Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase.
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.
Zhang
Chen
C
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA.
Cai
Zhaohui
Z
Kim
Young-Chan
YC
Kumar
Ranjith
R
Yuan
Fenghua
F
Shi
Pei-Yong
PY
Kao
Cheng
C
Luo
Guangxiang
G
D020365:RNA Helicases / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D017361:Viral Nonstructural Proteins / Q000378:metabolism* / Q000502:physiology; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D020365:RNA Helicases; D012367:RNA, Viral; 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; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase