Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.
Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.
复制标题
甲型肝炎病毒 RNA 5 非翻译区在允许猴肾细胞中指导帽独立翻译的效率低。
DOI：
10.1128/jvi.68.8.5253-5263.1994
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发表时间：
1994
影响因子：
5.4
通讯作者：
Lemon,SM
中科院分区：
文献类型：
--
作者：
Whetter,LE;Day,SP;Elroy-Stein,O;Brown,EA;Lemon,SM
为了在体内表征存在于甲型肝炎病毒（HAV）RNA的5'非翻译区（5' NTR）中的翻译控制元件，我们创建了HAV允许的猴肾细胞系（BT 7-H），该细胞系稳定表达T7 RNA聚合酶并从含有T7启动子的转染DNA进行无帽RNA的胞质转录。通过使用转录双顺反子RNA的BT 7-H细胞证实HAV的5 'NTR内存在内部核糖体进入位点（IRES），其中5' NTR位于顺反子间空间内，控制下游报告蛋白（细菌氯霉素乙酰转移酶）的翻译。然而，与含有脑心肌炎（EMC）病毒IRES或短的非小核糖核酸病毒5'非翻译前导序列的单顺反子转录物的翻译相比，在这些双顺反子转录物和其中HAV 5' NTR位于氯霉素乙酰转移酶编码序列上游的单顺反子T7转录物中由5 'NTR指导的翻译是非常低效的。HAV IRES内的大缺失（Δ 355-532）消除了双顺反子转录物中的IRES活性。相比之下，单顺反子转录物中IRES内的较大缺失（delta 1-354、delta 1-532、delta 1-633和delta 158-633）导致翻译增加4至14倍。在后一种情况下，这很可能是由于从IRES指导的翻译转变为5 '末端依赖性扫描的翻译起始。含有EMC病毒IRES或非小核糖核酸病毒前导序列的RNA的翻译通过将报告基因构建体与pEP 2A共转染而显著增强，pEP 2A指导含有与脊髓灰质炎病毒2Apro编码区融合的EMC病毒IRES的RNA的转录。这种2Apro对帽非依赖性翻译的增强表明，在2Apro介导的真核起始因子eIF-4F的p220亚基的切割和随后的5'帽依赖性翻译的关闭之后，限制性细胞翻译因子的可用性更高。相反，pEP 2A共转染导致HAV IRES在单顺反子或双顺反子转录物中指导的翻译的严重抑制。这种抑制是由于来自存在于pEP-2A转录物中的EMC病毒IRES的竞争，以及具有蛋白水解活性的2Apro的表达。2Apro介导的HAV翻译抑制在HAV IRES中含有大缺失的转录物（delta 158-633、delta 1-532或delta 1-633）中未观察到。这些数据表明，HAV IRES可能对完整的p220有独特的要求，或者它可能依赖于另一种细胞翻译因子的活性表达，这种因子通常以严格限制的数量存在。（400字处截断摘要）
To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)