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Nonstructural Proteins (nonstructural + protein)

Distribution by Scientific Domains

Chemistry	45%
Medical Sciences	41%

Selected Abstracts

Crystallization and preliminary X-ray diffraction studies of infectious bronchitis virus nonstructural protein 9

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
Yanlin Ma
Avian infectious bronchitis virus (IBV), which causes respiratory disease in infected birds, belongs to coronavirus group 3. IBV encodes 15 nonstructural proteins (nsp2,nsp16) which play crucial roles in RNA transcription and genome replication. Nonstructural protein 9 (nsp9) has been identified as a protein that is essential to viral replication because of its single-stranded RNA-binding ability. The gene segment encoding IBV nsp9 has been cloned and expressed in Escherichia coli. The protein has been crystallized and the crystals diffracted X-rays to 2.44,Å resolution. They belonged to the cubic space group I432, with unit-cell parameters a = b = c = 123.4,Å, , = , = , = 90°. The asymmetric unit appeared to contain one molecule, with a solvent content of 62% (VM = 3.26,Å3,Da,1). [source]

Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naïve patients,,§

HEPATOLOGY, Issue 6 2008
Thomas Kuntzen
Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000-fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment-naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT-C) in the population, we analyzed HCV genome sequences from 507 treatment-naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication-competent, drug-resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN-191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG-021541; and to the NS4A antagonist ACH-806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug-resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. Conclusion: Naturally occurring dominant STAT-C resistance mutations are common in treatment-naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin. (HEPATOLOGY 2008;48:1769,1778.) [source]

Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA,expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope

HEPATOLOGY, Issue 6 2001
Carine Brinster
The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.105 cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope. [source]

Detection of viruses identified recently in children with acute wheezing

JOURNAL OF MEDICAL VIROLOGY, Issue 8 2007
Ju-Young Chung
Abstract The etiologic role of recently identified respiratory viruses for acute wheezing in children is not yet clear. The purpose of this study was to investigate the prevalence of recently identified viruses, including human metapneumovirus (hMPV), human bocavirus (hBoV), human coronavirus NL63 (hCoV-NL63), and human coronavirus HKU1 (hCoV-HKU1) in children with acute wheezing. Viral etiology was identified in 231 children hospitalized with acute wheezing, aged from 1 month to 5 years. Viral antigens for common respiratory viruses were detected by IFA or multiplex PCR. RT-PCR was used to detect respiratory rhinoviruses, hCoV-NL63, hCoV-HKU1, and hMPV. PCR assays for hBoV DNA were performed using the primer sets for noncapsid protein (NP1) and nonstructural protein (NS1) genes. Viruses were found in 61.5% (142/231) of the study population and a single virus was detected in 45.5% (105/231) of the study population. Rhinovirus (33.3%), human respiratory syncytial virus (hRSV; 13.8%), and hBoV (13.8%) were the most frequently detected viruses. hMPV and hCoV-NL63 were detected in 7.8% and 1.3% of wheezing children, respectively. HCoV-HKU1 was not detected. In 16.0% of the study population, more than one virus was detected. In children with acute wheezing, rhinovirus, hRSV, and hBoV were most frequently detected. Further studies including healthy control subjects are needed to define the clinical significance of hBoV in acute wheezing. J. Med. Virol. 79: 1238,1243, 2007. © 2007 Wiley-Liss, Inc. [source]

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C

JOURNAL OF VIRAL HEPATITIS, Issue 4 2002
C.-H. Pan
summary.,The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH2 -related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH0 or TH2 -related cytokines, which might play a critical role in the chronicity of HCV infection. [source]

Discovery of the hepatitis C virus

LIVER INTERNATIONAL, Issue 2009
Michael Houghton
Abstract After nearly 6 years of intensive investigations between 1982 and 1988 in my laboratory at Chiron corporation, in which numerous molecular biological methods were used to investigate the viral aetiology of parenterally transmitted non-A, non-B viral hepatitis (NANBH), a single cDNA clone (5-1-1) was isolated that was shown to be derived from a new flavi-like virus, termed the hepatitis C virus (HCV). After screening hundreds of millions of bacterial cDNA clones derived from different liver and plasma samples obtained from experimentally infected chimpanzees, a single HCV clone was eventually isolated using a novel, blind immunoscreening method in which antibodies derived from a clinically diagnosed NANBH patient were used to identify a cDNA clone encoding an immunodominant epitope within HCV nonstructural protein 4. Its viral origin was demonstrated by its specific hybridization to a large single-stranded RNA molecule of ,10 000 nucleotides found only in NANBH-infected samples that shared distant sequence identity with flaviviruses. Further, HCV clone 5-1-1 was shown to be extrachromosomal and to encode an antigen eliciting antibody seroconversion only in NANBH-infected chimpanzees and humans. Subsequent work demonstrated that HCV was the principal cause of parenterally transmitted NANBH around the world, with an estimated 170 million global carriers and that blood screening tests detecting circulating HCV antibodies and viral RNA could effectively eradicate the transmission of transfusion-associated NANBH. Key viral-encoded enzymes essential to its life cycle are now the targets of vigorous, ongoing drug development activities, and the feasibility of successful vaccination strategies has been demonstrated using the valuable chimpanzee model, without which any progress on HCV would not have been possible. My colleagues and coworkers who made essential contributions to the discovery of HCV were George Kuo, who had his own laboratory at Chiron and who provided intellectual and practical input, Dan Bradley of the Centers for Disease Control and Prevention, who provided a large supply of well-characterized chimpanzee samples and knowledge of the NANBH field, and Qui-Lim Choo, in my own laboratory, who provided many years of outstandingly dedicated and precise molecular biology expertise. [source]

What can we learn by computing 13C, chemical shifts for X-ray protein models?

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009
Yelena A. Arnautova
The room-temperature X-ray structures of ubiquitin (PDB code 1ubq) and of the RNA-binding domain of nonstructural protein 1 of influenza A virus (PDB code 1ail) solved at 1.8 and 1.9,Å resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed 13C, chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and Rfree factors similar to those of the deposited X-ray structure, the 13C, chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed 13C, chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed 13C, chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed 13C, chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed 13C, chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with accuracy as good as the PDB structure. Therefore, generation of an ensemble is a necessary step to determine whether or not a single structure is sufficient for an accurate representation of both experimental X-ray data and observed 13C, chemical shifts in solution. [source]

Crystallization and preliminary X-ray diffraction studies of infectious bronchitis virus nonstructural protein 9

Expression, crystallization and preliminary crystallographic study of human coronavirus HKU1 nonstructural protein 9

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009
Wei Wang
Human coronavirus HKU1 (HCoV-HKU1) belongs to coronavirus group II and encodes 16 nonstructural proteins (nsps) which mediate genome replication and transcription. Among these nsps, nsp9 has been shown to possess single-stranded DNA/RNA-binding properties. The gene that encodes HCoV-HKU1 nsp9 was cloned and expressed in Escherichia coli and the protein was subjected to crystallization trials. The crystals diffracted to 2.7,Å resolution and belonged to space group P21212, with unit-cell parameters a = 83.5, b = 88.4, c = 31.2,Å, , = , = , = 90° and two molecules per asymmetric unit. [source]

CD8+ T-cell interaction with HCV replicon cells: Evidence for both cytokine- and cell-mediated antiviral activity

HEPATOLOGY, Issue 6 2003
Chen Liu
The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8+ T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with 51Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-,), anti-tumor necrosis factor , (TNF-,), and anti-FasL. Cytolytic activity was measured by 51Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8+ cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8+ antiviral effect was markedly reduced by blocking either IFN-, or FasL but was unaffected by blocking TNF-,. In conclusion, HCV-specific CD8+ cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes. [source]

Course and outcome of hepatitis C

HEPATOLOGY, Issue 5B 2002
31 Center Dr., Jay H. Hoofnagle Bldg. 3, Room 9A2
The hepatitis C virus (HCV) is a small enveloped RNA virus belonging to the family flaviviridae and genus hepacivirus. The HCV RNA genome is 9,600 nucleotides in length and encodes a single polyprotein that is post-translationally cleaved into 10 polypeptides including t3 structural (C, E1, and E2) and multiple nonstructural proteins ([NS] NS2 to NS5). The NS proteins include enzymes necessary for protein processing (proteases) and viral replication (RNA polymerase). The virus replicates at a high rate in the liver and has marked sequence heterogeneity. There are 6 genotypes and more than 90 subtypes of HCV, the most common in the United States being 1a and 1b (approximately 75%), 2a and 2b (approximately 15%), and 3 (approximately 7%). Acute hepatitis C is marked by appearance of HCV RNA in serum within 1 to 2 weeks of exposure followed by serum alanine aminotransferase (ALT) elevations, and then symptoms and jaundice. Antibody to HCV (anti-HCV) tends to arise late. In acute resolving hepatitis, HCV RNA is cleared and serum ALT levels fall to normal. However, 55% to 85% of patients do not clear virus, but develop chronic hepatitis C. Chronic hepatitis C is often asymptomatic, but is usually associated with persistent or fluctuating elevations in ALT levels. The chronic sequelae of hepatitis C include progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Extra-hepatic manifestations include sicca syndrome, cryoglobulinemia, glomerulonephritis, and porphyria cutanea tarda. Knowledge of the course and outcome of hepatitis C is important in developing approaches to management and therapy. [source]

Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India

JOURNAL OF MEDICAL VIROLOGY, Issue 4 2001
Nobumichi Kobayashi
Abstract Nucleotide sequences of RNA segments encoding structural proteins(VP4, VP6, and VP7) and nonstructural proteins(NSP1 and NSP3) of a human group B rotavirus CAL-1, which was detected in Calcutta, India, were determined and their relatedness with cognate genes of other group B rotaviruses was analyzed. The CAL-1 genes showed generally high sequence identities (more than 90%) to those of human group B rotavirus, adult diarrheal rotavirus (ADRV) in China, while identities with bovine, murine, and ovine viruses were considerably lower (58,73%). Among RNA segments analyzed, sequence identity of the VP6 gene was relatively high compared with other gene segments. In the CAL-1 VP7 sequence, many characteristics were shared by ADRV, but not by other animal group B rotaviruses. In contrast, VP4 and NSP3 of CAL-1 were single animo acid and 23 amino acids longer than those of ADRV strain, respectively, due to differences of a few nucleotides. These findings suggested that human group B rotaviruses CAL-1 and ADRV might have originated from a common ancestral virus distinct from animal group B rotaviruses reported so far, while some notable sequence differences indicated the distinct nature of these viruses. J. Med. Virol. 64:583,588, 2001. © 2001 Wiley-Liss, Inc. [source]

Antigenicity of chimeric and cyclic synthetic peptides based on nonstructural proteins of GBV-C/HGV

JOURNAL OF PEPTIDE SCIENCE, Issue 4 2006
T. Pérez
Abstract In this work, new putative epitopes located in nonstructural proteins of GBV-C/HGV were synthesized using solid-phase chemistry for their use in immunoassays. The antigens were obtained in linear, chimeric and cyclic forms with the main aim of improving the sensitivity of the enzyme immunoassays. Our results showed, on one hand, that the combination of different antigens seems to be necessary to ensure good sensitivity and more specificity and, on the other hand, that cyclic compounds show higher ability to recognize anti-GBV-C/HGV antibodies than its parent peptide. Furthermore, CD and FTIR have been used in conjunction to characterize the conformational changes therein with synthetic constructs that could explain their different antigenicity. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source]

HCV-specific T-cell responses in injecting drug users: evidence for previous exposure to HCV and a role for CD4+ T cells focussing on nonstructural proteins in viral clearance

JOURNAL OF VIRAL HEPATITIS, Issue 6 2008
T. A. Ruys
Summary., In order to understand the parameters associated with resolved hepatitis C virus (HCV)-infection, we analysed the HCV-specific T-cell responses longitudinally in 13 injecting drug-users (IDUs) with a prospectively identified acute HCV infection. Seven IDUs cleared HCV and six IDUs remained chronically infected. T-cell responses were followed in the period needed to resolve and a comparable time span in chronic carriers. Ex vivo T-cell responses were measured using interferon-, Elispot assays after stimulation with overlapping peptide pools spanning the complete HCV genome. CD4+ memory- T-cell responses were determined after 12-day stimulation with HCV proteins. The maximum response was compared between individuals. The T-cell responses measured directly ex vivo were weak but significantly higher in resolvers compared to chronic carriers, whereas the CD4+ memory -T-cell response was not different between resolvers and chronic carriers. However, HCV Core protein was targeted more often in chronic carriers compared to individuals resolving HCV infection. CD4+ T-cell responses predominantly targeting nonstructural proteins were associated with resolved HCV infection. Interestingly, observation of memory-T-cell responses present before the documented HCV-seroconversion suggests that reinfections in IDUs occur often. The presence of these responses however, were not predictive for the outcome of infection. However, a transition of the HCV-specific CD4+ memory -T-cell response from targeting Core to targeting nonstructural proteins during onset of infection was associated with a favourable outcome. Therefore, the specificity of the CD4+ memory -T-cell responses measured after 12-day expansion seems most predictive of resolved infection. [source]

Structure of the C-terminal domain of nsp4 from feline coronavirus

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009
Ioannis Manolaridis
Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26,31,kb) encodes 15,16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication,transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (,100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8,Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly ,-helical content displaying a unique fold that could be engaged in protein,protein interactions. [source]

Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
Xiaohang Tong
Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4,kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20,Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8,Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (VM = 2.16,Å3,Da,1). [source]

Crystallization and preliminary X-ray diffraction studies of infectious bronchitis virus nonstructural protein 9

Expression, crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of nsp2 from avian infectious bronchitis virus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009
Anqi Yang
Avian infectious bronchitis virus (IBV) is a prototype of the group III coronaviruses and encodes 15 nonstructural proteins which make up the transcription/replication machinery. The nsp2 protein from IBV has a unique and novel sequence and has no experimentally confirmed function in replication, whereas it has been proposed to be crucial for early viral infection and may inhibit the early host immune response. The gene that encodes a double-mutant IBV nsp2 N-terminal domain (residues 9,393 of the polyprotein, with mutations Q132L and L270F) was cloned and expressed in Escherichia coli and the protein was subjected to crystallization trials. The crystals diffracted to 2.5,Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 114.2, c = 61.0,Å, , = , = 90, , = 120°. Each asymmetric unit contained one molecule. [source]

Expression, crystallization and preliminary crystallographic study of human coronavirus HKU1 nonstructural protein 9

Hepatitis B and C virus-related carcinogenesis

CLINICAL MICROBIOLOGY AND INFECTION, Issue 11 2009
J. Fung
Abstract Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the most important causes of hepatocellular carcinoma (HCC), accounting for the majority of the cases worldwide. The geographical distribution of HCC therefore coincides with the distribution of HBV and HCV infections in those areas. Similar to nonviral liver diseases, HBV and HCV infection can cause chronic injury to the liver, with subsequent progression to severe fibrosis and cirrhosis. The presence of cirrhosis is a major risk factor for the development of HCC. However, HCC can occur in the absence of cirrhosis, suggesting that both HBV and HCV may be directly involved in hepatocarcinogenesis. Several HBV factors have been implicated in hepatocarcinogenesis, including the HBx gene, the pre-S2/S gene and the HBV spliced protein. Furthermore, HBV can be integrated into the host genome, leading to changes in genomic function or chromosomal instability. By contrast to HBV, HCV cannot integrate into the host genome. Various HCV proteins, including the core, envelope and nonstructural proteins, have been shown to have oncogenic properties. For HBV infection, antiviral therapy and vaccination have been shown to decrease the risk of HCC. Antiviral therapy for HCV can also reduce the risk of HCC. [source]