Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein.
We constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5'NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon alpha-2b in cell lines containing replicons derived from these two strains of HCV.
Yi
MinKyung
M
Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, 77555-1019, USA.
Bodola
Francis
F
Lemon
Stanley M
SM
D000469:Alkaline Phosphatase / Q000235:genetics*; D002460:Cell Line; D015696:Gene Products, tat / Q000235:genetics; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins; D012093:Replicon; D014162:Transfection
D015696:Gene Products, tat; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase