trans-complementation of flavivirus RNA polymerase gene NS5 by using kunjin virus replicon-expressing BHK cells
trans-complementation of flavivirus RNA polymerase gene NS5 by using kunjin virus replicon-expressing BHK cells
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DOI：
10.1128/jvi.72.9.7270-7279.1998
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发表时间：
1998-09-01
影响因子：
5.4
通讯作者：
Westaway, EG
中科院分区：
文献类型：
--
作者：
Khromykh, AA;Kenney, MT;Westaway, EG
持续表达 Kunjin (KUN) 病毒复制子 RNA 的 BHK 细胞系 (repBHK，类似于我们最近描述的 ME/76Neo BHK 细胞系 [A. A. Khromykh 和 E. G. Westaway, J. Virol. 71:1497-1505, 1997])用于拯救和繁殖 RNA 聚合酶基因 (NS5) 缺陷的 KUN 病毒。一种新的感染性全长 KUN 病毒 cDNA 克隆 FLSDX，由我们先前描述的 cDNA 克隆 pAKUN (A. A. Khromykh 和 E. G. Westaway,, J. Virol. 68:4580-4588, 1994)制备，并且具有比 pAKUN 相似的高 10 (5) 倍的特异性感染性，用于制备缺陷突变体。将预测的RNA聚合酶基序GDD（产生FLdGDD）和预测的甲基转移酶基序之一（S-腺苷甲硫氨酸[SAM]结合位点，产生FLdSAM）的缺失分别引入FLSDX中。将FLdGDD和FLdSAM RNA转录并转染到repBHK细胞中，但不转染到正常BHK细胞中，导致它们的复制和只能在repBHK细胞中复制的有缺陷的病毒的恢复。逆转录 PCR 和测序分析显示，恢复的病毒基因组中保留了引入的缺失。这些缺失的保留，以及在FLdGDD或FLdSAM转染的repBHK细胞长时间孵育（7天）后我们无法恢复能够在正常BHK细胞中复制的病毒，排除了转染RNA中存在的删除的缺陷NS5基因与repBHK细胞中存在的功能性NS5基因之间发生重组的可能性。在转染的repBHK细胞中还补充了GDD基序（FLGVD）点突变的RNA，并且在转染后第3天恢复了缺陷病毒。然而，与 FLdGDD 和 FLdSAM RNA 的结果相反，在正常 BHK 细胞中长时间（4 天或更长时间）孵育 FLGVD RNA 可以恢复病毒，其中 GVD 突变已通过单个碱基变化恢复为野生型 GDD 序列。总体而言，这些结果首次证明了缺陷黄病毒RNA与黄病毒RNA聚合酶基因NS5中有害缺失的反式互补。这里描述的互补系统可能被证明对于影响任何其他黄病毒非结构蛋白的功能域或基本二级结构的缺失和突变的体内互补是有用的。
A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497-1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway,, J. Virol. 68:4580-4588, 1994) and possessing similar to 10(5)-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLdGDD and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FLGVD) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days or more) incubation of FLGVD RNA in normal BHK cells allowed recovery of a virus in which the GVD mutation had reverted via a single base change to the wild-type GDD sequence. Overall, these results represent the first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.