Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure
Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure
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DOI：
10.1128/jvi.71.8.6183-6190.1997
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发表时间：
1997-08-01
影响因子：
5.4
通讯作者：
Baker, SC
中科院分区：
文献类型：
--
作者：
Rowe, CL;Fleming, JO;Baker, SC
冠状病毒RNA在持续感染期间在小鼠的中枢神经系统(CNS)中进化。这种进化可以通过检测一种病毒准种的尖峰缺失变体(SDVS)来监测(C.L.Rowe，S.C.Baker，M.J.Nathan和J.O.Fleming，J.Virol。71：2959-2969,1997)。我们和其他人发现，缺失集中在尖峰基因序列5‘端1,200到1,800个核苷酸的区域，称为’‘高变区’‘。为了解决SDVS可能发生的原因，我们生成了整个4,139-nT尖峰RNA序列的正链和负链的预测折叠结构。我们发现一个突出的、分离的茎环结构与每个结构中的高变区一致，为了确定这个预测的茎环是否是RNA重组的“热点”，我们重新评估了在从急性和持续感染的小鼠的中枢神经系统分离的病毒序列中，这一尖峰区域是否比其他三个选定的尖峰序列区域更频繁地缺失。利用克隆的SPEK逆转录-PCR产物的差异菌落杂交，我们检测到热点缺失的SDVS，但没有检测到SPEK序列其他区域完全缺失的SDVS。此外，对25种不同模式的SDVS的交叉位点的序列分析和定位表明，大多数交叉位点聚集在分离的茎环底部的两个区域，我们将这两个区域命名为高频重组位点1和2。有趣的是，SDVS的大多数左右交叉位点彼此直接交叉或邻近，表明这些SDVS可能是由分子内重组产生的。总体而言，我们的结果与尖峰RNA二级结构作为持续感染期间SDVS产生的促成因素的重要作用是一致的。
Coronavirus RNA evolves in the central nervous systems (CNS) of mice during persistent infection. This evolution can be monitored by detection of a viral quasispecies of spike deletion variants (SDVs) (C. L. Rowe, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-2969, 1997). We and others have Pound that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike gene sequence, termed the ''hypervariable'' region. To address how SDVs might arise, we generated the predicted folding structures of the positive- and negative-strand senses of the entire 4,139-nt spike RNA sequence. We found that a prominent, isolated stem-loop structure is coincident with the hypervariable region in each structure, To determine if this predicted stem-loop is a ''hot spot'' for RNA recombination, Re assessed whether this region of the spike is more frequently deleted than three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of the spike sequence were exclusively deleted. Furthermore, sequence analysis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-frequency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely generated by intramolecular recombination. Overall, our results are consistent with there being an important role for the spike RNA secondary structure as a contributing factor in the generation of SDVs during persistent infection.