Jia X Y, Tesar M, Summers D F, Ehrenfeld E
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717, USA.
J Virol. 1996 May;70(5):2861-8. doi: 10.1128/JVI.70.5.2861-2868.1996.
The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.
通过分析含有脑心肌炎病毒（EMCV）内部核糖体进入片段（IRES）以及甲型肝炎病毒（HAV）5'端不同长度（237、151或98个核苷酸[nt]）序列的嵌合RNA的翻译和复制情况，研究了5'非翻译区在甲型肝炎病毒（HAV）复制中的作用。所有截短后仅编码衣壳蛋白序列的嵌合RNA在兔网织红细胞裂解物中的翻译效率相同，并且比HAV IRES的翻译效率有显著提高。用亲本HAV RNA和嵌合RNA转染FRhK-4细胞产生了一种活病毒，该病毒在连续传代过程中保持稳定；然而，HAV 5'端超过151 nt的序列才是支持病毒复制所必需的。从亲本RNA转染以及转染含有HAV前237 nt下游EMCV IRES的RNA所回收病毒的单步生长曲线表明，它们的复制动力学和产量相似。当用编码这些病毒RNA的质粒或仅含有亲本或嵌合5'非翻译区下游HAV衣壳编码序列的亚克隆转染感染了产生SP6 RNA聚合酶以扩增HAV RNA的重组痘苗病毒的FRhK-4细胞时，从HAV IRES合成病毒衣壳抗原的效率等于或高于用EMCV IRES所达到的效率。这些数据表明，HAV IRES固有的翻译效率可能不是HAV生长缓慢表型的主要限制决定因素。
