Developing Efficient HCV Replication Cell Culture System

University Of Texas Medical Br Galveston, Galveston TX

Investigators

Abstract

DESCRIPTION (provided by applicant): No cell culture system exists that efficiently supports hepatitis C virus (HCV) replication and assembly; cloned, full-length HCV RNA cannot replicate efficiently and assemble into viral particles in today's cell lines. One cell line, Huh-7 cell line, however, supports continuous replication of HCV replicons. In an HCV replicon, NNeo, the neomycin phosphotransferase gene and the encephalomyocarditis virus internal ribosome entry site sequence replace the HCV structural protein genes and NS2 gene. NNeo RNA efficiently accumulated within the first 3 days after transfection of in vitro-synthesized NNeo RNA transcripts into Huh-7 cells. In marked contrast, full-length HCV RNA did not efficiently accumulate after transfection of the cloned, infectious RNA transcripts that are the replicon's parent. Determining the reason the full-length HCV RNA did not replicate well in the cell culture is at the heart of establishing an efficient HCV replication system in cell culture. The present application aims to understand why only a low level of RNA synthesis occurs after transfection of full-length HCV in Huh-7 cells. We will test whether replication of full-length HCV may induce cell death, thereby suppressing HCV RNA replication. We will examine whether the enormity of full-length HCV RNA might severely affect its replication efficiency. Furthermore, the possibility that an RNA element(s) that suppresses HCV RNA replication may be encoded in full-length HCV RNA will be explored. Information from the studies proposed here will answer why the full-length HCV clone does not replicate well in Huh 7 cells, and will let us reach our ultimate aim of developing an efficient HCV replication system in cell culture.