Restoration of a stem-loop structure required for potato virus X RNA accumulation indicates selection for a mismatch and a GNRA tetraloop.
Restoration of a stem-loop structure required for potato virus X RNA accumulation indicates selection for a mismatch and a GNRA tetraloop.
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马铃薯病毒 X RNA 积累所需的茎环结构的恢复表明对错配和 GNRA 四环的选择。
DOI：
10.1006/viro.1999.9843
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Hemenway,C
中科院分区：
文献类型：
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作者：
Miller,ED;Kim,KH;Hemenway,C
马铃薯X病毒（PVX）RNA的5′端含有一个茎环结构，即茎环1（SL 1），这是正链RNA有效积累所必需的。为了确定SL 1中单个元件的变化如何被病毒容纳，我们将含有末端四环（TL）、茎C（SC）和茎D（SD）区域修饰的PVX转录物接种到本氏烟草（Nicotiana benthamianaplants）上，并在一系列传代中分析后代RNA。几个后代RNA分离的TL突变体含有GAAA基序的第一个核苷酸的变化或删除的整个TL序列接种的植物被发现含有多个A插入的末端环区域内。野生型TL基序，GAAA，恢复所有的TL突变体的第二代，这表明该元件的序列和潜在的结构是至关重要的PVX感染。从接种突变体的SD和SC的中心区域的植物中分离的回复突变体RNA表明，增加的茎长是耐受的。SD长度恢复到野生型PVX RNA典型的4 bp，伴随着A插入环C。具有C55-C78错配转化为G-C对、在SC中心区域内重新定位该错配或缺失C55-C78的突变体不能感染原生质体和植物。相比之下，具有C55-C78错配到A-C错配的转换的突变体（其在原生质体中表现出低水平的PVX正链RNA）能够感染植物并迅速恢复到野生型C-C错配。这些数据表明，SL 1内的重要序列和二级结构元件是有效的病毒感染所必需的，并且TL和环C区域内的多个A插入，可能通过聚合酶口吃，伴随着SL 1结构的恢复。
The 5′ region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions ontoNicotiana benthamianaplants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55–C78 mismatch to a G–C pair, relocation of this mismatch within the central region of SC, or deletion of C55–C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55–C78 mismatch to an A–C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C–C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure.