Kusov Yuri, Kanda Tatsuo, Palmenberg Ann, Sgro Jean-Yves, Gauss-Müller Verena
Institute of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.
J Virol. 2006 Jun;80(11):5599-610. doi: 10.1128/JVI.01773-05.
Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.
甲型肝炎病毒（HAV）感染可导致急性肝炎，极少数情况下会引发暴发性肝衰竭，尤其是在慢性丙型肝炎病毒感染患者中。基于我们之前的观察，即小干扰RNA（siRNA）可使编码萤火虫荧光素酶的HAV复制子的翻译和复制沉默，我们现在利用这项技术来证明siRNA对Huh-7细胞中病毒感染的影响。用靶向HAV非结构基因中各个位点的siRNA转染新鲜感染和持续感染的细胞。与单次应用相比，连续转染靶向病毒基因组中多个序列的siRNA比单次转染产生更有效和持续的沉默效果。在大多数情况下，单次siRNA的多次应用导致出现具有突变靶位点的病毒逃逸突变体，使这些基因组对RNA干扰（RNAi）具有抗性。连续应用靶向计算机预测的发夹结构的siRNA后，实现了对病毒感染性的有效和持续抑制。这种siRNA有望成为治疗严重HAV感染病程的治疗工具。此外，这些结果为序列特异性RNAi的结构基础提供了新的见解。
