a new model to produce infectious hepatitis c virus without the replication requirement新模式产生感染丙型肝炎病毒的复制要求.pdfVIP

A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement 1¤ 1 1,2,3,4 Miriam Triyatni , Edward A. Berger , Bertrand Saunier * 1 Molecular Structure Section, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, Maryland, United States of America, 2 Paris-Descartes University, Faculty of Medicine, Paris, France, 3 Institut Cochin, Paris, France, 4 Inserm U1016, Paris, France Abstract Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/ replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 59 and 39 termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenom