Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas Sophias Ave, 11521 Athens, Greece.
Virus Res. 2011 Jan;155(1):213-20. doi: 10.1016/j.virusres.2010.10.007. Epub 2010 Oct 17.
The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.
丙型肝炎病毒拥有一个重叠核心基因的另类开放阅读框，其产物被称为核心+1 或另类阅读框（ARF）或 F 蛋白（s）。对 HCV-1a 基因型的广泛研究表明，核糖体移码支持核心+1 蛋白的合成，当核心密码子 9-11 中存在连续十个 As 时，而在没有这种模体的情况下，核心+1 ORF 的表达主要通过内部翻译起始来介导。然而，在 HCV-1b 中，尚未描述通过内部翻译起始产生的核心+1 同工型。使用包含先前描述的来自肝活检的 HCV-1b 临床分离物的核心/Core+1(342-770)区域的构建体，我们提供了在瞬时转染的哺乳动物细胞中通过核或细胞质表达系统进行内部翻译起始合成核心+1 蛋白的证据。定点突变分析表明：(a) 核心+1 蛋白的合成与多蛋白表达无关，因为我们观察到缺乏多蛋白翻译起始子的构建体中核心+1 蛋白表达增加，(b) 主要的 Core+1 产物是从 AUG(85)表达的，类似于 HCV-1a 的 Core+1/S 蛋白，(c) 核心+1 同工型的合成也介导自 GUG(58)或在某些条件下 GUG(26)内部密码子，但效率较低。最后，与 HCV-1a Core+1 蛋白类似，HCV-1b Core+1 产物受核心表达和蛋白酶体途径的负调控。HCV-1b 临床分离物中 Core+1 ORF 的表达和刺激其表达的翻译起始机制的保留鼓励研究这些蛋白在 HCV 发病机制中的作用。
