GROWTH-RESTRICTED DENGUE VIRUS MUTANTS CONTAINING DELETIONS IN THE 5' NONCODING REGION OF THE RNA GENOME
GROWTH-RESTRICTED DENGUE VIRUS MUTANTS CONTAINING DELETIONS IN THE 5' NONCODING REGION OF THE RNA GENOME
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DOI：
10.1006/viro.1995.1052
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发表时间：
1995-02-20
期刊：
影响因子：
3.7
通讯作者：
LAI, CJ
中科院分区：
文献类型：
--
作者：
CAHOUR, A;PLETNEV, A;LAI, CJ
登革4型病毒（DEN 4）RNA基因组含有101-nt的5'非编码（NC）序列，该序列被预测形成稳定的二级结构。使用可转录感染性RNA的DEN 4 cDNA来工程化5 ′ NC区中的缺失，用于RNA结构的功能分析和用于分离可被评估为用于活减毒疫苗的候选物的DEN 4突变体。在核苷酸18和98之间的DEN 4基因组区域中构建了11个不同的缺失;预测每个突变改变或破坏5' NC RNA结构中的局部碱基配对。从这些缺失突变体中的5个的RNA转录物中未回收感染性病毒。值得注意的是，4的5个明显致命的缺失位于一个5- 6-nt的碱基配对区域的预测长茎或邻近它。相反，有一个例外，突变体产生感染性病毒的缺失位于一个环或短茎区域。在体外检测缺失对病毒RNA转录物翻译效率的影响。不产生活病毒的缺失构建体的RNA转录物以野生型病毒转录物的40 - 160%的效率翻译。感染性RNA转录物的翻译效率也不同。与野生型病毒相比，从RNA转录物中回收的缺失突变体表现出低至中等的翻译效率，具有小的空斑形态，并且在猿猴LLC-MK（2）和蚊子C6/36细胞中表现出生长减少。在11个突变体构建体中，nts 82-87的缺失引起翻译效率的最大降低。然而，从用突变体d（82-87）的RNA转录物转染的LLC-MK（2）细胞中回收到感染性病毒。该突变体的后代在LLC-MK（2）细胞上产生小噬斑，并在这些细胞中生长至低滴度。与野生型DEN 4或其他测试的DEN 4缺失突变体不同，突变体d（82-87）不能在C6/36细胞上产生噬斑，并且在胸内接种后在埃及伊蚊和白纹伊蚊中也是复制缺陷的。(C)出版社：Academic Press
The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK(2) and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK(2) cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK(2) cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation. (C) 1995 Academic Press, Inc.