Hinton Tracey M, Crabb Brendan S
Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology, The University of Melbourne, Australia1.
The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, VIC 3050, Australia2.
J Gen Virol. 2001 Sep;82(Pt 9):2257-2269. doi: 10.1099/0022-1317-82-9-2257.
Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem-loop structures (H-L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189-920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem-loops H-L as well as four additional upstream stem-loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869-880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.
马鼻炎B病毒（ERBV）最近被归类为埃博病毒属，这是小RNA病毒科中的一个新属。ERBV与利用II型内部核糖体进入序列（IRES）起始翻译的心病毒属和口蹄疫病毒属成员关系较远。我们发现ERBV也拥有II型IRES的核心茎环结构（H-L）。使用双顺反子质粒对ERBV IRES的功能进行了表征，该质粒通过转染到BHK-21细胞以及在兔网织红细胞裂解物中进行体外转录和翻译来分析。在这两个系统中，多聚（C）序列下游核苷酸（nt）189 - 920所包含的区域对于最大程度的翻译是必需的。该序列包括茎环H-L以及另外四个上游茎环。nt 904对应于位于多嘧啶序列（核苷酸869 - 880）下游紧邻的三个框内AUG密码子中的第二个。定点诱变表明，尽管AUG1在多嘧啶序列下游16 nt处位置合适，但AUG2是主要的起始密码子。在直接的IRES竞争实验中，ERBV IRES能够与脑心肌炎病毒（EMCV）的IRES强烈竞争翻译因子。当在体外（IRES顺式或反式竞争）和体内（IRES顺式竞争）进行检测时都是如此。对几种IRES强度的比较分析表明，ERBV IRES与EMCV的IRES一样，是一种强大的翻译诱导剂，在哺乳动物表达系统中可能具有类似的应用潜力。
