Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

Beran, Rudolf K. F.; Pyle, Anna Marie

doi:10.1074/jbc.m804065200

Cited by 100 publications

(98 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The data presented in this work expand the known functions of HCV NS3 helicase. It has been shown previously that the activity of HCV NS3 helicase domain is modulated by other factors such as the protease domain of HCV NS3 [2] and HCV NS5B [5]. Although it is outside the scope of this article, in the future it would be of interest to see how these factors affect the activities described in this work.…”

Section: Resultsmentioning

confidence: 87%

HCV NS3 protein helicase domain assists RNA structure conversion

Huang

Wang

2010

FEBS Letters

View full text Add to dashboard Cite

Edited by Hans-Dieter KlenkKeywords: HCV NS3 helicase RNA structure conversion Strand annealing dsRNA unwinding RNA chaperone DExH/D-box protein a b s t r a c t NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle.

show abstract

Section: Resultsmentioning

confidence: 87%

HCV NS3 protein helicase domain assists RNA structure conversion

Huang

Wang

2010

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…The isolated domains of NS3, i.e., the protease and helicase domains are functional on their own. It has been reported that in the full-length enzyme the NS3/4A protease enhances RNA binding and unwinding by the helicase and that also the helicase enhances protease activity (7,8). In the past decade, HCV NS3/4A protease has emerged as an important drug target for treatment of HCV infection.…”

mentioning

confidence: 99%

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Schiering

D’Arcy

Villard

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed proteasehelicase domain assembly in solution.structure-based drug design | X-ray structure | solution scattering | medicinal chemistry C hronic hepatitis C virus (HCV) infection affects more than 3% of the world's population and is a leading cause of chronic liver diseases (1). Therapeutic options have been suboptimal, especially for HCV genotype 1, the most prevalent genotype in developed countries. Recently, the addition of direct-acting antiviral agents (DAAs) to the previous standard of care (combination therapy with pegylated interferon and ribavirin) have demonstrated considerable improvement in sustained virological response rates in patients infected with HCV genotype 1 (2). With the first DAAs now having been introduced into clinical practice, it is to be expected that in the near future standard therapy will change to a triple therapy including an HCV NS3/4A protease inhibitor in combination with pegylated interferon and ribavirin.The positive-strand RNA genome of HCV encodes a polyprotein precursor, which is proteolytically processed by host and viral proteases into 10 individual structural and nonstructural (NS) proteins. The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3, 4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairin...

show abstract

“…Each of the two domains can be expressed separately and retain activity (18). However, the activities are interdependent, since recently it was shown that the NS3 helicase domain stimulates the serine protease activity (19). Both protease and helicase activities are required for viral replication (20).…”

mentioning

confidence: 99%

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Jennings

Mackintosh

Harrison

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nM. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.Helicases are ubiquitous enzymes required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-5). These enzymes catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation. However, there is considerable variability in the quaternary structure of the active forms of helicases. Some helicases function effectively in unwinding activities as monomers, whereas others are active as dimers or oligomers. For example, bacteriophage T4 gp41 helicase and Escherichia coli DnaB helicase form hexameric structures that encircle and sequester single-stranded DNA (6, 7). Indeed, a large number of helicases form and function as hexameric structures (3). PcrA, a Gram-positive bacterial helicase, translocates on single-stranded DNA as a monomer (8) and has been proposed to unwind double-stranded DNA as a monomer (9). Bacteriophage T4 Dda helicase has activity in the monomeric form (10) but demonstrates more efficient unwinding activity under conditions where multiple helicase molecules bind a given substrate molecule (11). It has been shown that E. coli Rep helicase unwinds DNA as a dimer (12). UvrD has...

show abstract

Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase

Cited by 100 publications

References 38 publications

HCV NS3 protein helicase domain assists RNA structure conversion

HCV NS3 protein helicase domain assists RNA structure conversion

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Contact Info

Product

Resources

About