Ozdarendeli A, Ku S, Rochat S, Williams G D, Senanayake S D, Brian D A
Department of Microbiology, University of Tennessee, College of Veterinary Medicine, Knoxville, Tennessee 37996-0845, USA.
J Virol. 2001 Aug;75(16):7362-74. doi: 10.1128/JVI.75.16.7362-7374.2001.
Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5' and 3' coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (-17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5' end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.
冠状病毒中导致亚基因组mRNA（sgmRNA）合成的机制尚不清楚，但已知涉及一个七聚体信号基序，最初称为基因间序列。基因间序列被认为是RNA依赖性RNA聚合酶（RdRp）在不连续转录过程中的交叉区域（融合位点），该过程导致5'和3'共末端的sgmRNA。在牛冠状病毒中，mRNA 5合成的主要融合位点（GGUAGAC）在三个位置（下划线）不符合典型基序（UC[U,C]AAAC），但它仅位于这样一个序列（UCCAAAC）下游14个核苷酸处。通过计算机预测，不常用的典型序列埋藏在一个稳定发夹的茎中（-17.2千卡/摩尔）。在这里，我们通过酶切探测证明了这个茎的存在，并通过分析从突变的缺陷干扰RNA构建体在体内产生的转录本，研究了它以及相邻序列对融合位点异常选择的影响。我们了解到：（i）预计会以各种方式解开茎环的突变并没有将RdRp交叉点切换到上游典型位点；（ii）一个完全不符合的下游基序导致两个位点都没有可测量的转录；（iii）上游典型位点不能异位发挥作用以使下游非典型位点具有转录能力；（iv）以减少与病毒基因组5'端序列相似性的方式改变下游非典型基序下游的侧翼序列，会导致急剧切换到上游典型位点。这些结果表明，非典型位点下游的序列元件可以显著影响mRNA 5合成的融合位点选择，并被解释为最符合负链合成过程中相似性辅助的RdRp链切换机制。
