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 NS5A
Recommended Name Non-structural protein 5A
Other Names p56
EC 
OMIM 114550, 609532
euhcvdb protein link http://euhcvdb.ibcp.fr/euHCVdb/jsp/NS5A.jsp
GenBank (Location: 6258..7601) NC_004102
VBRC Accession 64601
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]. NS5A is phosphorylated in a basal form termed p56. p58 is an hyperphosphorylated form of p56. p56 and p58 coexist in the cell in roughly equivalent amounts. Hyperphosphorylation is dependent on the presence of NS4A. Human AKT1, RPS6KB1/p70S6K, MAP2K1/MEK1, MAP2K6/MKK6 and CSNK1A1/CKI-alpha kinases may be responsible for NS5A phosphorylation [PubMed: 10488152].
Uniprot Functions NS5A is a component of the replication complex involved in RNA-binding. Its interaction with Human VAPB may target the viral replication complex to vesicles. Down-regulates viral IRES translation initiation. Mediates interferon resistance, presumably by interacting with and inhibiting human EIF2AK2/PKR. Seems to inhibit apoptosis by interacting with BIN1 and FKBP8. The hyperphosphorylated form of NS5A is an inhibitor of viral replication. [PubMed: 16894197]
Uniprot Subunit Structure NS4B and NS5A form homodimers and seem to interact with all other non-structural (NS) proteins. NS5A also interacts with human EIF2AK2/PKR, FKBP8, GRB2, BIN1, PIK3R1, SRCAP, VAPB and with most Src-family kinases.
Uniprot Domain The N-terminus of NS5A acts as membrane anchor. The central part of NS5A seems to be intrinsically disordered and interacts with NS5B and host PKR. The C-terminus of NS5A contains a variable region called variable region 3 (V3). The SH3-binding domain of NS5A is involved in the interaction with human Bin1, GRB2 and Src-family kinases. [PubMed: 1658800 PubMed: 10729138]
Uniprot Subcellular Localization Host endoplasmic reticulum membrane; Peripheral membrane protein. Host cytoplasm › host perinuclear region. Host mitochondrion. Note: Host membrane insertion occurs after processing by the NS3 protease. [PubMed: 10702287, PubMed: 8982089, PubMed: 10729138, PubMed: 11907211, PubMed: 12692244, PubMed: 17192310, PubMed: 14752815]
Functions Phosphoprotein [PMID 17552023]; inhibition of IFN-alpha, inhibition of apoptosis [PMID 17455808]
Drug Development The structure of domain I of NS5A provides a framework for the rational design of small antiviral molecules. The other two domains of NS5A are less characterized [PMID 17552023]. Domain II has been proposed to be involved in inhibition of the interferon-induced double stranded RNA activated protein kinase PKR [PMID 9710605, PMID 17552023], and domain III is a less conserved region, which can tolerate insertions or partial deletions [PMID 15709040, PMID 15220413, PMID 17552023]. NS5A has attracted considerable interest because of its potential role in modulating the response to interferon alpha therapy [PMID 11050035, PMID 14752815]. Japanese studies suggested the existence of a discrete region of NS5A, tentatively named the interferon sensitivity determining region [PMID 8531962, PMID 14752815]
Description NS5A is a membrane-associated protein containing a unique amphipathic alpha-helix at its N-terminus, which serves as an in-plane membrane anchor [PMID 11744739, PMID 15247283]. Like most HCV proteins, NS5A is detected in association with ER or ER-derived membranes [PMID 11744739]. Besides its membrane anchor sequence, NS5A contains three distinct domains that are separated by low complexity sequences (LCs) I and I [PMID 15339921]. Recently, the x-ray crystal structure of domain I was solved [PMID 15902263]. It is composed of a basic N-terminal subdomain IA and a predominantly acidic C-terminal subdomain IB. In subdomain IA a zinc ion is coordinated by a unique motif of 4 fully conserved cysteine residues, which are absolutely essential for RNA replication [PMID 15339921, PMID 15902263]. In subdomain IB an unusual disulfide bond linking 2 cysteine residues near the C-terminal subdomain border was found. However, this disulfide bond does not seem to be essential for HCV RNA replication. Domain I forms homodimers via contacts near the N-terminal end of the molecules. This dimerization results in the formation of a basic groove facing the cytosol at the surface of the membrane. This ‘claw like’ structure is believed to provide an RNA binding site that might be involved in regulated genome targeting within the replication complex [PMID 15902263]. In line with this observation, NS5A has been shown to bind to HCV RNA and more specifically to the 3’-ends of HCV plus and minus strand RNAs, with a preference for the polypyrimidine tract in the 3’ non-translated region of positive strand RNA [PMID 16126720]. NS5A is a protein which is essential for genome re-plication [PMID 16407182, PMID 16154356]. Indeed, mutations that enhance RNA replication in cell culture map to the NS5A-coding sequence. In addition, NS5A has been shown to interact with NS5B, and this interaction is essential for maintenance of sub-genomic replicons in Huh-7 cells [PMID 14990694, PMID 11801599]. NS5A is expressed as a basally phosphorylated and a hyperphosphorylated forms [PMID 15530561]. The functional relevance of the different pho-sphorylated forms is unknown. However, mutations that reduce NS5A hyperphosphorylation can lead to a dra-matic ehancement of HCV genomic replication [PMID 15709040, PMID 15326295]. Furthermore, treatment of cells carrying non-adapted repli-cons with an inhibitor of the cellular kinase(s) responsible for NS5A hyperphosphorylation leads to an increase in HCV genomic replication [PMID 15542681]. In addition to its role in HCV genomic replication, NS5A has initially attracted considerable interest because of its potential role in modu-lating the interferon response [PMID 11352662]. NS5A has also been shown to interact with components of numerous cellular signaling pathways [PMID 12160863, PMID 12065893, PMID 15302943]. Among the potential cellular partners identified for NS5A, human vesicle-associated membrane protein-associated protein A (hVAP-A) is of particular interest because it is regulated by NS5A phosphorylation [PMID 15326295, PMID 10544080]. Indeed, NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication. VAP-A is a protein found on ER and Golgi membranes, which is involved in intracellular vesicle trafficking. It remains however to be determined why NS5A hijacks hVAP-A at some step of its life cycle. Another potentially important host cell factor interacting with NS5A is the geranylgeranylated protein FBL-2 [PMID 15893726]. In line with this observation, it has been shown that inhibition of geranylgeranylation in cells abolishes HCV RNA replication [PMID 14668447]. (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. NS5A plays a vital role in HCV replication and is emerging as an important target for the therapy of HCV infection. BMS-824 is a small molecule which targets NS5A and inhibits hyperphosphorylation or p58 formation [PMID 19812153]. Inhibitors that disrupt phosphorylation with a concomitant decrease in virus replication are indeed very attractive and should be developed further. Another molecule, zinc mesoporphyrin (a synthetic heme analogue with a central zinc of the mesoporphyin macrocycle) causes a dramatic downregulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent degradation. Zinc mesoporphyrin holds immense potential as a novel drug to treat HCV infection [PMID 19909748]. (Text taken from: Sharma, S. D. (2010) Hepatitis C virus: molecular biology & current therapeutic options, Indian J Med Res 131, 17-34.)
Uniprot P27958, Q0MR49, Q0MR50, Q0MR51
Protein Type Nonstructural Protein
Genome Single-stranded positive sense RNA
Family Flaviviridae
Genus Hepacivirus
Strain Name HCV-1a-H77-NS5a
Molecule Type mature protein
Amino Acid Length 448 aa
pI 5.35
Gene Size 1344 bp
Molecular Mass by SDS-Page 56 - 58 kDa [PMID 17552023]
Molecular Weight 49 kDa
Proteolytic Enzymes NS3 protease
NCBI CDD Pfam pfam01506, pfam08300, pfam08301
Entrez Gene ID 951475
GI 26053629
NCBI RefSeq NP_751927.1
Pfam Pf01506, Pf08300, Pf08301
Interpro IPR013193, IPR013192, IPR002868
PDB 1zh1, 1r7c, 1r7d, 1r7e, 1r7f, 1r7g
Recommended PubMed Readings PUBMED:18985028, PUBMED:12691456, PUBMED:19286138, PUBMED:14752815, PUBMED:17455808, PUBMED:2827459, PUBMED:17552023, PUBMED:9710605, PUBMED:9143277, PUBMED:15902263, PUBMED:15339921, PUBMED:15902263, PUBMED:15902263, PUBMED:11744739, PUBMED:17192310, PUBMED:15302943, PUBMED:10488152
Gene Ontology GO:0006915, GO:0030683, GO:0045449, GO:0003968, GO:0004197, GO:0004252, GO:0008270, GO:0017111, GO:0005576, GO:0005789, GO:0016021, GO:0046774, GO:0006412, GO:0046782, GO:0046786, GO:0045071, GO:0006917, GO:0006926, GO:0045930, GO:0010389, GO:0006417, GO:0005635, GO:0005783, GO:0019034, GO:0005634, GO:0005737, GO:0003677, GO:0042803, GO:0003723
 
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