Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The Hepatitis C virus is a positive-stranded RNA virus which is the causal agent for a chronic liver infection afflicting more than 170,000,000 people world-wide. The HCV genome is approximately 9.6 kb in length and the proteome encoded is a polyprotein of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into structural and non-structural proteins. The functions of most of these proteins have been established by analogy to other viruses and by sequence homology to known proteins, as well as subsequent biochemical analysis. Two of the non-structural proteins, NS4b and NS5a, are still of unknown function. The development of antivirals for this infectious agent has been hampered by the lack of robust and economical cell culture and animal infection systems. Recent progress in the molecular virology of HCV has come about due to the definition of molecular clones, which are infectious in the chimpanzee, the development of a subgenomic replicon system in Huh7 cells, and the description of a transgenic mouse model for HCV infection. Recent progress in the structural biology of the virus has led to the determination of high resolution three-dimensional structures of a number of the key virally encoded enzymes, including the NS3 protease, NS3 helicase, and NS5b RNA-dependent RNA polymerase. In some cases these structures have been determined in complex with substrates, co-factors (NS4a), and inhibitors. Finally, a variety of techniques have been used to define host factors, which may be required for HCV replication, although this work is just beginning.
J Mol Biol 2001 Oct 26
PMID:Recent advances in the molecular biology of hepatitis C virus. 1167 30

Hepatitis C virus (HCV) NS3 protein is a multifunctional enzyme, possessing protease, NTPase and helicase activities within a single polypeptide of 625 amino acid residues. These activities are essential for the virus life cycle and are considered attractive targets for anti-HCV chemotherapy. Beside ATP, the NS3 protein has the ability to utilise deoxynucleoside triphosphates (dNTPs) as the energy source for nucleic acid unwinding. We have performed an extensive analysis of the substrate specificities of both NS3 NTPase and helicase activities with respect to all four dNTPs as well as with dideoxynucleoside triphoshate (ddNTP) analogs, including both d-(beta) and l-(beta)-deoxy and dideoxy-nucleoside triphosphates. Our results show that almost all dNTPs and ddNTPs tested were able to inhibit hydrolysis of ATP by the NTPase activity, albeit with different efficiencies. Moreover, this activity showed almost no stereoselectivity, being able to recognise both d-(beta), l-(beta)-deoxy and ddNTPs. On the contrary, the helicase activity had more strict substrate selectivity, since, among d-(beta)-nucleotides, only ddTTP and its analog 2',3'-didehydro-thymidine triphosphate could be used as substrates with an efficiency comparable to ATP, whereas among l-(beta)-nucleotides, only l-(beta)-dATP was utilised. Comparison of the steady-state kinetic parameters for both reactions, suggested that dATP, l-(beta)-dCTP and l-(beta)-dTTP, specifically reduced a rate limiting step present in the helicase, but not in the NTPase, reaction pathway. These results suggest that NS3-associated NTPase and helicase activities have different sensitivities towards different classes of deoxy and dideoxy-nucleoside analogs, depending on a specific step in the reaction, as well as show different enantioselectivity for the d-(beta) and l-(beta)-conformations of the sugar ring. These observations provide an essential mechanistic background for the development of specific nucleotide analogs targeting either activity as potential anti-HCV agents.
J Mol Biol 2001 Nov 02
PMID:Hepatitis C virus NS3 NTPase/helicase: different stereoselectivity in nucleoside triphosphate utilisation suggests that NTPase and helicase activities are coupled by a nucleotide-dependent rate limiting step. 1169 97

The NS3 protein of the hepatitis C virus (HCV) is a 631 amino acid residue bifunctional enzyme with a serine protease localized to the N-terminal 181 residues and an RNA helicase located in the C-terminal 450 residues. The HCV NS3 RNA helicase consists of three well-defined subdomains which all contribute to its helicase activity. The second subdomain of the HCV helicase is flexibly linked to the remainder of the NS3 protein and could undergo rigid-body movements during the unwinding of double-stranded RNA. It also contains several motifs that are implicated in RNA binding and in coupling NTP hydrolysis to nucleic acid unwinding and translocation. As part of our efforts to use NMR techniques to assist in deciphering the enzyme's structure-function relationships and developing specific small molecule inhibitors, we have determined the solution structure of an engineered subdomain 2 of the NS3 RNA helicase of HCV, d(2Delta)-HCVh, and studied the backbone dynamics of this protein by (15)N-relaxation experiments using a model-free approach. The NMR studies on this 142-residue construct reveal that overall subdomain 2 of the HCV helicase is globular and well structured in solution even in the absence of the remaining parts of the NS3 protein. Its solution structure is very similar to the corresponding parts in the X-ray structures of the HCV NS3 helicase domain and intact bifunctional HCV NS3 protein. Slow hydrogen-deuterium exchange rates map to a well-structured, stable hydrophobic core region away from the subdomain interfaces. In contrast, the regions facing the subdomain interfaces in the HCV NS3 helicase domain are less well structured in d(2Delta)-HCVh, show fast hydrogen-deuterium exchange rates, and the analysis of the dynamic properties of d(2Delta)-HCVh reveals that these regions of the protein show distinct dynamical features. In particular, residues in motif V, which may be involved in transducing allosteric effects of nucleotide binding and hydrolysis on RNA binding, exhibit slow conformational exchange on the milli- to microsecond time-scale. The intrinsic conformational flexibility of this loop region may facilitate conformational changes required for helicase function.
J Mol Biol 2001 Nov 30
PMID:Solution structure and backbone dynamics of an engineered arginine-rich subdomain 2 of the hepatitis C virus NS3 RNA helicase. 1184 66

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.
J Biochem Mol Biol 2002 Mar 31
PMID:In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates. 1229 31

We have designed small focused combinatorial library of hexapeptide inhibitors of NS3 serine protease of the hepatitis C virus (HCV) by structure-based molecular design complemented by combinatorial optimisation of the individual residues. Rational residue substitutions were guided by the structure and properties of the binding pockets of the enzyme's active site. The inhibitors were derived from peptides known to inhibit the NS3 serine protease by using unusual amino acids and alpha-ketocysteine or difluoroaminobutyric acid, which are known to bind to the S1 pocket of the catalytic site. Inhibition constants (Ki) of the designed library of inhibitors were predicted from a QSAR model that correlated experimental Ki of known peptidic inhibitors of NS3 with the enthalpies of enzyme-inhibitor interaction computed via molecular mechanics and the solvent effect contribution to the binding affinity derived from the continuum model of solvation. The library of the optimised inhibitors contains promising drug candidates-water-soluble anionic hexapeptides with predicted Ki* in the picomolar range.
J Mol Graph Model 2004 Jan
PMID:Structure-based design of inhibitors of NS3 serine protease of hepatitis C virus. 1462 79

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer and is a common cancer type worldwide. Many aetiological factors have been related to HCC development, such as liver cirrhosis, hepatitis viruses and alcohol consumption. Inactivation of the p53 tumour suppressor gene is one of the most common abnormalities in many tumours, including HCC. p53 is of crucial importance for the regulation of the cell cycle and the maintenance of genomic integrity. In HCC, hepatitis B and C virus (HBV and HCV) effect carcinogenic pathways, independently leading to anomalies in p53 function. Several authors have reported that some HCV proteins, such as the core, NS5A and NS3 proteins, interact with p53 and prevent its correct function. The mechanisms of action of these HCV proteins in relation to p53 are not completely clear, but they might cause its cytoplasmic retention or accumulation in the perinuclear region where the protein is not functional. The identification of the interactions between p53 and HCV proteins is of great importance for therapeutic strategies aimed at reducing the chronicity and/or carcinogenicity of the virus.
Expert Rev Mol Med 2003 Nov 19
PMID:Hepatocellular carcinoma: molecular interactions between hepatitis C virus and p53 in hepatocarcinogenesis. 1498 3

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.
J Mol Biol 2005 Apr 15
PMID:HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen. 1578 58

Intracellular viral infection is detected by the cytoplasmic RNA helicase RIG-I, which has an essential role in initiating the host antiviral response. The adaptor molecule that connects RIG-I sensing of incoming viral RNA to downstream signaling and gene activation has recently been elucidated by four independent research groups, and has been ascribed four different names: MAVS, IPS-1, VISA and Cardif. The fact that MAVS/IPS-1/VISA/Cardif localizes to the mitochondrial membrane suggests a link between viral infection, mitochondrial function and development of innate immunity. Furthermore, the hepatitis C virus NS3/4A protease specifically cleaves MAVS/IPS-1/VISA/Cardif as part of its immune-evasion strategy. These studies highlight a novel role for the mitochondria and for caspase activation and recruitment domain (CARD)-containing proteins in coordinating immune and apoptotic responses.
Trends Mol Med 2006 Feb
PMID:MasterCARD: a priceless link to innate immunity. 1640 12

The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.
Nat Struct Mol Biol 2006 Apr
PMID:Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. 1653 6

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.
J Mol Biol 2006 May 12
PMID:Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. 1656 13


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