Wakita T, Moradpour D, Tokushihge K, Wands J R
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown 02129, USA.
J Med Virol. 1999 Mar;57(3):217-22.
We developed approaches using antisense RNA to inhibit hepatitis C virus (HCV) RNA translation and HCV core protein expression. An HCV genotype 1b cDNA comprising nt 1-1321 or a fusion construct consisting of HCV (nt 1-584) and luciferase cDNAs were inserted downstream of T7 and CMV promoter sequences and used to generate HCV RNA target molecules. Such constructs will produce HCV core or HCV coreluciferase fusion proteins in vitro or within transfected cells. Seven different antisense RNA constructs were designed to target the highly conserved 5' region of HCV RNA at nt positions 1-402. For in vitro experiments, synthesized HCV RNA target sequences and antisense RNAs were mixed at various molar ratios and subsequently translated in a rabbit reticulocyte lysate system. In cell culture studies, the HCV core-luciferase fusion cDNA was co-transfected with antisense RNA-producing constructs into human hepatocellular carcinoma (HCC) cells. Luciferase activity in cell lysates was measured to determine quantitatively antiviral effects within the cell. It was found that translation of HCV RNAs was efficiently inhibited by antisense RNA in vitro. The specificity of this inhibition was confirmed using control target RNA sequences or nonrelevant antisense RNA constructs. Co-transfection studies demonstrated that antisense RNA inhibited HCV core-luciferase fusion protein expression by 41-57% in HuH-7 HCC cells. These studies indicate that antisense RNA will find viral target RNA sequences in HuH-7 cells and inhibit HCV RNA translation. More important, these studies have defined critical viral RNA target sequences susceptible to antisense inhibitory effects within the cell.
我们开发了利用反义RNA抑制丙型肝炎病毒（HCV）RNA翻译和HCV核心蛋白表达的方法。将包含核苷酸1至1321的HCV 1b基因型cDNA或由HCV（核苷酸1至584）和荧光素酶cDNA组成的融合构建体插入T7和CMV启动子序列下游，并用于生成HCV RNA靶分子。此类构建体将在体外或转染细胞内产生HCV核心或HCV-荧光素酶融合蛋白。设计了七种不同的反义RNA构建体，以靶向HCV RNA在核苷酸位置1至402的高度保守的5'区域。对于体外实验，将合成的HCV RNA靶序列和反义RNA以各种摩尔比混合，随后在兔网织红细胞裂解物系统中进行翻译。在细胞培养研究中，将HCV核心-荧光素酶融合cDNA与产生反义RNA的构建体共转染到人肝癌（HCC）细胞中。测量细胞裂解物中的荧光素酶活性，以定量确定细胞内的抗病毒作用。结果发现，反义RNA在体外可有效抑制HCV RNA的翻译。使用对照靶RNA序列或不相关的反义RNA构建体证实了这种抑制的特异性。共转染研究表明，反义RNA在HuH-7 HCC细胞中可将HCV核心-荧光素酶融合蛋白的表达抑制41%至57%。这些研究表明，反义RNA可在HuH-7细胞中找到病毒靶RNA序列并抑制HCV RNA翻译。更重要的是，这些研究确定了细胞内易受反义抑制作用影响的关键病毒RNA靶序列。
