A cell-based model of HCV-negative-strand RNA replication utilizing a chimeric hepatitis C virus/reporter RNA template.
The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric 'HCV-like' RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5' and 3' untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5' HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4alpha that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.
King
Robert W
RW
The Experimental Station, Bristol-Myers Squibb, Wilmington, Del., USA. robert.king@bms.com
Zecher
Marianne
M
Jeffries
Matthew W
MW
Carroll
Denise R
DR
Parisi
Joseph M
JM
Pasquinelli
Claudio
C
D002460:Cell Line; D017930:Genes, Reporter; D006367:HeLa Cells / Q000821:virology; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012093:Replicon; D013698:Templates, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D008156:Luciferases; D020365:RNA Helicases; D016372:RNA, Antisense; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus