Nucleic Acid Translocation By Hepatitis C Virus Helicase NS3h Is Dependent on Sugar and Base Moieties (original) (raw)
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Journal of Molecular Biology, 2010
The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3′-to-5′ superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s− 1 (oligo(dT)), 35 nt s− 1 (oligo(dU)), and 42 nt s− 1 (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are as follows: 3.3 ± 0.4 μM nucleotide (poly(dT)), 27 ± 2 μM nucleotide (poly(dU)), and 36 ± 2 μM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS0 are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (∼ 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.
Enzymatic properties of hepatitis C virus NS3-associated helicase
The Journal of general virology, 2000
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 str...
Fuel specificity of the hepatitis C virus NS3 helicase
2009
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Mutational analysis of hepatitis C virus NS3-associated helicase
2000
Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N- terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational-functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has
Nucleic Acids Research, 2004
Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK a s. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK a s in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK a values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK a s including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK a s shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.
Journal of Virology, 2003
Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU) 8 has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative -facial hydrogen bond between the 2-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the -facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.
2003
The NS3 ATPase/helicase was isolated and characterized from three different infectious clones of hepatitis C virus (HCV). One helicase was from a genotype that normally responds to therapy (Hel-2a), and the other two were from more resistant genotypes, 1a (Hel-1a) and 1b (Hel-1b). Although the differences among these helicases are generally minor, all three enzymes have distinct properties. Hel-1a is less selective for nucleoside triphosphates, Hel-1b hydrolyzes nucleoside triphosphates less rapidly, and Hel-2a unwinds DNA more rapidly and binds DNA more tightly than the other two enzymes. Unlike related proteins, different nucleic acid sequences stimulate ATP hydrolysis by HCV helicase at different maximum rates and with different apparent efficiencies. This nucleic acid stimulation profile is conserved among the enzymes, but it does not result entirely from differential DNA-binding affinities. Although the amino acid sequences of the three proteins differ by up to 15%, one variant amino acid that is critical for helicase action was identified. NS3 residue 450 is a threonine in Hel-1a and Hel-1b and is an isoleucine in Hel-2a. A mutant Hel-1a with an isoleucine substituted for threonine 450 unwinds DNA more rapidly and binds DNA more tightly than the parent protein.
Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in1756-0500-3-196
Background: About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings: Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions: Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.
Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action
2003
The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The roles of two such motifs are examined here. The first motif (YRGXDV) forms a loop that connects SF2 helicase motifs 4 and 5, at the tip of which is Arg 393. When Arg 393 is changed to Ala, the resulting protein (R393A) retains a nucleic acid stimulated ATPase but cannot unwind RNA. R393A also unwinds DNA more slowly than wild type and translocates poorly on single-stranded DNA (ssDNA). DNA and RNA stimulate ATP hydrolysis catalyzed by R393A like the wild type, but the mutant protein binds ssDNA more weakly both in the presence and absence of the nonhydrolyzable ATP analog ADP(BeF 3). The second motif (DFSLDPTF) forms a loop that connects two anti-parallel sheets between SF2 motifs 5 and 6. When Phe 444 in this Phe-loop is changed to Ala, the resulting protein (F444A) is devoid of all activities. When Phe 438 is changed to Ala, the protein (F438A) retains nucleic acidstimulated ATPase, but does not unwind RNA. F438A unwinds DNA and translocates on ssDNA at about half the rate of the wild type. Equilibrium binding data reveal that this uncoupling of ATP hydrolysis and unwinding is due to the fact that the F438A mutant does not release ssDNA upon ATP binding like the wild type. A model is presented explaining the roles of the Arg-clamp and the Phe-loop in the unwinding reaction.