The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate

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

doi:10.1074/jbc.m707165200

Cited by 104 publications

(124 citation statements)

References 40 publications

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“…Moreover, as the polynucleotide stimulator for ATP hydrolysis and the substrate for dsRNA unwinding were not derived from HCV RNA, NS3H is able to act on non-HCV RNA. These findings correspond with the observations from earlier reports [3,13,14]. mNS3H did not catalyze ATP hydrolysis (Fig.…”

Section: Resultssupporting

confidence: 93%

HCV NS3 protein helicase domain assists RNA structure conversion

Huang

Wang

2010

FEBS Letters

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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.

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Section: Resultssupporting

confidence: 93%

HCV NS3 protein helicase domain assists RNA structure conversion

Huang

Wang

2010

FEBS Letters

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“…5 and 6). Although the NS3 protease domain likely modulates nucleic acid substrate binding to increase duplex unwinding, it is not essential for the helicase activity (29,32). Thus, the fundamental mode of structural transitions involved in the motion of the HCV helicase is likely independent of the protease.…”

Section: Resultsmentioning

confidence: 99%

“…The NS3 protease domain, which is positioned to capture the C-terminal tail of the helicase in the structure of an engineered single-chain NS3-4A protein (28), may rearrange its interactions with NS3h to enhance RNA substrate binding (29,30). .…”

Section: Resultsmentioning

confidence: 99%

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

Gu¹,

Rice

2009

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

215

348

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A virally encoded superfamily-2 (SF2) helicase (NS3h) is essential for the replication of hepatitis C virus, a leading cause of liver disease worldwide. Efforts to elucidate the function of NS3h and to develop inhibitors against it, however, have been hampered by limited understanding of its molecular mechanism. Here we show x-ray crystal structures for a set of NS3h complexes, including ground-state and transition-state ternary complexes captured with ATP mimics (ADP · BeF 3 and ADP · AlF − 4 ). These structures provide, for the first time, three conformational snapshots demonstrating the molecular basis of action for a SF2 helicase. Upon nucleotide binding, overall domain rotation along with structural transitions in motif V and the bound DNA leads to the release of one base from the substrate base-stacking row and the loss of several interactions between NS3h and the 3′ DNA segment. As nucleotide hydrolysis proceeds into the transition state, stretching of a "spring" helix and another overall conformational change couples rearrangement of the (d)NTPase active site to additional hydrogen-bonding between NS3h and DNA. Together with biochemistry, these results demonstrate a "ratchet" mechanism involved in the unidirectional translocation and define the step size of NS3h as one base per nucleotide hydrolysis cycle. These findings suggest feasible strategies for developing specific inhibitors to block the action of this attractive, yet largely unexplored drug target.antivirals | motor protein | step size | superfamily 2 | transition state H epatitis C virus (HCV) is a 9.6 kb positive-sense, singlestranded RNA (ssRNA) virus of the Flaviviridae family. It is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). A protective vaccine is not available and the existing treatment is frequently ineffective. Like other positive-strand RNA viruses, HCV replicates in close association with modified intracellular membranes (2), where the viral replicase complex catalyzes accumulation of progeny RNA genomes through a negative-strand intermediate. The helicase domain of viral nonstructural protein 3 (NS3h) is an enzymatic component of this replication apparatus and is essential for HCV propagation; the precise role of the helicase, however, remains obscure [reviewed in (2, 3)].NS3h is classified as a superfamily-2 (SF2) DExH helicase. It has polynucleotide-stimulated (d)NTPase activity (4) and can unwind both RNA and DNA in a 3′-5′ direction (5, 6). Structures of NS3h in complex with single-stranded DNA (ssDNA) have shown that DNA binds in a groove between domain 3 and the RecA-like domains, with the bases stacked in a row between two "bookend" residues (V432 in the domain 2 β-hairpin and W501 in domain 3) (7,8). The structure of the substrate DNA remains ambiguous, however, as the pucker conformations assigned to the sugar groups differ between reports. Whereas ATP binding has been suggested between NS3h domains 1 and 2 (7), the atomic details of nucleotide binding and hydrolysis have...

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“…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.

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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...

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The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate

Cited by 104 publications

References 40 publications

HCV NS3 protein helicase domain assists RNA structure conversion

HCV NS3 protein helicase domain assists RNA structure conversion

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

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

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