Hertz J M, Huang H V
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
J Virol. 1992 Feb;66(2):857-64. doi: 10.1128/JVI.66.2.857-864.1992.
We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.
我们使用辛德毕斯病毒（一种甲病毒）作为模型来研究病毒顺式作用序列识别的进化。在甲病毒的生命周期中，会产生一条全长负链RNA，它作为基因组RNA复制和亚基因组mRNA转录的模板。转录起始于一个内部启动子位点，即连接序列，以产生亚基因组mRNA。甲病毒的连接序列高度保守，但确实存在一些碱基差异。这些差异在进化过程中可能基本上是中性突变，以至于任何一种当代序列都能被任何一种甲病毒有效识别。或者，这些变化可能导致了显著的功能分歧，使得当代病毒不再能将异源连接序列识别为启动子。为了区分这些可能性，我们构建了带有两个亚基因组mRNA启动子的辛德毕斯病毒衍生物。一个是用于表达结构蛋白的野生型辛德毕斯病毒启动子。另一个是最小的辛德毕斯病毒启动子或来自其他甲病毒的相应连接序列，它们位于细菌氯霉素乙酰转移酶（CAT）基因的上游。通过RNA分析来确定各种连接序列的相对启动子强度。结果表明，除了两个序列外，所有序列都被辛德毕斯病毒识别为启动子。利用CAT酶分析来测量以异源连接序列作为启动子转录的mRNA所产生的CAT蛋白的积累量。大多数病毒表达的CAT酶量彼此相差在10倍以内。两个连接序列未被识别为启动子的病毒没有产生显著的CAT表达。我们得出结论，就辛德毕斯病毒而言，连接序列在功能上是保守的；也就是说，连接序列中大多数当代核苷酸差异是中性或近中性突变。功能保守性表明顺式作用序列及其转录因子的同源结合位点都不能独立改变。顺式作用序列与其同源病毒蛋白结合位点之间的这种协同进化类型可能是一种普遍现象。例如，这就解释了RNA病毒各家族中保守顺式作用序列的普遍存在。这一假说对抗病毒药物的设计具有启示意义。
