To see the other types of publications on this topic, follow the link: Ns3.
Journal articles on the topic 'Ns3'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Ns3.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
VERMA, RAKESH KUMAR, YASHBIR SINGH SHIVAY, MUKESH CHOUDHARY, PRAKASH CHAND GHASAL, and RAGHAVENDRA MADAR. "Nutrient mobilization and crop assimilation as influenced by nutrient management strategies under direct seeded basmati rice (Oryza sativa) – based cropping systems." Indian Journal of Agricultural Sciences 90, no. 10 (December 4, 2020): 1894–901. http://dx.doi.org/10.56093/ijas.v90i10.107891.
Full textAbstract:
The field experiment was carried out for two consecutive years (2014–2016) in split-plot design to investigate the effect of integrated nutrient management and crop diversification through inclusion of legume and vegetable crops in direct seeded basmati rice (Oryza sativa L.)–based cropping systems (DSRB) on nutrient availability for crop uptake. The study involved four cropping systems (CS) in main plots (DSBR‒wheat‒fallow (CS1), DSBR‒wheat‒greengram (CS2), DSBR‒cabbage‒greengram (CS3) and DSBR‒cabbage‒onion (CS4) and four nutrient management strategies under subplots (unfertilized (NS0), 100% recommended dose of fertilizers (RDF) (NS1), 50% RDF + 25% recommended dose of nitrogen (RDN) through leaf compost (LC) + biofertilizer (NS2), 50% RDF + 25% RDN through vermicompost (VC) + biofertilizer (NS3)). The results revealed that diversification of rice–wheat system with legume (greengram) or vegetable (cabbage and onion) crops and integrated nutrient management strategies had positive effect on nutrient uptake and available nutrient status in the soil. Significantly higher uptake of N, P and K in all crops and Zn, Fe, Mn and Cu in rice and wheat were observed with NS2 and NS3 as compared to NS0. Available N, P and K status were significantly higher in NS2 and NS3 as against NS0 and NS1. Inclusion of cereal crops in the cropping systems showed a negative apparent N balance, but inclusion of vegetable crops in the cropping systems exhibited positive apparent N balance under different nutrient management strategies except NS0. The highest positive apparent N balance was observed in NS1 treatment. The apparent P balance was found to be positive in all the cropping systems with all the nutrients sources except NS0. Apparent K balance was found negative in all the cropping systems under different nutrient management strategies. Thus, cropping systems with summer greengram, cabbage and onion (CS2, CS3 and CS4) under integrated nutrient management practices (NS2 and NS3) were found more sustainable after two years of cropping cycle and can be advocated by the farmers of IGP.
2
Isken, Olaf, Thomas Walther, Luis Wong-Dilworth, Dirk Rehders, Lars Redecke, and Norbert Tautz. "Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity." PLOS Pathogens 18, no. 6 (June 21, 2022): e1010644. http://dx.doi.org/10.1371/journal.ppat.1010644.
Full textAbstract:
Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3.
3
Anderson, Jenna, Emmanuel Bréard, Karin Lövgren Bengtsson, Kjell-Olov Grönvik, Stéphan Zientara, Jean-Francois Valarcher, and Sara Hägglund. "Purification, Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals." Clinical and Vaccine Immunology 21, no. 3 (January 22, 2014): 443–52. http://dx.doi.org/10.1128/cvi.00776-13.
Full textAbstract:
ABSTRACTBluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus orEscherichia coliexpression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or −80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n= 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.
4
Phan, Tung, Rudolf K. F. Beran, Christopher Peters, Ivo C. Lorenz, and Brett D. Lindenbach. "Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes." Journal of Virology 83, no. 17 (June 10, 2009): 8379–95. http://dx.doi.org/10.1128/jvi.00891-09.
Full textAbstract:
ABSTRACT The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.
5
Ilyas, Fizza, Muhammad Jamsahid, Irfan Bashir, Rabia Aslam, Tooba Mehboob, Naila Tabassam, and Muhammad Nadeem Alvi. "Solvent Diffusion Method: An Effective Approach to Formulate Nanosponges Loaded with Naproxen Sodium." RADS Journal of Pharmacy and Pharmaceutical Sciences 8, no. 2 (November 11, 2020): 74–80. http://dx.doi.org/10.37962/jpps.v8i2.338.
Full textAbstract:
Objective: Solubility of naproxen sodium is limited. In conventional dosage form it causes different gastro intestinal problems. To overcome these difficulties naproxen sodium loaded nano sponges were designed.
Methodology: Nanosponges were formulated by using emulsion solvent evaporation technique. To obtain dispersion of nanosponges, homogenization of active drug, with specified quantities of polyvinyl alcohol, dichloromethane, ethyl cellulose and distilled, water was done. Compatibility among excipients and active drug was checked by FTIR and results didn’t show any interaction between them. 11 trial formulations were tested for poly dispersity, zeta potential, particle size and viscosity.
Results: Results showed all formulations except NS9, NS10 and NS11 were in nano range. Formulation NS1 to NS6 fall in category of “mid poly dispersity” and formulation NS7 to NS11 were in the category of “very poly dispersity”. Values of Zeta potential of all formulations were in negative range -0.106 to -9.75 mV. The value of viscosity of all formulations were 0.8872. NS2 and NS3 were selected for further testing like Franz cell diffusion study, stability testing and drug loading efficiency. In Franz cell diffusion study, drug release for NS2= 89.62%, for NS3= 89.10% at 50 minutes’ time. Stability studies performed for the 21 days, NS2 and NS3 revealed slight change in percentage drug content at 4°C and 25°C, and major changes were observed at 45°C temperature. Drug loading efficiency was found in NS2= 97.659 % and for NS3= 98.901%.
Conclusion: Nanosponges formulations loaded with naproxen sodium have successfully been prepared.
6
Yi, MinKyung, Yinghong Ma, Jeremy Yates, and Stanley M. Lemon. "Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus." Journal of Virology 81, no. 2 (November 1, 2006): 629–38. http://dx.doi.org/10.1128/jvi.01890-06.
Full textAbstract:
ABSTRACT There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction (“H-NS2/NS3-J”) and at a site of natural, intergenotypic recombination within NS2 [“H-(NS2)-J”] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.
7
Juhász, Evelin Kármen, and Andrea Balláné Kovács. "The effect of sulphur and nitrogen supply on the growth and nutrient content of spring wheat (Triticum aestivum L.)." Acta Agraria Debreceniensis, no. 74 (June 30, 2018): 65–70. http://dx.doi.org/10.34101/actaagrar/74/1666.
Full textAbstract:
Sulphur is an essential element for plants. Decreasing sulphur deposition from the air, and the use of more concentrated phosphate fertilizers, which contain no sulphur, has led to reports of sulphur deficiencies for wheat. Sulphur deficiency significantly affects yield and also the quality of wheat. The pot experiment was set up on calcareous chernozem soil at Látókép, Hungary, test plant was spring wheat (Triticum aestivum L). Seven treatments were used where nitrogen and sulphur were supplied as soil fertilizers in increasing rates (NS1, NS2, NS3) and in foliar fertilizer as well (NS1+fol., NS2+fol., NS3+fol.). Plant aboveground biomass production was determined in samples taken in the stages of development BBCH 29-30, 51-59, 61-69, 89. The nitrogen and sulphur content of straw and grain were measured. N/S ratios of grain and straw were calculated. The weights of grain were ranging between 8.6–16.1 g/pot. NS2 and NS2+fol. treatments produced the highest values. Foliar fertilizer had no further effect on grain. Analysing the values of the straw, it was observed that tendencies were similar to values of grain. The NS2 treatment produced the highest weight of straw and the NS3 rate already decreased that amount. The obtained results show the unfavourable effect of excessively high rate applied in NS3 treatment. The supplementary foliar fertilizer had no significant influence on the weight of straw. Both N and S-uptake of plant was very intensive at the stem elongation stage, then the N and S-content of plant continuously decreased in time in all treatments. The N-content of grain ranged between 2.215–2.838%.
The N-content of grain slightly increased with increasing of nitrogen doses. In the higher doses (NS2, NS3) foliar fertilization slightly increased the nitrogen content of grain, although this effect was not statistically proved. The N-content of straw varied from 0.361 to 0.605%. The growing dose of soil fertilizer also considerably increased the nitrogen content of straw. Foliar fertilization further increased the nitrogen content of straw. The S-content of grain ranged between 0.174–0.266%. The lowest fertilizer dose (NS1) significantly increased the sulphur content of grain. The further increasing fertilizer doses (NS2, NS3) did not cause additional enhance in sulphur content of grain.
The foliar fertilizer also did not change the sulphur value of plant. The increasing amount of soil fertilizer and the supplementary foliar fertilizer had no effect on the sulphur content of straw. The treatments influenced the N/S ratios of grain and straw. On the basis of experimental results it can be concluded that the examined nitrogen and sulphur containing soil fertilizer had positive effect on the growth and yield of spring wheat grown on the calcareous chernozem soil. The soil fertilizer application enhanced the grain nitrogen and sulphur content. The highest rate of fertilizer (600 kg ha-1) proved to have decreasing effect on the yield. The sulphur and nitrogen containing foliar fertilizer did not have significant effect on the yield parameters but slightly increased the nitrogen content of plant.
8
Klemens, O., D. Dubrau, and N. Tautz. "Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses." Journal of Virology 89, no. 22 (September 9, 2015): 11668–80. http://dx.doi.org/10.1128/jvi.01646-15.
Full textAbstract:
ABSTRACTA peculiarity of theFlaviviridaeis the critical function of nonstructural (NS) proteins for virus particle formation. For pestiviruses, like bovine viral diarrhea virus (BVDV), uncleaved NS2-3 represents an essential factor for virion morphogenesis, while NS3 is an essential component of the viral replicase. Accordingly, in natural pestivirus isolates, processing at the NS2-3 cleavage site is not complete, to allow for virion morphogenesis. Virion morphogenesis of the related hepatitis C virus (HCV) shows a major deviation from that of pestiviruses: while RNA replication also requires free NS3, virion formation does not depend on uncleaved NS2-NS3. Recently, we described a BVDV-1 chimera based on strain NCP7 encompassing the NS2-4B*-coding region of strain Osloss (E. Lattwein, O. Klemens, S. Schwindt, P. Becher, and N. Tautz, J Virol 86:427–437, 2012, doi:10.1128/JVI.06133-11). This chimera allowed for the production of infectious virus particles in the absence of uncleaved NS2-3. The Osloss sequence deviates in the NS2-4B* part from NCP7 in 48 amino acids and also has a ubiquitin insertion between NS2 and NS3. The present study demonstrates that in the NCP7 backbone, only two amino acid exchanges in NS2 (E1576V) and NS3 (V1721A) are sufficient and necessary to allow for efficient NS2-3-independent virion morphogenesis. The adaptation of a bicistronic virus encompassing an internal ribosomal entry site element between the NS2 and NS3 coding sequences to efficient virion morphogenesis led to the identification of additional amino acids in E2, NS2, and NS5B that are critically involved in this process. The surprisingly small requirements for approximating the packaging schemes of pestiviruses and HCV with respect to the NS2-3 region is in favor of a common mechanism in an ancestral virus.IMPORTANCEFor positive-strand RNA viruses, the processing products of the viral polyprotein serve in RNA replication as well as virion morphogenesis. For bovine viral diarrhea virus, nonstructural protein NS2-3 is of critical importance to switch between these processes. While free NS3 is essential for RNA replication, uncleaved NS2-3, which accumulates over time in the infected cell, is required for virion morphogenesis. In contrast, the virion morphogenesis of the related hepatitis C virus is independent from uncleaved NS2-NS3. Here, we demonstrate that pestiviruses can adapt to virion morphogenesis in the absence of uncleaved NS2-3 by just two amino acid exchanges. While the mechanism behind this gain of function remains elusive, the fact that it can be achieved by such minor changes is in line with the assumption that an ancestral virus already used this mechanism but lost it in the course of adapting to a new host/infection strategy.
9
Yen, Yu-Ting, and Betty Wu-Hsieh. "Dengue viral component triggering reactive nitrogen and oxygen species production leads to endothelial cell apoptosis (45.33)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 45.33. http://dx.doi.org/10.4049/jimmunol.182.supp.45.33.
Full textAbstract:
Abstract Vascular leakage is one of the life-threatening complications occurring in dengue patients, however, the pathogenic mechanism is not well understood. In dissecting the complex interplay between the virus and the host, we found that dengue virus productively infects the endothelial cells and causes apoptotic cell death through triggering the production of reactive nitrogen (RNS) and oxygen radicals (ROS). To determine the dengue virus component(s) that is responsible for the cause of cell death, we transfected human microvascular endothelial cell HMEC with pCR3.1 plasmids encoding Flag-tagged viral components Core, PrM, NS1, NS2A, NS2B, NS3, NS2B-NS3, NS4A, NS4B, and NS5, separately. Apoptotsis in cells transfected with NS2B-NS3 was 10-fold higher and that in cells transfected with NS1, NS3 and NS5 or core was only between 4- to 6-fold higher than cells transfected with vector alone. Confocal microscopy revealed that while NS2B expression remained in the cytosol, NS3 and NS2B-NS3 in both the cytosol and the nucleus at 24 h after transfection. Interestingly, the effect of NS2B-NS3 on endothelial cells was reversed in the presence of NO and ROS inhibitors. These results demonstrate that NS2B-NS3, being the major component that induces endothelial cell apoptosis, interacts with subcellular components in both the cytosol and the nucleus, and induces the production of RNS and ROS to cause apoptotic outcome.
10
Kümmerer, Beate M., Dieter Stoll, and Gregor Meyers. "Bovine Viral Diarrhea Virus Strain Oregon: a Novel Mechanism for Processing of NS2-3 Based on Point Mutations." Journal of Virology 72, no. 5 (May 1, 1998): 4127–38. http://dx.doi.org/10.1128/jvi.72.5.4127-4138.1998.
Full textAbstract:
ABSTRACT Bovine viral diarrhea virus (BVDV) isolates can either be cytopathogenic (cp) or noncytopathogenic (noncp). While both biotypes express the nonstructural protein NS2-3, generation of NS3 strictly correlates with the cp phenotype. The production of NS3 is usually caused by cp specific genome alterations, which were found to be due to RNA recombination. Molecular analyses of the cp BVDV strain Oregon revealed that it does not possess such genome alterations but nevertheless is able to generate NS3 via processing of NS2-3. The NS3 serine protease is not involved in this cleavage, which, according to protein sequencing, occurs between amino acids 1589 and 1590 of the BVDV Oregon polyprotein. Transient-expression studies indicated that important information for the cleavage of NS2-3 is located within NS2. This was verified by expression of chimeric constructs containing cDNA fragments derived from BVDV Oregon and a noncp BVDV. It could be shown that the C-terminal part of NS2 plays a crucial role in NS2-3 cleavage. These data, together with results obtained by site-specific exchanges in this region, revealed a new mechanism for NS2-3 processing which is based on point mutations within NS2.
11
Umareddy, Indira, Alex Chao, Aruna Sampath, Feng Gu, and Subhash G. Vasudevan. "Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA." Journal of General Virology 87, no. 9 (September 1, 2006): 2605–14. http://dx.doi.org/10.1099/vir.0.81844-0.
Full textAbstract:
Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3.
12
. "NS3." Huisarts en Wetenschap 50, no. 12 (December 2007): 835. http://dx.doi.org/10.1007/bf03085357.
Full text
13
Boyer, Audrey, Julie Dreneau, Amélie Dumans, Julien Burlaud-Gaillard, Anne Bull-Maurer, Philippe Roingeard, and Jean-Christophe Meunier. "Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly." Cells 8, no. 5 (May 22, 2019): 487. http://dx.doi.org/10.3390/cells8050487.
Full textAbstract:
During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein–protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.
14
Mendez, Ernesto, Nicolas Ruggli, Marc S. Collett, and Charles M. Rice. "Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity." Journal of Virology 72, no. 6 (June 1, 1998): 4737–45. http://dx.doi.org/10.1128/jvi.72.6.4737-4745.1998.
Full textAbstract:
ABSTRACT Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.
15
Liu, Hailiang, Sajjad Hussain, Jehoon Lee, Dhanasekaran Vikraman, and Jungwon Kang. "Ultrasonically Processed WSe2 Nanosheets Blended Bulk Heterojunction Active Layer for High-Performance Polymer Solar Cells and X-ray Detectors." Materials 14, no. 12 (June 10, 2021): 3206. http://dx.doi.org/10.3390/ma14123206.
Full textAbstract:
Two-dimensional (2D) tungsten diselenide (WSe2) has attracted considerable attention in the field of photovoltaic devices owing to its excellent structure and photoelectric properties, such as ordered 2D network structure, high electrical conductivity, and high mobility. For this test, we firstly prepared different sizes (NS1–NS3) of WSe2 nanosheets (NSs) through the ultrasonication method and characterized their structures using the field emission scanning electron microscope (FE-SEM), Raman spectroscopy, and X-ray powder diffraction. Moreover, we investigated the photovoltaic performance of polymer solar cells based on 5,7-Bis(2-ethylhexyl)benzo[1,2-c:4,5-c′]dithiophene-4,8-dione(PBDB-T):(6,6)-phenyl-C71 butyric acid methyl ester (PCBM) with different WSe2 NSs as the active layer. The fabricated PBDB-T:PCBM active layer with the addition of NS2 WSe2 NSs (1.5 wt%) exhibited an improved power conversion efficiency (PCE) of 9.2%, which is higher than the pure and NS1 and NS3 WSe2 blended active layer-encompassing devices. The improved PCE is attributed to the synergic enhancement of exciton dissociation and an improvement in the charge mobility through the modified active layer for polymer solar cells. Furthermore, the highest sensitivity of 2.97 mA/Gy·cm2 was achieved for the NS2 WSe2 NSs blended active layer detected by X-ray exposure over the pure polymer, and with the NS1 and NS2 WSe2 blended active layer. These results led to the use of transition metal dichalcogenide materials in polymer solar cells and X-ray detectors.
16
Neddermann, Petra, Angelica Clementi, and Raffaele De Francesco. "Hyperphosphorylation of the Hepatitis C Virus NS5A Protein Requires an Active NS3 Protease, NS4A, NS4B, and NS5A Encoded on the Same Polyprotein." Journal of Virology 73, no. 12 (December 1, 1999): 9984–91. http://dx.doi.org/10.1128/jvi.73.12.9984-9991.1999.
Full textAbstract:
ABSTRACT The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.
17
Lean, Fabian Z. X., Jean Payne, Jennifer Harper, Joanne Devlin, David T. Williams, and John Bingham. "Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses." Microorganisms 8, no. 8 (August 7, 2020): 1207. http://dx.doi.org/10.3390/microorganisms8081207.
Full textAbstract:
The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.
18
Ibuot, Aniefon Alphonsus, Iniobong Ime James, Nsikan Samuel Udoekong, Mayen Ben, Christiana Utibe Etuk, and Imaobong James. "CORRELATION BETWEEN CLASSROOM POPULATION, VENTILATION BACTERIAL LOADS AND THIER ANTIMICROBIAL PATTERNS IN SCHOOLS WITHIN IKOT EKPENE, NIGERIA." Bacterial Empire 2, no. 4 (October 9, 2019): 86. http://dx.doi.org/10.36547/be.2019.2.4.86-90.
Full textAbstract:
Indoor air of classroom in eight schools (4 nursery; NS1, NS2, NS3 and NS4, and 4 secondaries; SS1, SS2, SS3 and SS4) within Ikot Ekpene, Akwa Ibom State, Nigeria, were analyzed at ambient and populated sampling conditions using natural sedimentation on nutrient agar medium. The results revealed varying ventilation patterns in each of the classrooms, and the following airborne bacterial counts; NS1 (16.6 cfu/m3), NS2 (13.3 cfu/m3), NS3 (23.3 cfu/m3), NS4 (33.3 cfu/m3), SS1 (6.6 cfu/m3), SS2 (6.6 cfu/m3), SS3(28.3 cfu/m3) and SS4 (15 cfu/m3) at ambient sampling and 40 cfu/m3, 41.6 cfu/m3, 58.3 cfu/m3, 68.3 cfu/m3, 6.6 cfu/m3, 31.6 cfu/m3, 56.6 cfu/m3 and 25 cfu/m3 respectively at populated sampling. Bacterial isolates identified were Lactobacillus, Staphylococcus, Bacillus, Rothia, Kurthia, Corynebacterium, Pseudomonas, Brevibacterium, and Flavobacterium. Statistical analysis of the results revealed negative relationships between class area and aerobic plate counts (p>0.05), class population and aerobic plate count (p>0.805), and significant increase in aerobic plate counts at populated conditions over that at ambient conditions (p<0.05). The results therefore point to the dimensions of classrooms, ventilation and population of the classrooms as important factors in determining the bacterial air quality, and invariably affecting the health condition of students.
19
Shen, Weiran, Xuefeng Deng, Wei Zou, Fang Cheng, John F. Engelhardt, Ziying Yan, and Jianming Qiu. "Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1." Journal of Virology 89, no. 19 (July 29, 2015): 10097–109. http://dx.doi.org/10.1128/jvi.01374-15.
Full textAbstract:
ABSTRACTHuman bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1′ and D1′, in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1′ and smD1′ at the A1′ and D1′ splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1′ mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1′ mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI.IMPORTANCEHuman bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported.In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus.
20
Lackner, T., A. Müller, A. Pankraz, P. Becher, H. J. Thiel, A. E. Gorbalenya, and N. Tautz. "Temporal Modulation of an Autoprotease Is Crucial for Replication and Pathogenicity of an RNA Virus." Journal of Virology 78, no. 19 (October 1, 2004): 10765–75. http://dx.doi.org/10.1128/jvi.78.19.10765-10775.2004.
Full textAbstract:
ABSTRACT Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.
21
Kümmerer, Beate M., and Charles M. Rice. "Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles." Journal of Virology 76, no. 10 (May 15, 2002): 4773–84. http://dx.doi.org/10.1128/jvi.76.10.4773-4784.2002.
Full textAbstract:
ABSTRACT Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aα, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK↓T within NS2A. Exchange of serine for lysine at this site (QKT→QST) blocks the production of both NS2Aα and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aα. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aα was replaced with serine. In addition to true reversions, NS2Aα cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aα cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aα site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles.
22
Isken, Olaf, Minh Tu Pham, Hella Schwanke, Felicia Schlotthauer, Ralf Bartenschlager, and Norbert Tautz. "Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis." PLOS Pathogens 18, no. 10 (October 10, 2022): e1010895. http://dx.doi.org/10.1371/journal.ppat.1010895.
Full textAbstract:
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
23
Belyaev, Alexander S., Susan Chong, Alexander Novikov, Ana Kongpachith, Frank R. Masiarz, Moon Lim, and Jungsuh P. Kim. "Hepatitis G Virus Encodes Protease Activities Which Can Effect Processing of the Virus Putative Nonstructural Proteins." Journal of Virology 72, no. 1 (January 1, 1998): 868–72. http://dx.doi.org/10.1128/jvi.72.1.868-872.1998.
Full textAbstract:
ABSTRACT The genome of a recently identified virus, hepatitis G virus (HGV), shows considerable homology to hepatitis C virus (HCV). Two HGV proteases similar to nonstructural proteins NS2 and NS3 of HCV were identified, and their cleavage site specificity was investigated. Amino acids essential for the protease activities were determined by mutation analysis. NS4A of HGV was demonstrated to be a cofactor for NS3-mediated proteolysis, with a region critical for activity residing between Leu1561 and Ala1598.
24
Haeni, Linlin, and Beti Ernawati Dewi. "ANALISIS GENETIK GEN NONSTRUKTURAL 3 DENGUE VIRUS SEROTYPE 4 STRAIN INDONESIA." Medika Kartika Jurnal Kedokteran dan Kesehatan, Volume 3 No 1 (October 31, 2019): 13–24. http://dx.doi.org/10.35990/mk.v3n1.p13-24.
Full textAbstract:
Demam Berdarah Dengue (DBD) adalah penyakit yang disebabkan virus dengan vektor nyamuk yang paling cepat menyebar di dunia. Penyebab DBD adalah virus RNA famili flaviviridae yang disebut virus dengue (DENV). Genom DENV terdiri dari tiga protein struktural yaitu capsid (C), protein membran (prM), dan protein envelop (E) serta tujuh gen protein nonstuktural yaitu NS1, NS2a, NS2b, NS3, NS4a, NS4b, dan NS5. Protein NS3 mengandung epitop yang dapat dikenali oleh sistem imun humoral maupun selular oleh karena itu protein NS3 merupakan target potensial bagi pengembangan vaksin dengue. Penelitian ini diawali dengan sekuensing pada gen NS3 DENV-4 IDS 96/10. Dari hasil sekuensing dilakukan analisis filogenetik dan analisis epitop. Analisis filogenetik menunjukkan gen NS3 IDS 96 /10 berada dalam satu clade dengan strain yang diisolasi dari Cina (2010), Singapura (2010) dan Thailand (2000). Pada gen NS3 DENV-4 IDS 96/10 terdapat epitop yang dapat dikenali oleh sel limfosit T CD4+ yaitu epitop #3 pada posisi asam amino (213-227) , #9A(243-257), #4(251-265), #5(258-272), # 6(266-280), #7(273-287) yang mempunyai urutan asam amino sama antar strain yang dibandingkan. Pada posisi epitop #8(281-295) terdapat variasi urutan asam amino. Asam amino pada posisi 500-508 dikenali oleh sel limfosit T CD8+ mempunyai urutan yang sama antar strain yang dibandingkan, dan asam amino pada posisi 526-531yang dikenali oleh limfosit B mempunyai urutan asam amino yang sama antar strain yang dibandingkan. Pengenalan epitop-epitop tersebut oleh limfosit T dan limfosit B menjadi dasar pengembangan vaksin khususnya vaksin yang khusus untuk strain Indonesia
25
Zhang, Chen, Zhaohui Cai, Young-Chan Kim, Ranjith Kumar, Fenghua Yuan, Pei-Yong Shi, Cheng Kao, and Guangxiang Luo. "Stimulation of Hepatitis C Virus (HCV) Nonstructural Protein 3 (NS3) Helicase Activity by the NS3 Protease Domain and by HCV RNA-Dependent RNA Polymerase." Journal of Virology 79, no. 14 (July 2005): 8687–97. http://dx.doi.org/10.1128/jvi.79.14.8687-8697.2005.
Full textAbstract:
ABSTRACT Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.
26
Deng, Lin, Motoko Nagano-Fujii, Motofumi Tanaka, Yuki Nomura-Takigawa, Masanori Ikeda, Nobuyuki Kato, Kiyonao Sada, and Hak Hotta. "NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner." Journal of General Virology 87, no. 6 (June 1, 2006): 1703–13. http://dx.doi.org/10.1099/vir.0.81735-0.
Full textAbstract:
The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.
27
Valdés, Katia, Mayling Alvarez, Maritza Pupo, Susana Vázquez, Rayner Rodríguez, and María G. Guzmán. "Human Dengue Antibodies against Structural and Nonstructural Proteins." Clinical Diagnostic Laboratory Immunology 7, no. 5 (September 1, 2000): 856–57. http://dx.doi.org/10.1128/cdli.7.5.856-857.2000.
Full textAbstract:
ABSTRACT Antibodies against dengue virus type 2 and 4 proteins in acute-phase sera of 10 primary and 10 secondary dengue fever and dengue hemorrhagic fever patients were studied by Western blotting. In the first group the immune response was barely detectable, while in the second group more proteins were detected, with a very strong reaction. Anti-NS1 and -NS3 antibodies were detected mainly in secondary cases. Anti-E, -NS3, and -NS5 antibodies were detected in a high number of cases. The possibility of implementing early diagnostic assays for antigen detection is suggested.
28
Prikhod'ko, Grigori G., Elena A. Prikhod'ko, Alexander G. Pletnev, and Jeffrey I. Cohen. "Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis." Journal of Virology 76, no. 11 (June 1, 2002): 5701–10. http://dx.doi.org/10.1128/jvi.76.11.5701-5710.2002.
Full textAbstract:
ABSTRACT The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.
29
Raffeiner, B., F. Ometto, D. Astorri, C. Botsios, and G. Pulga. "AB1315-HPR HYPERBARIC OXYGEN THERAPY IN FIBROMYALGIA PATIENTS – DOUBLE-BLIND PROSPECTIVE CLINICAL TRIAL." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1947.2–1948. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5927.
Full textAbstract:
Background:Hyperbaric Oxygen Therapy (HOT) proved effective in improving of symptoms of patients affected by fibromyalgia syndrome (FMS) [1].Objectives:The objective of the present study was to evaluate the effectiveness of HOT compared to hyperbaric treatment with no oxygen therapy (PBO) in the symptoms and working ability in FMS.Methods:We conducted a prospective trial in employed patients with FMS, randomly assigned to HOT or PBO. Patients and evaluating clinicians were blinded to the treatment. HOT arm comprised 40 sessions, 5 days/week, 120 minutes, 100% oxygen at 2ATA; PBO comprised the same sessions without oxygen. Evaluations were at baseline, after 4 (T1) and 8 weeks (T2). Parameters considered were: socio-demographics, biochemistry, clinical evaluation and patient-reported outcomes (PROs). Baseline assessment included questions BELIEF (how much do you hope to improve with this treatment) and HOPE (how much do you expect to improve with this treatment), with VAS response. Spearman’s, Mann-Whitney’s, Kruskal-Wallis and Fisher’s Exact test were used.Results:12 patients were included and completed the study, 6 in each arm (Tab. 1). No significant difference was observed in clinical measures or PROs at T1 and T2 between HOT and PBO arms, except for Working Productivity and Activity Impairment Questionnaire (WPAI) (result III) (Tab. 2). In both arms, disease duration was associated with worse PROs (Widespread pain index r=0.59,p=0.037, Severity Score r=0.81,p=0.025); higher BMI with improvement in function at T2 (r=0.63,p=0.027); higher baseline scores in BELIEF with reduction symptoms number (r=-0.67, p=0.021), higher scores in HOPE with reduction in Health Assessment Questionnaire (r=-0.057, p=0.039)Table 1.Patients characteristicsAllHOTPBONumber1266Age*55,5 (44;59,75)55,5 (47,75;60)51 (41;58,75)Females**6 (100)6 (100)6 (100)Disease duration *10 (8,25;26,75)9,5 (7,5;20,75)15 (10;26,75)BMI*25,5 (22,25;31)25 (22,75;28)28,5 (23;31,75)Smoke**0,5 (0;1)0 (0;1)1 (0,25;1)HOPE score*0 (0;1)0,5 (0;1)0 (0;0)>=80**9 (64,3)5 (62,5)4 (66,7)BELIEF score*70 (60;80)75 (67,5;83,75)62,5 (60;68,75)>=70**8 (57,1)6 (75)2 (33,3)*median (IQR); **number (%)Table 2.Change from baseline in clinical measures and PROs at T2.Medians (IQR)P value≧20 Percentage amelioration No. (%)P valueAllHOTPBOAllHOTPBOShort Form-36 - Physical1 (-1;4)3 (-1;5,5)0,5 (-0,75;3,25)ns0 (0)0 (0)0 (0)nsSF-36 - Mental4 (-1;8)6 (2,5;9,5)1 (-1,75;5,25)ns0 (0)0 (0)0 (0)nsSeverity score - total-13 (-15;-2)-15 (-17;-13,5)-3 (-5,5;-0,5)ns2 (18,2)2 (40)0 (0)nsNumber symptoms-3 (-4;0)-4 (-4,5;-1,5)-2 (-3;-0,25)ns2 (18,2)2 (40)0 (0)nsSASP score-1 (-3;0)-2 (-3,5;-0,5)-1 (-1,75;-0,25)ns1 (9,1)0 (0)1 (16,7)nsWidespread pain index0 (-2;0)0 (-2;0)-0,5 (-1,75;0)ns2 (18,2)2 (40)0 (0)nsTender Points Count (0-18-2 (-3;-0,5)-2 (-3,5;-1)-1,25 (-2;-0,13)ns3 (27,3)2 (40)1 (16,7)NsHealth Assessment Questionnaire0 (0;0)0 (-0,5;0,5)0 (0;0)ns3 (27,3)1 (20)2 (33,3)nsFibromyalgia Impact Questionnaire10 (0;15)10 (7,5;15)2,5 (-3,75;12,5)ns1 (9,1)0 (0)1 (16,7)nsWPAI result-2-0,5 (-1,25;-0,13)-1,5 (-2;-0,5)-0,25 (-0,5;0,19)ns3 (21,4)0 (0)3 (37,5)nsWPAI result-30 (-2,38;0)-2,75 (-3,88;-2)0 (0;0)ns5 (35,7)0 (0)5 (62,5)P=0.008WPAI result-1-1 (-3,25;-0,38)-3,5 (-4,75;-1,5)-0,5 (-1;0,38)ns6 (42,9)2 (33,3)4 (50)nsConclusion:8-week HOT treatment does not substantially improve symptoms in FMS compared to PBO. All patients on hyperbaric treatment may experience amelioration of symptoms: other factors should be considered, including beliefs and expectations on the treatment.References:[1]DOI:10.1371/journal.pone.0127012Disclosure of Interests:None declared
30
Rahim, Md Niaz, Mohammed Selman, Patricia J. Sauder, Nicole E. Forbes, William Stecho, Wanhong Xu, Mark Lebar, Earl G. Brown, and Kevin M. Coombs. "Generation and characterization of a new panel of broadly reactive anti-NS1 mAbs for detection of influenza A virus." Journal of General Virology 94, no. 3 (March 1, 2013): 593–605. http://dx.doi.org/10.1099/vir.0.046649-0.
Full textAbstract:
Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for virus replication and may be a good target for IAV diagnosis. To generate broadly cross-reactive NS1-specific mAbs, mice were immunized with A/Hong Kong/1/1968 (H3N2) 6×His-tagged NS1 and hybridomas were screened with glutathione S-transferase-conjugated NS1 of A/Puerto Rico/8/1934 (H1N1). mAbs were isotyped and numerous IgG-type clones were characterized further. Most clones specifically recognized NS1 from various H1N1 and H3N2 IAV types by both immunoblot and immunofluorescence microscopy in mouse M1, canine Madin–Darby canine kidney and human A549 cells. mAb epitopes were mapped by overlapping peptides and selective reactivity to the newly described viral NS3 protein. These mAbs detected NS1 in both the cytoplasm and nucleus by immunostaining, and some detected NS1 as early as 5 h post-infection, suggesting their potential diagnostic use for tracking productive IAV replication and characterizing NS1 structure and function. It was also demonstrated that the newly identified NS3 protein is localized in the cytoplasm to high levels.
31
van Niekerk, M., V. van Staden, A. A. van Dijk, and H. Huismans. "Variation of African horsesickness virus nonstructural protein NS3 in southern Africa." Journal of General Virology 82, no. 1 (January 1, 2001): 149–58. http://dx.doi.org/10.1099/0022-1317-82-1-149.
Full textAbstract:
NS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36·3% across serotypes and 27·6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2·3% and 9·7%. NS3 of field isolates within a serotype differed up to 11·1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.
32
Qu, Lin, Laura K. McMullan, and Charles M. Rice. "Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity." Journal of Virology 75, no. 22 (November 15, 2001): 10651–62. http://dx.doi.org/10.1128/jvi.75.22.10651-10662.2001.
Full textAbstract:
ABSTRACT Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.
33
Cooper, Lyndsey, Sian C. Davies, Jonathan R. Dilworth, David L. Hughes, Marcin Konkol, Raymond L. Richards, J. Roger Sanders, and Piotr Sobota. "Iron complexes of the N(CH2CH2S)33(NS3) ligand with isocyanide co-ligands." Canadian Journal of Chemistry 79, no. 5-6 (May 1, 2001): 490–94. http://dx.doi.org/10.1139/v00-198.
Full textAbstract:
The complexes NEt4[Fe(NS3)(CNR)] (1), [Fe{Fe(NS3)(CNR)}2-S,S' ] (2), [Co{Fe(NS3)(CNMe)}2-S,S' ] (3), and [Ni{Fe(NS3)(CNMe)}2-S,S' ] (4) (R = Me, t-Bu, or C6H11(Cy)) have been prepared and the X-ray crystal structures of NEt4[Fe(NS3)(CNMe)] (1a) and [Fe{Fe(NS3)(CNCy)}2-S,S' ] (2c) have been determined. Mössbauer and IR spectroscopic data and room temperature magnetic moments for these complexes are reported.Key words: iron, cobalt, nickel, thiolate, isocyanide.
34
Ueno, Takamasa, Satoru Misawa, Yoichi Ohba, Mitsuhiro Matsumoto, Makiko Mizunuma, Nobuhiro Kasai, Kouhei Tsumoto, Izumi Kumagai, and Hideya Hayashi. "Isolation and Characterization of Monoclonal Antibodies That Inhibit Hepatitis C Virus NS3 Protease." Journal of Virology 74, no. 14 (July 15, 2000): 6300–6308. http://dx.doi.org/10.1128/jvi.74.14.6300-6308.2000.
Full textAbstract:
ABSTRACT A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (Ki = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependentcis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent transprocessing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.
35
Xu, Shan, Yali Ci, Leijie Wang, Yang Yang, Leiliang Zhang, Caimin Xu, Chengfeng Qin, and Lei Shi. "Zika virus NS3 is a canonical RNA helicase stimulated by NS5 RNA polymerase." Nucleic Acids Research 47, no. 16 (July 30, 2019): 8693–707. http://dx.doi.org/10.1093/nar/gkz650.
Full textAbstract:
Abstract Zika virus is a positive single-strand RNA virus whose replication involved RNA unwinding and synthesis. ZIKV NS3 contains a helicase domain, but its enzymatic activity is not fully characterized. Here, we established a dsRNA unwinding assay based on the FRET effect to study the helicase activity of ZIKV NS3, which provided kinetic information in real time. We found that ZIKV NS3 specifically unwound dsRNA/dsDNA with a 3′ overhang in the 3′ to 5′ direction. The RNA unwinding ability of NS3 significantly decreased when the duplex was longer than 18 base pairs. The helicase activity of NS3 depends on ATP hydrolysis and binding to RNA. Mutations in the ATP binding region or the RNA binding region of NS3 impair its helicase activity, thus blocking viral replication in the cell. Furthermore, we showed that ZIKV NS5 interacted with NS3 and stimulated its helicase activity. Disrupting NS3-NS5 interaction resulted in a defect in viral replication, revealing the tight coupling of RNA unwinding and synthesis. We suggest that NS3 helicase activity is stimulated by NS5; thus, viral replication can be carried out efficiently. Our work provides a molecular mechanism of ZIKV NS3 unwinding and novel insights into ZIKV replication.
36
Rho, Jaerang, Seeyoung Choi, Young Rim Seong, Joonho Choi, and Dong-Soo Im. "The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1." Journal of Virology 75, no. 17 (September 1, 2001): 8031–44. http://dx.doi.org/10.1128/jvi.75.17.8031-8044.2001.
Full textAbstract:
ABSTRACT The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg1493 in the QRR1488GRTGR1493G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg1488 is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.
37
Tijssen, P., Y. Li, M. El-Far, J. Szelei, M. Letarte, and Z. Zádori. "Organization and Expression Strategy of the Ambisense Genome of Densonucleosis Virus of Galleria mellonella." Journal of Virology 77, no. 19 (October 1, 2003): 10357–65. http://dx.doi.org/10.1128/jvi.77.19.10357-10365.2003.
Full textAbstract:
ABSTRACT The expression strategy of parvoviruses of the Densovirus genus has as yet not been reported. Clones were obtained from the densonucleosis virus of Galleria mellonella (GmDNV) that yielded infectious virus upon transfection into LD652 cells. Its genome was found to be the longest (6,039 nucleotides [nt]), with the largest inverted terminal repeats (ITRs) (550 nt) among all parvoviruses. The distal 136 nt could be folded into hairpins with flop or flip sequence orientations. In contrast to vertebrate parvoviruses, the gene cassettes for the nonstructural (NS) and structural (VP) proteins were found on the 5′ halves of the opposite strands. The transcripts for both cassettes started 23 nt downstream of the ITRs. The TATA boxes, as well as all upstream promoter elements, were localized in the ITRs and, therefore, identical for the NS and VP transcripts. These transcripts overlapped for 60 nt at the 3′ ends (antisense RNAs) at 50 m.u. The NS cassette consisted of three genes of which NS2 was contained completely within NS1 but from a different reading frame. Most of the NS transcripts were spliced to remove the upstream NS3, allowing leaky scanning translation of NS1 and NS2, similar to the genes of RNA-6 of influenza B virus. NS3 could be translated from the unspliced transcript. The VP transcript was not spliced and generated four VPs by a leaky scanning mechanism. The 5′-untranslated region of the VP transcript was only 5 nt long. Despite the transcription and translation strategies being radically different from those of vertebrate parvoviruses, the capsid was found to have phospholipase A2 activity, a feature thus far unique for parvoviruses.
38
Zhang, Zhu-Xu, Una Lazdina, Margaret Chen, Darrell L. Peterson, and Matti Sällberg. "Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein." Clinical Diagnostic Laboratory Immunology 7, no. 1 (January 1, 2000): 58–63. http://dx.doi.org/10.1128/cdli.7.1.58-63.2000.
Full textAbstract:
ABSTRACT We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.
39
Kundharapu, Satyamurthy, and Tirumala Kumar Chowdary. "Dengue Virus NS4b N-Terminus Disordered Region Interacts with NS3 Helicase C-Terminal Subdomain to Enhance Helicase Activity." Viruses 14, no. 8 (August 3, 2022): 1712. http://dx.doi.org/10.3390/v14081712.
Full textAbstract:
Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b–NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.
40
Gao, Xiaopan, Kaixiang Zhu, Justyna Aleksandra Wojdyla, Pu Chen, Bo Qin, Ziheng Li, Meitian Wang, and Sheng Cui. "Crystal structure of the NS3-like helicase from Alongshan virus." IUCrJ 7, no. 3 (April 10, 2020): 375–82. http://dx.doi.org/10.1107/s2052252520003632.
Full textAbstract:
Alongshan virus (ALSV) is an emerging human pathogen that was identified in China and rapidly spread to the European continent in 2019, raising concerns about public health. ALSV belongs to the distinct Jingmenvirus group within the Flaviviridae family with segmented RNA genomes. While segments 2 and 4 of the ALSV genome encode the VP1–VP3 proteins of unknown origin, segments 1 and 3 encode the NS2b–NS3 and NS5 proteins, which are related to Flavivirus nonstructural proteins, suggesting an evolutionary link between segmented and unsegmented viruses within the Flaviviridae family. Here, the enzymatic activity of the ALSV NS3-like helicase (NS3-Hel) was characterized and its crystal structure was determined to 2.9 Å resolution. ALSV NS3-Hel exhibits an ATPase activity that is comparable to those measured for Flavivirus NS3 helicases. The structure of ALSV NS3-Hel exhibits an overall fold similar to those of Flavivirus NS3 helicases. Despite the limited amino-acid sequence identity between ALSV NS3-Hel and Flavivirus NS3 helicases, structural features at the ATPase active site and the RNA-binding groove remain conserved in ALSV NS3-Hel. These findings provide a structural framework for drug design and suggest the possibility of developing a broad-spectrum antiviral drug against both Flavivirus and Jingmenvirus.
41
Gwack, Yousang, Hyouna Yoo, Inyoung Song, Joonho Choe, and Jang H. Han. "RNA-Stimulated ATPase and RNA Helicase Activities and RNA Binding Domain of Hepatitis G Virus Nonstructural Protein 3." Journal of Virology 73, no. 4 (April 1, 1999): 2909–15. http://dx.doi.org/10.1128/jvi.73.4.2909-2915.1999.
Full textAbstract:
ABSTRACT Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed inEscherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the otherFlaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3′-to-5′ direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5′ and 3′ overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.
42
Tseng, Alanna C., Vivek R. Nerurkar, Kabi R. Neupane, Helmut Kae, and Pakieli H. Kaufusi. "Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication." Viruses 13, no. 2 (January 31, 2021): 216. http://dx.doi.org/10.3390/v13020216.
Full textAbstract:
West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.
43
Kullappan, Malathi, Balakrishnan Anna Benedict, Anusha Rajajagadeesan, Padmasini Baskaran, Nanthini Devi Periadurai, Jenifer Mallavarpu Ambrose, Sri Harshini Gandhamaneni, et al. "Ellagic Acid as a Potential Inhibitor against the Nonstructural Protein NS3 Helicase of Zika Virus: A Molecular Modelling Study." BioMed Research International 2022 (August 21, 2022): 1–15. http://dx.doi.org/10.1155/2022/2044577.
Full textAbstract:
Zika virus is a member of the Flaviviridae family and genus Flavivirus, which has a phylogenetic relationship with spondweni virus. It spreads to humans through a mosquito bite. To identify potential inhibitors for the Zika virus with biosafety, we selected natural antiviral compounds isolated from plant sources and screened against NS3 helicase of the Zika virus. The enzymatic activity of the NS3 helicase is associated with the C-terminal region and is concerned with RNA synthesis and genome replication. It serves as a crucial target for the Zika virus. We carried out molecular docking for the target NS3 helicase against the selected 25 phytochemicals using AutoDock Vina software. Among the 25 plant compounds, we identified NS3 helicase-ellagic acid (-9.9 kcal/mol), NS3 helicase-hypericin (-9.8 kcal/mol), and NS3 helicase-pentagalloylglucose (-9.5 kcal/mol) as the best binding affinity compounds based on their binding energies. To understand the stability of these complexes, molecular dynamic simulations were executed and the trajectory analysis exposed that the NS3 helicase-ellagic acid complex possesses greater stability than the other two complexes such as NS3 helicase-hypericin and NS3 helicase-pentagalloylglucose. The ADMET property prediction of these compounds resulted in nontoxicity and noncarcinogenicity.
44
Chang, Yu-Shiu, Ching-Len Liao, Chang-Huei Tsao, Mei-Chieh Chen, Chiu-I. Liu, Li-Kuang Chen, and Yi-Ling Lin. "Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus." Journal of Virology 73, no. 8 (August 1, 1999): 6257–64. http://dx.doi.org/10.1128/jvi.73.8.6257-6264.1999.
Full textAbstract:
ABSTRACT Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or α-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducibleEscherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or β-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.
45
Shiryaev, Sergey A., Boris I. Ratnikov, Alexei V. Chekanov, Sergey Sikora, Dmitri V. Rozanov, Adam Godzik, Jun Wang, et al. "Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase." Biochemical Journal 393, no. 2 (December 23, 2005): 503–11. http://dx.doi.org/10.1042/bj20051374.
Full textAbstract:
Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9–12-mer peptides are potent inhibitors of WNV NS3 with Ki values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
46
Yu, Hong, Hui Huang, Jim Xiang, Lorne A. Babiuk, and Sylvia van Drunen Littel-van den Hurk. "Dendritic cells pulsed with hepatitis C virus NS3 protein induce immune responses and protection from infection with recombinant vaccinia virus expressing NS3." Journal of General Virology 87, no. 1 (January 1, 2006): 1–10. http://dx.doi.org/10.1099/vir.0.81423-0.
Full textAbstract:
Infections with Hepatitis C virus (HCV) pose a serious health problem worldwide. In this study, the hypothesis that adoptive transfer of dendritic cells (DCs) pulsed with HCV NS3 protein and matured with an oligodeoxynucleotide (ODN) containing CpG motifs (CpG) ex vivo would initiate potent HCV-specific protective immune responses in vivo was tested. NS3 protein was efficiently transduced into DCs and treatment of DCs with CpG ODN induced phenotypic maturation and specifically increased the expression of CD40. DCs matured with CpG ODN produced higher interleukin 12 levels and a stronger allogeneic T-cell response compared with untreated DCs. Notably, there were no differences between NS3-pulsed DCs and DCs pulsed with a control protein with respect to phenotype, cytokine production or mixed lymphocyte reaction, indicating that transduction with NS3 protein did not impair DC functions. Compared with the untreated NS3-pulsed DCs, the NS3-pulsed DCs matured with CpG ODN induced stronger cellular immune responses including enhanced cytotoxicity, higher interferon-γ production and stronger lymphocyte proliferation. Upon challenge with a recombinant vaccinia virus expressing NS3, all mice immunized with NS3-pulsed DCs showed a significant reduction in vaccinia virus titres when compared with mock-immunized mice. However, the NS3-pulsed DCs matured with CpG ODN induced higher levels of protection compared with the untreated NS3-pulsed DCs. These data are the first to show that NS3-pulsed DCs induce specific immune responses and provide protection from viral challenge, and also demonstrate that CpG ODNs, which have a proven safety profile, would be useful in the development of DC vaccines.
47
De Francesco, Raffaele, Antonello Pessi, and Christian Steinkühler. "The Hepatitis C Virus NS3 Proteinase: Structure and Function of a Zinc-Containing Serine Proteinase." Antiviral Therapy 3, no. 3_suppl (April 1998): 99–109. http://dx.doi.org/10.1177/135965359800303s01.
Full textAbstract:
The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral memebrane protein that acts as an activator of the IMS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A β-strand contributed by NS4A is clamped between two β-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4’), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottm by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P, position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the Km values of the respective substrates and follow the order NS4A<NS5A<NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.
48
Grassmann, Claus W., Olaf Isken, and Sven-Erik Behrens. "Assignment of the Multifunctional NS3 Protein of Bovine Viral Diarrhea Virus during RNA Replication: an In Vivo and In Vitro Study." Journal of Virology 73, no. 11 (November 1, 1999): 9196–205. http://dx.doi.org/10.1128/jvi.73.11.9196-9205.1999.
Full textAbstract:
ABSTRACT 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 intrans 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.
49
Clemente Dias, Renieidy Flávia. "Identificação de potenciais inibidores da enzima NS2B-NS3 do Zika Vírus." Revista Eletrônica Científica da UERGS 8, no. 3 (December 23, 2022): 258–66. http://dx.doi.org/10.21674/2448-0479.83.258-266.
Full textAbstract:
O vírus Zika (ZIKV) são flavivírus pertencentes à família flaviviridae, que são transmitidos pela picada do vetor infectado; nesse caso, mosquito do gênero Aedes aegypti. A replicação viral é função da proteína não estrutural NS3-pro que atua em associação com NS2B, aumentando a eficiência enzimática. Assim, esse domínio da protease N2B-NS3 apresenta um alvo atrativo para o planejamento de novos fármacos antivirais. Diante disto, este artigo buscou abordar a importância farmacológica dos heterocíclicos benzotiazóis e trouxe exemplos de derivados benzotiazóis como novos candidatos a fármacos capazes de inibirem a protease N2B-NS3 do ZIKV.
Palavras-chave: NS2B-NS3; ZIKV; Aedes aegypti.
Abstract
Identification of potential inhibitors of the Zika Virus NS2B-NS3 enzyme
The Zika virus (ZIKV) are flaviviruses belonging to the flaviviridae family, which are transmitted by the bite of an infected vector, in this case, a mosquito of the genus Aedes aegypti. Viral replication is a function of the non-structural protein NS3-pro that works in association with NS2B, increasing enzymatic efficiency. Thus, this domain of the N2B-NS3 protease presents an attractive target for the design of new antiviral drugs. Therefore, this article sought to address the pharmacological importance of heterocyclic benzothiazoles and brought examples of benzothiazole derivatives as new drug candidates capable of inhibiting the N2B-NS3 protease of ZIKV.
Keywords: NS2B-NS3; ZIKV; Aedes aegypti.
Resumen
Identificación de posibles inhibidores de la enzima NS2B-NS3 del virus del Zika
El virus Zika (ZIKV) son flavivirus pertenecientes a la familia flaviviridae, que se transmiten por la picadura de un vector infectado, en este caso, un mosquito del género Aedes aegypti. La replicación viral es una función de la proteína no estructural NS3-pro que trabaja en asociación con NS2B, ampliando la eficiencia enzimática. Por ello, este dominio de la proteasa N2B-NS3 presenta un objetivo atractivo para el diseño de nuevos fármacos antivirales. Frente a esto, el artículo buscó abordar la importancia farmacológica de los benzotiazoles heterocíclicos y trajo ejemplos de derivados de benzotiazol como nuevos candidatos a fármacos capaces de inhibir la proteasa N2B-NS3 del ZIKV.
Palavras clave: NS2B-NS3; ZIKV; Aedes aegypti.
50
Wahaab, Abdul, Ke Liu, Muddassar Hameed, Muhammad Naveed Anwar, Lei Kang, Chenxi Li, Xiaochun Ma, et al. "Identification of Cleavage Sites Proteolytically Processed by NS2B-NS3 Protease in Polyprotein of Japanese Encephalitis Virus." Pathogens 10, no. 2 (January 21, 2021): 102. http://dx.doi.org/10.3390/pathogens10020102.
Full textAbstract:
Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.