Pogue G P, Hall T C
Department of Biology, Texas A & M University, College Station 77843-3258.
J Virol. 1992 Feb;66(2):674-84. doi: 10.1128/JVI.66.2.674-684.1992.
Sequences with strong similarity to internal control regions 1 and 2 (ICR1 and -2; A and B boxes) of tRNA genes are found at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other plant viruses. The functionality of these motifs was studied by introducing point mutations into the ICR2-like sequence of pRNA-2 M/S, a BMV RNA-2 deletion mutant that replicates in the presence of RNAs-1 and -2 but does not encode a functional viral protein. The accumulation of positive-strand progeny from pRNAs bearing single and double base substitutions was 70 to 91% lower than that of the wild type, while the addition of single bases at position 8 of this motif reduced replication by 80%. These dramatic decreases in positive-strand synthesis paralleled decreases in transcription seen (C. Traboni, G. Ciliberto, and R. Cortese, Cell 36:179-187, 1984) from a tRNAPro gene containing similar mutations, suggesting comparable functions for the ICR regions in protein factor binding and demonstrating that a wild-type composition of the virus ICR2-like motif is required for proper RNA replication. Substitutions introduced at bases surrounding the ICR2 motif yielded levels of pRNA replication that differed, depending on the maintenance of a putative 5' stem-loop structure in the positive strand of the viral genome. Mutations disrupting this positive-strand stem-loop while maintaining the integrity of the complementary negative-strand structure reduced pRNA replication by 85 to 97%. In contrast, disruption of the negative-strand structure while maintaining the positive-strand stem-loop did not reduce pRNA replication. Similar positive-strand structures can be predicted to form from 5' sequences of cucumber mosaic virus (strain Q) and cowpea chlorotic mottle virus RNAs-1 and -2, supporting the concept that such structures comprise an integral part of virus genomic positive-strand promoters. The requirement of a stem-loop structure present on the positive-strand provided the basis for a new model describing how these sequence and structural elements act in the production of virus positive-strand RNA.
在雀麦花叶病毒（BMV）及其他植物病毒的基因组RNA的5'末端，发现了与tRNA基因的内部控制区1和2（ICR1和-2；A盒和B盒）具有高度相似性的序列。通过将点突变引入pRNA-2 M/S的ICR2样序列来研究这些基序的功能，pRNA-2 M/S是一种BMV RNA-2缺失突变体，在RNA-1和-2存在的情况下进行复制，但不编码功能性病毒蛋白。携带单碱基和双碱基替换的pRNA产生的正链子代积累量比野生型低70%至91%，而在该基序的第8位添加单碱基会使复制减少80%。正链合成的这些显著减少与（C. Traboni、G. Ciliberto和R. Cortese，《细胞》36:179 - 187，1984）从含有类似突变的tRNAPro基因中观察到的转录减少相似，这表明ICR区域在蛋白质因子结合方面具有类似功能，并证明病毒ICR2样基序的野生型组成是正确RNA复制所必需的。在ICR2基序周围的碱基处引入的替换产生了不同水平的pRNA复制，这取决于病毒基因组正链中假定的5'茎环结构的维持情况。破坏这种正链茎环同时保持互补负链结构完整性的突变使pRNA复制减少了85%至97%。相反，在保持正链茎环的同时破坏负链结构并不会减少pRNA复制。可以预测，黄瓜花叶病毒（Q株系）以及豇豆褪绿斑驳病毒RNA-1和-2的5'序列也会形成类似的正链结构，这支持了这样一种概念，即这些结构是病毒基因组正链启动子的一个组成部分。正链上存在茎环结构的要求为一个新模型提供了基础，该模型描述了这些序列和结构元件在病毒正链RNA产生过程中的作用方式。
