Reconstitution of hepatitis C virus protease activities in yeast.
The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed in yeast with their natural substrates fused to a ligand-dependent transcriptional activator, the retinoic acid receptor (RARbeta). RARbeta can activate transcription in yeast cells in response to retinoic acids. We hypothesized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RARbeta, thereby activating transcription of a reporter gene. Our results from Western blot analyses and reporter gene activation indicate that the wild-type NS2/3 and NS3 enzymes are catalytically active in yeast cells, whereas mutations in the catalytic domain of NS2(C993V) and NS3(S1165A) lead to inactive enzymes. We conclude that HCV NS2/3 and NS3 protease activities can be reconstituted in yeast.
Mak
P
P
Molecular Biology and Virology Section, Wyeth-Ayerst Research, 401 N. Middletown Road, Pearl River, NY 10965, USA. makp@war.wyeth.com
Palant
O
O
Labonté
P
P
Plotch
S
S
D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D016174:Hepacivirus / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D017931:DNA Primers; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases