Raman Sharmila, Bouma Peter, Williams Gwyn D, Brian David A
Department of Microbiology, University of Tennessee, College of Veterinary Medicine, Knoxville, Tennessee 37996-0845, USA.
J Virol. 2003 Jun;77(12):6720-30. doi: 10.1128/jvi.77.12.6720-6730.2003.
Higher-order structures in the 5' untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5' UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of -9.1 kcal/mol in the positive strand and -3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that its associated intra-5' UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.
在许多情况下，正链RNA病毒5'非翻译区（UTR）中的高阶结构在RNA翻译、复制或转录中起顺式作用元件的功能。在此，我们描述了支持茎环III结构及其在缺陷干扰（DI）RNA复制中的顺式作用功能的证据，茎环III是在32 kb牛冠状病毒（BCoV）基因组210个核苷酸（nt）的5'UTR内预测的四个高阶结构中的第三个。茎环III位于第97至116位核苷酸处，正链的计算自由能为-9.1 kcal/mol，负链为-3.0 kcal/mol，并且从第100位核苷酸开始与之相关联的是一个开放阅读框（ORF），可能编码一个8氨基酸的肽。茎环III被推测在正链中起作用，但其作用链尚未确定。茎环III（i）在第2组冠状病毒中显示出系统发育保守性，并且在第1组和第3组冠状病毒中似乎有一个同源物，（ii）在所有已知序列的冠状病毒中都有一个紧密相关的短的、由AUG起始的5'UTR内ORF，（iii）得到BCoV RNA中酶结构探测证据的支持，（iv）在BCoV DI RNA中DI RNA复制必须保持茎的完整性，并且（v）在BCoV DI RNA中短ORF的维持与最大DI RNA积累之间显示出正相关。这些结果表明，BCoV 5'UTR中的茎环III是DI RNA复制的顺式作用元件，并且其相关的5'UTR内ORF可能起到增强复制的作用。据推测，这两个元件在病毒基因组中功能相似。
