Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system.
The hepatitis C virus (HCV) NS3 protein possesses three enzymatic activities: an N-terminal serine protease activity, a C-terminal RNA-stimulated NTPase activity, and an RNA helicase activity. To characterize them, the full-length NS3(631)/4A and three C-terminal truncated proteases (NS3(201)/4A, NS3(181)/4A, and NS3(155)/4A were expressed in mammalian cells with HSV amplicon-defective viruses. Our results revealed that all of the NS3/4A proteins produced in mammalian cells (except NS3(155)/4A) are active in processing both cis and trans cleavage sites. Temperature optimization studies revealed that the protease is more active at temperatures ranging from 4 to 25 degrees C and is completely inactive at 42 degrees C. The RNA-stimulated ATPase activity was characterized with a partially purified NS3(631)/4A fraction and has a higher optimal temperature at 37 to 42 degrees C. The effects of detergents on both NS3 protease and RNA-stimulated ATPase were similar. Nonionic detergents such as Triton X-100, Nonidet P-40 and Tween 20 did not affect the activities, while anionic detergents such as sodium dodecyl sulfate and deoxycholic acid were inhibitory. Zwitterionic detergent such as 3-[(3-cholamidopropyl)- dimethyl-ammoniol-1-propanesulfonate (CHAPS) inhibited protease activity at a concentration of 0.5% (8 mM), which had no effect on ATPase activity. Finally, RNA-unwinding activity was demonstrated in the NS3(631)/4A fraction but not in the similarly purified NS3(181)/4A and NS3(201)/4A fractions. NS(363)/4A unwinds RNA duplexes with 3' but not 5' single-stranded overhangs, suggesting that the NS3 RNA helicase functions in a 3'-to-5' direction.
Hong
Z
Z
Antiviral Chemotherapy and Structural Chemistry Departments, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.zhi.hong@spcorp.com
Ferrari
E
E
Wright-Minogue
J
J
Chase
R
R
Risano
C
C
Seelig
G
G
Lee
C G
CG
Kwong
A D
AD
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003673:Defective Viruses / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D003902:Detergents; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C027900:RNA-dependent ATPase