Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors.
OBJECTIVE
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV METHODS: Active NS3 protease domains of genotype 1-3 (representing five subtypes: la, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B.


RESULTS
Our data revealed that NS3 proteases cross-interacted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner.


CONCLUSIONS
Our study suggests that the NS4A cofactor function is well conserved among HCV It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.
Wright-Minogue
J
J
Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
Yao
N
N
Zhang
R
R
Butkiewicz
N J
NJ
Baroudy
B M
BM
Lau
J Y
JY
Hong
Z
Z
D019143:Evolution, Molecular; D018067:Flaviviridae / Q000378:metabolism; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D010446:Peptide Fragments; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C080106:NS4A protein, flavivirus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase