Vidalin O, Major M E, Rayner B, Imbach J L, Trépo C, Inchauspé G
Institut National de la Santé et de la Recherche Médicale U271, Lyon, France.
Antimicrob Agents Chemother. 1996 Oct;40(10):2337-44. doi: 10.1128/AAC.40.10.2337.
We have explored different domains within the hepatitis C virus (HCV) 5' noncoding region as potential targets for inhibition of HCV translation by antisense oligodeoxynucleotides (ODNs). Inhibition assays were performed with two different cell-free systems, rabbit reticulocyte lysate and wheat germ extract, and three types of chemical structures for the ODNs were evaluated: natural phosphodiesters (beta-PO), alpha-anomer phosphodiesters (alpha-PO), and phosphorothioates (PS). A total of six original ODNs, displaying sequence-specific inhibition ranging from 62 to 96%, that mapped in the pyrimidine-rich tract (nucleotides [nt] 104 to 127) and in the initiator AUG codon (nt 338 to 357) were identified. Two ODNs, which were targeted at the initiatory AUG (nt 341 to 367 and 351 to 377) and which had been previously described as active against genotype 1b and 2a sequences, were shown to exhibit inhibition of expression (> 95%) of a type 1a sequence. Control experiments with the irrelevant chloramphenicol acetyltransferase sequence as a marker and randomized ODNs demonstrated that levels of inhibition associated with the use of PS compounds (of as much as 94%) were mainly due to nonspecific effects. Both alpha- and beta-PO ODNs were found equally active, and no difference could be seen in the activity of beta-PO when it was tested in either rabbit reticulocyte lysate or wheat germ extract, suggesting that RNase H-independent mechanisms may be involved in the inhibitions observed. However, specific RNA cleavage products generated from beta-PO inhibition experiments could be identified, indicating that, with these compounds, control of translation also involves RNase H-dependent mechanisms. This study further delimits the existence of favorable target sequences for the action of ODNs within the HCV 5' noncoding region and indicates the possibility of using nuclease-resistant alpha-PO compounds in cellular studies.
我们探索了丙型肝炎病毒（HCV）5'非编码区内的不同区域，作为反义寡脱氧核苷酸（ODN）抑制HCV翻译的潜在靶点。使用两种不同的无细胞系统（兔网织红细胞裂解物和小麦胚芽提取物）进行抑制试验，并评估了ODN的三种化学结构类型：天然磷酸二酯（β-PO）、α-异头磷酸二酯（α-PO）和硫代磷酸酯（PS）。共鉴定出六个原始ODN，其序列特异性抑制范围为62%至96%，这些ODN定位于富含嘧啶的区域（核苷酸[nt]104至127）和起始AUG密码子（nt 338至357）。两个靶向起始AUG（nt 341至367和351至377）且先前已被描述对1b型和2a型序列有活性的ODN，显示出对1a型序列表达的抑制作用（>95%）。以无关的氯霉素乙酰转移酶序列作为标记的对照实验和随机ODN表明，与使用PS化合物相关的抑制水平（高达94%）主要是由于非特异性效应。发现α-PO和β-PO ODN具有同等活性，并且在兔网织红细胞裂解物或小麦胚芽提取物中测试β-PO时，其活性没有差异，这表明不依赖RNase H的机制可能参与了观察到的抑制作用。然而，可以鉴定出β-PO抑制实验产生的特异性RNA裂解产物，这表明对于这些化合物，翻译控制也涉及依赖RNase H的机制。本研究进一步界定了HCV 5'非编码区内ODN作用的有利靶序列的存在，并表明在细胞研究中使用抗核酸酶的α-PO化合物的可能性。
