A CIS-ACTING MUTATION IN THE SINDBIS VIRUS JUNCTION REGION WHICH AFFECTS SUBGENOMIC RNA-SYNTHESIS
A CIS-ACTING MUTATION IN THE SINDBIS VIRUS JUNCTION REGION WHICH AFFECTS SUBGENOMIC RNA-SYNTHESIS
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DOI：
10.1128/jvi.63.12.5216-5227.1989
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发表时间：
1989-12-01
影响因子：
5.4
通讯作者：
RICE, CM
中科院分区：
文献类型：
--
作者：
GRAKOUI, A;LEVIS, R;RICE, CM
辛德比斯病毒负链以及基因组和亚基因组RNA的合成被认为需要模板RNA中的特定顺式作用序列或结构以及病毒特异性蛋白质和反式作用的宿主成分的组合。连接区约 21 个核苷酸的保守序列包含亚基因组 RNA 的起始位点，已被认为可作为负链模板上的启动子来合成亚基因组 RNA (J.-H. Ou, C. M., Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79：5235-5239，1982）。我们在此序列中引入了一个三碱基插入，它还在辛德比斯病毒的 cDNA 克隆中的 nsP4 COOH 末端附近插入了一个氨基酸，从中可以生成感染性 RNA 转录本。这种名为 Toto1100CR4.1 的突变体的表型通过鸡胚成纤维细胞或 BHK 细胞的感染性 RNA 转染进行了研究。该突变导致亚基因组 RNA 水平急剧下降，但不会改变 RNA 的起始位点。可能是由于结构蛋白合成抑制的结果，很少有子代病毒粒子被释放，并且突变体即使在长时间孵育后也会产生微小或模糊的斑块。该突变的顺式作用效果通过将野生型或突变体连接区掺入有缺陷的干扰RNA中并在野生型辅助病毒存在的情况下检查体内有缺陷的干扰RNA衍生的亚基因组RNA的相对合成来证明。这些结果表明，连接区被尚未鉴定的 firal 反式作用成分识别，用于亚基因组 RNA 合成。当被尚未鉴定的病毒反式作用成分识别用于亚基因组 RNA 合成时。当Toto1100CR4.1突变体在培养物中传代时，很容易出现噬菌斑形态变异。总共分离出 24 个独立回复体，并对 16 个进行了详细表征。所有分析的回复体均显示亚基因组 RNA 合成水平有所增加。连接区的序列分析表明，所有这些都是假回复突变体，只有两个在连接区含有潜在的补偿性变化。开发了一种检测方法来鉴定在参与亚基因组 RNA 合成的反式作用病毒成分中具有第二位点变化的回复体。至少鉴定出两种这样的回复突变体。这些和其他第二位点补偿突变的图谱可以提供关于哪些病毒特异性蛋白负责与保守连接区域相互作用以促进亚基因组 RNA 合成的遗传线索。
The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been propoed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M., Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of sindbis virus from which infectious RNA transctripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied infectious RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction regions into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified firal trans-acting components for subgenomic RNA synthesis. When the recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independence revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.