XAS Characterization of the Zn Site of Non-structural Protein 3 (NS3) from Hepatitis C Virus

2007 ◽  
Author(s):  
I. Ascone ◽  
G. Nobili ◽  
M. Benfatto ◽  
A. Congiu-Castellano
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Protein

Enzymatic properties of hepatitis C virus NS3-associated helicase

Microbiology ◽  
2000 ◽  
Vol 81 (5) ◽  
pp. 1335-1345 ◽  
Author(s):  
Chantal Paolini ◽  
Raffaele De Francesco ◽  
Paola Gallinari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Structure ◽  
Structural Protein ◽  
Single Cycle ◽  
Detailed Characterization ◽  
Stem Loop ◽  
Hybrid Substrates ◽  
Serine Protease Activity

The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3′ to 5′ direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem–loop RNA structure (SL I) within the 3′-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.

Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli

2004 ◽  
Vol 37 (1) ◽  
pp. 144-153 ◽  
Author(s):  
Luyun Huang ◽  
Elena V Sineva ◽  
Michele R.S Hargittai ◽  
Suresh D Sharma ◽  
Mehul Suthar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Protein ◽  
Purification And Characterization

Comparative characterization of two DEAD-box RNA helicases in superfamily II: human translation-initiation factor 4A and hepatitis C virus non-structural protein 3 (NS3) helicase

2002 ◽  
Vol 363 (1) ◽  
pp. 147-155 ◽  
Author(s):  
Mark X. DU ◽  
Robert B. JOHNSON ◽  
Xin-Lai SUN ◽  
Kirk A. STASCHKE ◽  
Joseph COLACINO ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Helicase Domain ◽  
Dna Duplexes ◽  
Structural Protein ◽  
Comparative Characterization ◽  
Specific Inhibitors

Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase and is homologous to the non-structural protein 3 (NS3) helicase domain encoded by hepatitis C virus (HCV). Reported here is the comparative characterization of human eIF4A and HCV NS3 helicase in an effort to better understand viral and cellular helicases of superfamily II and to assist in designing specific inhibitors against HCV infections. Both eIF4A and HCV NS3 helicase domain were expressed in bacterial cells as histidine-tagged proteins and purified to homogeneity. Purified eIF4A exhibited RNA-unwinding activity and acted on RNA or RNA/DNA but not DNA duplexes. In the absence of cellular cofactors, eIF4A operated unwinding in both the 3′ to 5′ and 5′ to 3′ directions, and was able to unwind blunt-ended RNA duplex, suggesting that bidirectionality is an intrinsic property of eIF4A. In contrast, HCV NS3 helicase showed unidirectional 3′ to 5′ unwinding of RNA and RNA/DNA, as well as of DNA duplexes. With respect to NTPase activity, eIF4A hydrolysed only ATP or dATP in the presence of RNAs, whereas HCV NS3 helicase could hydrolyse all ribo- and deoxyribo-NTPs in an RNA-independent manner. In parallel, only ATP or dATP could drive the unwinding activity of eIF4A whereas HCV NS3 could function with all eight standard NTPs and dNTPs. The observed differences in their substrate specificity may prove to be useful in designing specific inhibitors targeting HCV NS3 helicase but not human eIF4A.

Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus

1999 ◽  
Vol 13 (1-2) ◽  
pp. 61-69 ◽  
Author(s):  
Peter Borowski ◽  
Reinhard Kühl ◽  
Rainer Laufs ◽  
Julian Schulze zur Wiesch ◽  
Max Heiland
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Binding Site ◽  
Structural Protein ◽  
Histone Binding ◽  
Identification And Characterization
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