Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategy in vitro to isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6-16 and G6-19 RNA, were found to inhibit NS3 in vitro. Kinetic studies of the inhibition revealed that the aptamer G6-16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 microM. We also analyzed aptamers G6-16 and G6-19 for their action with a longer protein substrate (amino acid region 2203-2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6-16 and G6-19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.
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PMID:Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA. 935 39

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.
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PMID:Mutational analysis of the hepatitis C virus RNA helicase. 937

The Ilheus (ILH) virus has long been known to belong to group B of the arboviruses. Previous attempts to relate it to existing serogroups within the Flavivirus genus using conventional serological techniques such as hemagglutination inhibition, neutralization and complement fixation tests have been inconclusive. We have first used denaturing gel electrophoresis to estimate the molecular weight of immunoprecipitated radiolabeled viral proteins and the cross-reactivity among ILH proteins and hyperimmune sera to several flaviviruses only from the mosquito-borne encephalitis virus serogroups. The estimated molecular weight for the three proteins was in the same order of magnitude, as previously established, for mosquito-borne flaviviruses. Cross-immunoprecipitation tests showed that NS3 protein is the most cross-reactive. Partial nucleotide sequence analyses of the NS3 gene, corresponding to an area linking the helicase and the RNA triphosphatase domains, revealed that ILH virus is very closely related to the Japanese encephalitis virus complex confirming earlier serological data.
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PMID:Ilheus virus (Flaviviridae, Flavivirus) is closely related to Japanese encephalitis virus complex. 961 22

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and beta-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.
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PMID:Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein. 965 15

The hepatitis C virus (HCV) was identified as the major causative agent of posttransfusion and community-acquired non-A, non-B hepatitis throughout the world. It is an enveloped virus with a plus-strand RNA genome encoding a polyprotein of about 3,010 amino acids. This polyprotein is cleaved co- and posttranslationally into mature viral proteins by host cell signal peptidases and 2 viral enzymes designated the NS2-3 proteinase and the NS3/4A proteinase complex. It is assumed that virus replication takes place in a membrane-associated complex containing at least 2 viral enzymatic activities: the NS3 nucleoside triphosphatase (NTPase)/helicase and the NS5B RNA-dependent RNA polymerase (RdRp). Based on their important role for the viral life cycle and the wealth of information available for related cellular and viral proteins, the NS3/4A serine-type proteinase complex, the NS3 NTPase/helicase and the NS5B RdRp are the most attractive targets for development of HCV-specific antiviral therapies. This review will summarize our current knowledge about structure and function of these proteins and describe approaches pursued to identify effective antiviral compounds.
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PMID:Candidate targets for hepatitis C virus-specific antiviral therapy. 967 42

The NS3 protein of hepatitis C virus (HCV) is thought to be essential for viral replication. The N-terminal domain of the protein contains protease activity and the C-terminal domain contains nucleotide triphosphatase and RNA helicase activity. The RNA helicase domain of HCV NS3 protein was purified by using affinity-column chromatographic methods, and crystallized by using the microbatch crystallization method under oil at 277 K. The crystals belong to primitive trigonal space group P3121 or P3221 with cell dimensions of a = b = 93.3, c = 104.6 A. The asymmetric unit contains one molecule of the helicase domain, with the crystal volume per protein mass (Vm) of 2.50 A3 Da-1 and solvent content of about 50.8% by volume. A native data set to 2.3 A resolution was obtained from a frozen crystal indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.
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PMID:Crystallization and preliminary X-ray crystallographic analysis of the helicase domain of hepatitis C virus NS3 protein. 976 31

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.
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PMID: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. 1007 62

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.
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PMID:Structure-based mutagenesis study of hepatitis C virus NS3 helicase. 1048 34

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.
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PMID:Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3. 1049 48

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products-negative-strand intermediate and progeny positive-strand RNA-in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein-in particular the ATPase/RNA helicase-play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.
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PMID:Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study. 1051 27


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