Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication.
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The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold. (+info)
The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.
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NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity. (+info)
Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein.
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The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity. (+info)
Nucleolar localization of murine nuclear DNA helicase II (RNA helicase A).
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Nuclear DNA helicase II (NDH II) is a highly conserved member of the DEXH superfamily of eukaryotic helicases, whose physiological role is still unclear. To explore the function of NDH II, we studied the intracellular distribution of NDH II of different mammalian species by immunofluorescence and compared these findings with the known role of the Drosophila homologue MLE that is involved in sex-specific gene dosage compensation. NDH II displayed an apparent nucleolar localization in murine cells, whereas in cells from all other mammalian species examined so far the protein was confined to the nucleoplasm and apparently excluded from the nucleoli. The nucleolar localization of mouse NDH II strongly suggests a role in ribosomal RNA biosynthesis. Immunoelectron microscopic studies revealed that the mouse NDH II was found at the dense fibrillar components of the nucleoli, and a significant percentage of NDH II molecules colocalized with the RNA polymerase I (Pol I) transcription factor UBF (upstream binding factor). Additionally, the nucleolar localization of NDH II coincided with a preferential immunolabeling pattern of nascent transcripts with bromouridine (BrUMP). Furthermore, mouse NDH II redistributed in mitosis in a manner highly correlated with Pol I activity. Conditions leading to the inhibition of Pol I activity in the interphase decreased the amount of NDH II in the nucleoli that diffused into the nucleoplasm and the cytosol. Contrary to the effect of inhibiting rRNA synthesis, treatment of mouse cells with the translation inhibitor cycloheximide did not compromise the nucleolar localization of murine NDH II. (+info)
Nucleoside triphosphatase and RNA helicase activities associated with GB virus B nonstructural protein 3.
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GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Flaviviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase. The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease. In this report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity. This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities. Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) "AXXXXGK(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Kinetic parameters (K(m) and k(cat)) for hydrolysis of various NTPs or dNTPs were obtained. The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3' unpaired regions (RNAs with 5' unpaired regions only or with blunt ends are not suitable substrates for this enzyme). This indicates that GBV-B NS3 RNA helicase unwinds dsRNA in the 3' to 5' direction. Direct interaction of the GBV-B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay. Finally, a homology model of GBV-B NS3 RNA helicase domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed. (+info)
Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity.
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The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rNSP2). As shown by gel mobility shift assays, the rNSP2 multimers bound to RNA in discrete cooperative steps to form higher-order RNA-protein complexes. The RNA-binding activity of the rNSP2 multimers was determined to be nonspecific and to have a strong preference for single-stranded RNA over double-stranded RNA, for which it displayed little affinity. Enzymatic analysis revealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg(2+) catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicating that the hydrolysis of NTP resulted in the covalent linkage of the gamma-phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 expressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NTPase activity. (+info)
Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus.
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The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain. (+info)
Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein.
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We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein. (+info)