Hepatitis C Virus Protein Interaction Database
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Interaction details
Details Molecule A
Details Molecule B
Interaction details

Molecule A (HCV protein information)
Short Name NS3
Recommended Name HCV NS3 protein
Other Names Serine protease, NTPase, Hepacivirin, NS3P, p70
EC 3.6.1.15, 3.6.1.-, 3.4.21.98
OMIM 114550, 609532
euhcvdb protein link http://euhcvdb.ibcp.fr/euHCVdb/jsp/NS3.jsp
GenBank (Location: 3420..5312) NC_004102
VBRC Accession 64598
Uniprot Post-translational modification Specific enzymatic cleavages in vivo yield mature proteins. The structural proteins, core, E1, E2 and p7 are produced by proteolytic processing by host signal peptidases. The core protein is synthesized as a 21 kDa precursor which is retained in the ER membrane through the hydrophobic signal peptide. Cleavage by the signal peptidase releases the 19 kDa mature core protein. The other proteins (p7, NS2-3, NS3, NS4A, NS4B, NS5A and NS5B) are cleaved by the viral protease. [PubMed: 1658800 PubMed: 10729138]
Uniprot Functions NS3 displays three enzymatic activities: serine protease, NTPase and RNA helicase. NS3 serine protease, in association with NS4A, is responsible for the cleavages of NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B. NS3/NS4A complex also prevents phosphorylation of human IRF3, thus preventing the establishment of dsRNA induced antiviral state. NS3 RNA helicase binds to RNA and unwinds dsRNA in the 3` to 5` direction, and likely RNA stable secondary structure in the template strand. Cleaves and inhibits the host antiviral protein MAVS. [PubMed: 16894197]
Uniprot Subunit Structure NS4A interacts with NS3 serine protease and stabilizes its folding. NS3-NS4A complex is essential for the activation of the latter and allows membrane anchorage of NS3. NS3 interacts with human TANK-binding kinase TBK1 and MAVS.
Uniprot Domain The N-terminal one-third of serine protease NS3 contains the protease activity. This region contains a zinc atom that does not belong to the active site, but may play a structural rather than a catalytic role. This region is essential for the activity of protease NS2-3, maybe by contributing to the folding of the latter. The helicase activity is located in the C-terminus of NS3. [PubMed: 1658800 PubMed: 10729138]
Uniprot Subcellular Localization Host endoplasmic reticulum membrane; Peripheral membrane protein. Note: NS3 is associated to the ER membrane through its binding to NS4A. [PubMed: 10702287, PubMed: 8982089, PubMed: 10729138, PubMed: 11907211, PubMed: 12692244, PubMed: 17192310, PubMed: 14752815]
Functions N-terminal proteinase domain, C-terminal NTPase, helicase domain [PMID 17552023]; NS2/3 proteinase, NS3/4 proteinase, NTPase, RNA helicase, RNA binding [PMID 17455808]
Drug Development The serine proteinase activity of NS3 is an attractive target for new drugs that could block viral replication efficiently [PMID 14752815]. Despite abundant structural data obtained by X-ray crystallography, the mechanism of action of the helicase domain and its precise role during replication are not fully understood. Nevertheless, this activity remains an important potential target for new drugs, which could be combined with other drugs targeting the viral proteases and the RNA-dependent RNA polymerase [PMID 14752815]. And also the design of appropriate protease inhibitors is challenging because the NS3-NS4A protease pocket is uniquely shallow, requiring long interaction surfaces with substrates [PMID 17552023].
Description NS3 AND NS4A: NS3 is a multifunctional protein with an N-terminal serine-type protease domain and a C-terminal RNA helicase/NTPase domain. The NS3 protease domain has a typical chymotrypsin-like fold and is composed of two beta-barrel domains [PMID 8861917, PMID 8861916]. The protease activity of NS3 is enhanced by the NS4A cofactor. Indeed, NS4A contributes one beta-strand to the N-terminal protease domain and thereby allows its complete folding [PMID 8861917]. In addition, it induces a conformational change that leads to a repositioning of the catalytic triad. NS3 by itself has no transmembrane domain, but it associates non-covalently with the central domain of NS4A, which is a membrane protein. When co-expressed with NS4A, NS3 is found in association with ER or ER-like membranes whereas it is diffusely distributed in the cytoplasm and nucleus when expressed alone [PMID 10666260]. Deletion analyses have revealed that the hydrophobic N-terminal domain of NS4A is required for ER targeting of NS3. Interestingly, NS4A also stabilizes the protease against proteolytic degradation. The NS3-4A protease has an unusually shallow substrate-binding pocket and therefore requires rather long interaction surfaces with the substrate (reviewed in [PMID 14752815, PMID 15530561]). This made the design of efficient inhibitors of this protease challenging [PMID 16107835]. The NS3-4A protease is responsible for the polyprotein cleavage in the region downstream of NS3, and this activity is essential for the generation of components of the viral RNA replication complex [95]. It is therefore not surprising that this protease has been the first target for the development of new anti-HCV molecules [PMID 16107835]. In addition to its role in the processing of the poly-protein, the NS3-4A protease activity is also involved in blocking the ability of the host cell to mount an innate antiviral response [PMID 12702807]. The NS3-4A has indeed been shown to interfere with double-stranded RNA signaling pathways. It disrupts the cellular RNA helicase retinoic acid-inducible gene I (RIG-I) pathway through proteolysis of a newly discovered essential adaptor protein of interferon regulatory factor-3 (IRF-3) activation [PMID 16177806]. Due to its recent simultaneous discovery by four different groups, this adaptor protein has received four different names: IPS-1, Cardif, VISA and MAVS [PMID 16239922]. NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif results in its dissociation from the mitochondrial membrane and disruption of signaling to the antiviral immune response [PMID 16301520]. NS3-4A also cleaves the TRIF (also called TICAM-1) adaptor protein to ablate Toll-like receptor-3 (TLR-3) signaling of IRF-3 activation by extracellular double-stranded RNA [PMID 15710891]. However, this pathway has a minimal role in triggering the interferon antiviral response [PMID 16731946]. The C terminus of NS3 encodes a DexH/D-box RNA helicase [PMID 8970970]. Enzymes of this superfamily are capable of unwinding RNA-RNA duplexes in an ATP-dependent manner. The crystal structure of the HCV helicase shows a Y-shaped molecule composed of 3 nearly equally sized subdomains [PMID 9493270, PMID 9187654]. Although monomeric NS3 can bind RNA with high affinity, RNA unwinding requires an NS3 dimer [PMID 15269774]. Kinetic analyses indicate that this enzyme undergoes highly coordinated cycles of fast double-stranded RNA unwinding [PMID 15269774, PMID 15806107, PMID 11867545]. More recently, it has been reported that the cyclic movement of NS3 helicase is coordinated by ATP in discrete steps of 11 base pairs, and that actual unwinding occurs in rapid smaller sub-steps of 2 to 5 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm [PMID 16397502]. The NS3 helicase activity can be modulated by interactions between the serine protease and helicase domains. Indeed the kinetics of duplex RNA unwinding is slower for the isolated helicase domain as compared with the full-length NS3 protein [PMID 14585830]. In addition, the presence of NS4A enhances productive RNA binding of a full-length NS3-4A complex [PMID 11867545]. The function of the NS3 helicase in the HCV life cycle is not known. It may be involved in initiation of RNA replication by unwinding stable stem-loop structures at the termini of positive and/or negative strand of HCV RNA. It may also contribute to the process of the replicase complex by removing stable RNA secondary structures and/or by displacing bound proteins that might interfere with RNA synthesis. Finally, it may also be required for dissociation of the replicative form. Due to its enzymatic activity, the helicase domain of NS3 is another potential target for the development of anti-HCV molecules. The NS3 protein has been reported to interact with several cellular proteins [PMID 12160863], and it has been proposed to be involved in carcinogenesis [PMID 16690937]. However, the relevance of these interactions needs to be confirmed in the context of the recently developed cell culture system for HCV [PMID 15947137, PMID 15939869, PMID 15939869]. (Text taken from: Dubuisson, J. (2007) Hepatitis C virus proteins, World J Gastroenterol 13, 2406-2415).
Current State of Antiviral At present, there is no approved (HCV specific) antiviral therapy. HCV has developed complex mechanisms to use the variety and intricacy of the host lipidome. A promising candidate for anti-HCV therapy emerged with the recent development of statins, a drug that interferes with lipid metabolism. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Statins have been approved clinically for the treatment of hypercholesterolaemia. These cause a reduction in intracellular mevulonate, LDL and geranylgeraniol, thereby inhibiting cholesterol biosynthesis and also causing a decrease in prenylated proteins [PMID 18410471, PMID 17928739]. This drug has also been used to further enhance the antiviral effects of INF [PMID 17928739]. An interesting study utilizing replicon system showed that statins (mevastatin or simvastatin) in combination with polymerase or protease inhibitors could effectively clear the cultures from HCV replicons. In addition, mevastatin was found to prevent selection of replicons resistant to the non nucleoside inhibitor HCV-796 [PMID 19437494]. These cell culture studies suggest that a combination of specific viral inhibitors with statins may delay or prevent the development of resistance to viral inhibitors. However, this remains to be tested in clinical trials. MicroRNAs (miR) are (~22 nucleotide) naturally occurring noncoding RNAs. These belong to a family of small RNA molecules that perform and regulate critical cellular functions. MicroRNAs serve as gene regulators controlling the cell cycle and tissue differentiation [PMID 17679093]. These are also implicated in apoptosis and oncogenesis [PMID 17665990]. The miRNA (miR-122) is required for HCV replication and development of antiviral therapies targeting on specific miRNA has great potential for exploration. However, a better understanding of the role for miR-122 binding to the HCV is required for effective design of antiviral therapies. Attempts are underway utilizing RNAi technology and one such attempt is the designing of TT-033, which contains three separate RNAi molecules to shut down replication of all strains of the hepatitis C virus. This strategy sounds promising and could potentially stop the generation of viral escape mutants. Current efforts to develop antiviral therapeutics are haunted by the unique nature of the HCV, i.e., the error-prone nature of NS5B, generation of resistance mutants which presents a selective growth advantage. To circumvent this major hurdle in HCV therapy, the current research efforts are aimed at combination therapy targeting multiple proteins both viral and host. Numerous novel HCV specific inhibitors such as nucleoside and non nucleoside polymerase inhibitors, protease inhibitors, as well as non HCV specific compounds with antiviral activity such as nitazoxanide, cyclophilin inhibitors (Debio 025), silibinin are under development in clinical trials. An immunotherapeutic vaccine along with the current therapy may also be beneficial especially in immunocompromised patients. In the meantime, combination therapy utilizing PEG-INF and ribavirin will remain standard of care. A recent estimate projects the current treatment market for HCV to be around US$ 3 billion per year. This market is expected to grow rapidly and reach around US$ 8 billion by 2010 [PMID 17001802]. (Text taken from: Sharma, S. D. (2010) Hepatitis C virus: molecular biology & current therapeutic options, Indian J Med Res 131, 17-34.)
Antiviral Progress Wide varieties of inhibitors or directly acting antiviral agents are at different phases of development and some are currently in clinical trials. These inhibitors target HCV receptors, HCV-IRES, NS3/4A, NS5A and NS5B. In addition molecules targeting host factors which play a role in HCV replication, are also being pursued. The significance of targeting NS3-4A protease is clearly evident from its vital function, required to process viral polyprotein. Targeting NS3-4A has additional advantage, since NS3-4A protease also functions to inhibit host innate response by cleaving IFN-beta promoter stimulator-1 (IPS-1) and TRIF [PMID 15767257]. The latter activity of NS3 affects signaling of the RIG-I and Toll-like-receptor-3 pathways, thereby perturbing the interferon response. A specific inhibitor of NS3, telaprevir (VX-950) was put to clinical test recently with some success. The current thought in the field being that a combination therapy along with inhibitors which simultaneously inhibit multiple steps in the virus lifecycle (protease and replicase functions) may yield better SVR. Another interesting candidate for a peptide vaccine is the HCV-specific HLA-A2-restricted NS3 (1073) epitope [PMID 18582999]. As with any RNA virus, rapidly mutating genomes such as HCV pose a huge obstacle to antiviral/vaccine development and represents a major challenge to the field. In addition, HCV acquires a lipid envelope, which originates from host membranes. Thus, the lipid composition of the HCV envelope resembles that of the host cell membrane and this clever mimicry may allow HCV to avoid detection by the immune system [PMID 19638285]. (Text taken from: Sharma, S. D. (2010) Hepatitis C virus: molecular biology & current therapeutic options, Indian J Med Res 131, 17-34.)
Uniprot P27958, P26664, Q5I2N3, Q9WMX2, P26663, P26662, P29846, Q81258, Q99IB8
Protein Type Nonstructural Protein
Genome Single-stranded positive sense RNA
Family Flaviviridae
Genus Hepacivirus
Strain Name HCV-1a-H77-NS3
Molecule Type mature protein
Amino Acid Length 631 aa
pI 6.99
Gene Size 1893 bp
Molecular Mass by SDS-Page 69 kDa [PMID 17552023]
Molecular Weight 67 kDa
Proteolytic Enzymes NS3 protease, NS3/NS4A protease
NCBI CDD Pfam pfam02907
Entrez Gene ID 951475
GI 28921568
NCBI RefSeq NP_803144.1
Pfam PF02907
Interpro IPR004109
PDB 1a1q, 1a1r, 1bt7, 1cu1, 1dxp, 1dxw, 1dy8, 1dy9, 1jxp, 1n1l, 1ns3, 1rgq, 1rtl, 1w3c, 2a4g, 2a4q, 2a4r, 2f9u, 2f9v, 2fm2, 1hei
Recommended PubMed Readings PUBMED:18985028, PUBMED:12691456, PUBMED:19286138, PUBMED:14752815, PUBMED:17455808, PUBMED:2827459, PUBMED:17552023, PUBMED:9568891, PUBMED:8861916, PUBMED:9083052, PUBMED:7575585, PUBMED:8386278, PUBMED:8389908, PUBMED:17263146, PUBMED:12692242, PUBMED:8861917, PUBMED:9493270
Gene Ontology GO:0008236, GO:0019087, GO:0006508, GO:0010389, GO:0008166, GO:0045930, GO:0051260, GO:0008283, GO:0032479, GO:0019034, GO:0033202, GO:0031315, GO:0005783, GO:0004252, GO:0004004, GO:0017151, GO:0003677, GO:0017111, GO:0004170, GO:0003678, GO:0003723
 
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