突变的产生和分析。我们的结果表明，3AB的这个区域的疏水性是维持正常病毒RNA合成所必需的。然而，突变蛋白的体外膜关联实验并没有建立3AB膜关联与病毒RNA合成之间的直接关联。我们设计的一些致死突变产生了由于多蛋白正常切割顺序的改变而具有异常P2和p3加工能力的多蛋白。对这些突变体的详细分析表明，P2不是多肽2A和2BC的主要前体，P2蛋白产物来源于含有P2- p3的前体（最有可能是P2- p3或P2- 3ab）。这些前体很可能是由初级多蛋白裂解事件引起的，该事件启动了先前未记载的蛋白水解级联反应。我们的研究结果还表明，3AB的疏水结构域所提供的功能在反式中是无法提供的。我们讨论了这些结果对有限扩散复制复合物形成的影响，作为隔离RNA合成和蛋白质加工所需的P2和p3区域多肽的手段。
The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.