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

Du, Mark; Johnson, Robert B.; Sun, Xin-Lai; Staschke, Kirk A.; Colacino, Joseph M.; Wang, Q. May

doi:10.1042/0264-6021:3630147

Cited by 37 publications

(36 citation statements)

References 28 publications

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“…We also looked at the effect of pH on catalysis. It has been previously shown with other helicases that acidic conditions decrease the binding affinity of the enzyme to magnesium (24), resulting in a general loss of activity (25,26). In our experiments, the ATPase activity of the isolated RIG-Ih domain was consistently sensitive to low pH in all tested conditions and peaked between pH 7.0 and 7.5 (Fig.…”

Section: Atpase Activity Is Important For Cires-mediated Activation Osupporting

confidence: 55%

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Gee

Chua

Gevorkyan

et al. 2008

Journal of Biological Chemistry

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Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-␤ expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.Pathogen recognition receptors constitute one of the first lines of human host cell defense against viral infections. Among them, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) helicases are specialized sensors of viral nucleic acid. RIG-I was shown to be involved in the recognition of Sendai virus, Hepatitis C virus (HCV), 2 and vesicular stomatitis virus, whereas MDA-5 mediates the antiviral response to picornaviruses (1-3). Both RIG-I and MDA-5 are modular proteins of ϳ920 residues that contain an ATPase/helicase domain for RNA binding and two caspase activation and recruitment domains (CARDs) located in tandem at the N-terminal end of the protein (4). The CARDs, by way of homotypic interactions, are required for the signaling function between RIG-I and the newly discovered interferon-␤ promoter stimulator-1 (IPS-1), also called Cardif, MAVS, and VISA (for review see Ref. 5). The latter serves as an intermediate to the cascade leading to the activation of NF-B and IRF-3/7, which results in the production of antiviral cytokines such as type-1 interferons (6 -9). The receptor function of RIG-I is non-redundant and therefore essential to coordinate the signaling cascade leading to antiviral response (4), as confirmed by knock-out studies (10). Notably, the Huh7.5 cell line of hepatocytes is particularly permissive to HCV and vesicular stomatitis virus infection as the result of a defective RIG-I protein bearing a single mutation in its first CARD (11); the antiviral sig...

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Section: Atpase Activity Is Important For Cires-mediated Activation Osupporting

confidence: 55%

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Gee

Chua

Gevorkyan

et al. 2008

Journal of Biological Chemistry

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“…The phenylalanine of the Q-motif stacks with the adenine base that is also contacted by an aromatic or hydrophobic side chain from motif VI (Bono et al, 2006). Only the 39-OH group of the ribose is contacted, rationalizing the weak discrimination between ATP and dATP (Du et al, 2002). In addition to direct and indirect contacts to the nucleotides, the conserved motifs from both RecA-like domains form an intricate interaction network around the ATPase site.…”

Section: Nucleotide Bindingmentioning

confidence: 99%

“…Its highly conserved glutamine interacts with the adenine base and provides specificity for adenine nucleotides. Other nucleotides might compete for the binding site but are not efficiently hydrolyzed and do not stimulate RNA unwinding by DEAD box proteins (Du et al, 2002;Franca et al, 2007;Garcia and Uhlenbeck, 2008). It has therefore been suggested that the Q-motif also contributes to positioning of the nucleotide for hydrolysis (Tanner, 2003;Tanner et al, 2003).…”

Section: Nucleotide Bindingmentioning

confidence: 99%

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

Hilbert

Karow²,

Klostermeier³

2009

BCHM

143

171

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DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecAlike domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysisand unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

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“…The truncated helicase proteins lacking protease function (Fig. 1A) were based on those previously analyzed and include versions with N-terminal (10 -12) and C-terminal (7,(13)(14)(15)(16) polyhistidine tags (His tags) and a version in which the protease is replaced with a glutathione Stransferase (GST) protein (17). The presence of an intact protease domain dramatically enhances the ability of the protein to unwind RNA.…”

mentioning

confidence: 99%

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

Frick

Rypma

Lam

et al. 2004

Journal of Biological Chemistry

103

128

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The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3 -5 helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.The epidemic caused by infection by the hepatitis C virus (HCV) 1 is still a global crisis despite recent therapeutic advancements (1). Because HCV cannot be conventionally cultivated in cell culture and the only other host is the chimpanzee, the enzymes encoded by HCV have been studied intensely as targets for rational drug design. One key viral enzyme is the multifunctional nonstructural protein 3 (NS3), which possesses a serine protease activity and an ATPase function that fuels the ability of the protein to unwind RNA and DNA duplexes. Although it is clear that both the protease and helicase functions are necessary for viral replication (2), it is not clear whether the two functions, which reside in independent protein domains, cooperate in any manner (3).To examine possible effects of the NS3 protease on its helicase function, the activities of the full-length NS3 protein were rigorously compared with the same protein lacking the protease and also with recombinant proteins in which the protease is replaced with other non-HCV peptides. The experiments were designed to uncover effects of the NS3 protease domain on its helicase function which are either specific or nonspecific. Nonspecific effects are defined as those that can be duplicated by peptides not derived from HCV NS3, whereas specific effects cannot.All recombinant proteins used in this study ( Fig. 1) were derived from the same infectious clone of HCV genotype 1a (4). NS3 spans amino acids 1027-1659 of the ϳ3,000-amino acid long polyprotein encoded by HCV. At the N terminus resides the pro...

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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

Cited by 37 publications

References 28 publications

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

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