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1
Zou, Jing, Xuping Xie, Qing-Yin Wang, Hongping Dong, Michelle Yueqi Lee, Congbao Kang, Zhiming Yuan, and Pei-Yong Shi. "Characterization of Dengue Virus NS4A and NS4B Protein Interaction." Journal of Virology 89, no. 7 (January 7, 2015): 3455–70. http://dx.doi.org/10.1128/jvi.03453-14.
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ABSTRACTFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimatedKd(dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, orin vitrowith recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.
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Muñoz-Jordán, Jorge L., Maudry Laurent-Rolle, Joseph Ashour, Luis Martínez-Sobrido, Mundrigi Ashok, W. Ian Lipkin, and Adolfo García-Sastre. "Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses." Journal of Virology 79, no. 13 (July 1, 2005): 8004–13. http://dx.doi.org/10.1128/jvi.79.13.8004-8013.2005.
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ABSTRACT Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-α/β) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-α/β. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.
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Konan, Kouacou V., Thomas H. Giddings, Masanori Ikeda, Kui Li, Stanley M. Lemon, and Karla Kirkegaard. "Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus." Journal of Virology 77, no. 14 (July 15, 2003): 7843–55. http://dx.doi.org/10.1128/jvi.77.14.7843-7855.2003.
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ABSTRACT The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a “membranous web” (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.
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Roosendaal, Jojanneke, Edwin G. Westaway, Alexander Khromykh, and Jason M. Mackenzie. "Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein." Journal of Virology 80, no. 9 (May 1, 2006): 4623–32. http://dx.doi.org/10.1128/jvi.80.9.4623-4632.2006.
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ABSTRACT A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.
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Paredes, Anne M., and Keril J. Blight. "A Genetic Interaction between Hepatitis C Virus NS4B and NS3 Is Important for RNA Replication." Journal of Virology 82, no. 21 (August 20, 2008): 10671–83. http://dx.doi.org/10.1128/jvi.00875-08.
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ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.
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Koupriyanov, V. V., L. I. Nikolaeva, A. A. Zykova, P. I. Makhnovskiy, R. Y. Kotlyarov, A. V. Vasilyev, and N. V. Ravin. "IMMUNOGENIC PROPERTIES OF RECOMBINANT MOZAIC PROTEINS BASED ON ANTIGENS NS4A AND NS4B OF HEPATITIS C VIRUS." Problems of Virology, Russian journal 63, no. 3 (June 20, 2018): 138–43. http://dx.doi.org/10.18821/0507-4088-2018-63-3-138-143.
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The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.
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Stone, Michelle, Shuaizheng Jia, Won Do Heo, Tobias Meyer, and Kouacou V. Konan. "Participation of Rab5, an Early Endosome Protein, in Hepatitis C Virus RNA Replication Machinery." Journal of Virology 81, no. 9 (February 14, 2007): 4551–63. http://dx.doi.org/10.1128/jvi.01366-06.
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ABSTRACT Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce “membranous web” formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.
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Klaitong, Paeka, and Duncan R. Smith. "Roles of Non-Structural Protein 4A in Flavivirus Infection." Viruses 13, no. 10 (October 15, 2021): 2077. http://dx.doi.org/10.3390/v13102077.
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Infections with viruses in the genus Flavivirus are a worldwide public health problem. These enveloped, positive sense single stranded RNA viruses use a small complement of only 10 encoded proteins and the RNA genome itself to remodel host cells to achieve conditions favoring viral replication. A consequence of the limited viral armamentarium is that each protein exerts multiple cellular effects, in addition to any direct role in viral replication. The viruses encode four non-structural (NS) small transmembrane proteins (NS2A, NS2B, NS4A and NS4B) which collectively remain rather poorly characterized. NS4A is a 16kDa membrane associated protein and recent studies have shown that this protein plays multiple roles, including in membrane remodeling, antagonism of the host cell interferon response, and in the induction of autophagy, in addition to playing a role in viral replication. Perhaps most importantly, NS4A has been implicated as playing a critical role in fetal developmental defects seen as a consequence of Zika virus infection during pregnancy. This review provides a comprehensive overview of the multiple roles of this small but pivotal protein in mediating the pathobiology of flaviviral infections.
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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.
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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.
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Martin, Morgan M., Stephanie A. Condotta, Jeremy Fenn, Andrea D. Olmstead, and François Jean. "In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile." Biological Chemistry 392, no. 10 (October 1, 2011): 927–35. http://dx.doi.org/10.1515/bc.2011.076.
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AbstractThe need to identify anti-Flaviviridaeagents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.
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Selimović, Denis, and Mohamed Hassan. "Inhibition of Hepatitis C virus (HCV) Core protein- induced Cell Growth by Non-structural Protein 4A (NS4A) is Mediated by Mitochondrial Dysregulation." Bosnian Journal of Basic Medical Sciences 8, no. 1 (February 20, 2008): 4–11. http://dx.doi.org/10.17305/bjbms.2008.2988.
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Hepatitis C virus (HCV) is a significant health problem facing the world. More than 170 million people are infected with HCV worldwide. HCV encodes a large polyprotein precursor that is processed into at least 10 distinct products including structural (core, E1 and E2) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Besides its importance in virus replication, NS4A functions as a cofactor for NS3 and contributes to viral pathogenesis by influencing cellular functions. Here, we investigated the effect of NS4A protein on the growth rate induced by core protein in liver cells. Using our established tetracycline inducible system, we demonstrated the ability of NS4A protein to inhibit core protein-induced cell growth in Hepatoma cell line, HepG2. Induction of both core and NS4A proteins in HepG2- core/NS4A transfectants inhibited core-induced growth advantage in HepG2-core transfectants and blocked NS4A protein-induced cell growth inhibition in HepG2-NS4A transfectants. Using both immune fluorescence staining and Western blot analysis, we confirmed the localization of NS4A protein to the mitochondria in HepG2-NS4A transfectants expressing NS4A protein. Data obtained from flow cytometry analysis, using JC-1 demonstrated the loss of mitochondrial membrane potential (ΔΨ^ by the expression of NS4A protein in HepG2-NS4A transfectants, but not by the expression of core protein in HepG2-core transfectants. Whereas, the induction of the expression of both core and NS4A proteins in HepG2-core/NS4A transfectants blocked NS4A-induced loss of ΔΨm in HepG2 cells. Taken together, our data suggest an important role for mitochondria in the modulation HCV NS4A-induced inhibition of HCV core-mediated cell growth.
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Lundin, Marika, Magnus Monné, Anders Widell, Gunnar von Heijne, and Mats A. A. Persson. "Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B." Journal of Virology 77, no. 9 (May 1, 2003): 5428–38. http://dx.doi.org/10.1128/jvi.77.9.5428-5438.2003.
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ABSTRACT Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.
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Kaufusi, Pakieli H., Alanna C. Tseng, James F. Kelley, and Vivek R. Nerurkar. "Selective Reactivity of Anti-Japanese Encephalitis Virus NS4B Antibody Towards Different Flaviviruses." Viruses 12, no. 2 (February 14, 2020): 212. http://dx.doi.org/10.3390/v12020212.
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Studies investigating West Nile virus (WNV) NS4B protein function are hindered by the lack of an antibody recognizing WNV NS4B protein. Few laboratories have produced WNV NS4B antibodies, and none have been shown to work consistently. In this report, we describe a NS4B antibody against Japanese encephalitis virus (JEV) NS4B protein that cross-reacts with the NS4B protein of WNV but not of dengue virus (DENV). This JEV NS4B antibody not only recognizes WNV NS4B in infected cells, but also recognizes the NS4B protein expressed using transfection. It is evident from this data that the JEV NS4B antibody is specific to NS4B of WNV but not to NS4B of the four DENV serotypes. The specificity of this antibody may be due to the notable differences that exist between the amino acid sequence identity and antigenic relationships within the NS4B protein of the WNV, DENV, and JEV.
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Yu, Guann-Yi, Ki-Jeong Lee, Lu Gao, and Michael M. C. Lai. "Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein." Journal of Virology 80, no. 12 (June 15, 2006): 6013–23. http://dx.doi.org/10.1128/jvi.00053-06.
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ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.
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Chagnon, Fanny, Alain Lamarre, Claude Lachance, Michelle Krakowski, Trevor Owens, Jean-François Laliberté, and Pierre J. Talbot. "Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E." Canadian Journal of Microbiology 44, no. 10 (October 1, 1998): 1012–17. http://dx.doi.org/10.1139/w98-089.
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Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice to evaluate whether myelin-viral protein cross-reactive antibody responses could be generated. Each immunogen induced specific but not cross-reactive antibodies. We conclude that ns4b is expressed in infected cells and is immunogenic, although this does not involve amino acids shared with a self protein, at least in the experimental conditions used.Key words: human coronavirus 229E, nonstructural protein, ns4b protein, expression, immunogenicity.
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Gummow, Jason, Yanrui Li, Wenbo Yu, Tamsin Garrod, Danushka Wijesundara, Amelia J. Brennan, Ranajoy Mullick, Ilia Voskoboinik, Branka Grubor-Bauk, and Eric J. Gowans. "A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice." Journal of Virology 89, no. 15 (May 27, 2015): 7991–8002. http://dx.doi.org/10.1128/jvi.00803-15.
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ABSTRACTThere are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising “multiantigen” vaccine that elicits robust CMI.IMPORTANCESince their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.
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Vajdy, Michael, Mark Selby, Angelica Medina-Selby, Doris Coit, John Hall, Laura Tandeske, David Chien, et al. "Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses." Journal of General Virology 87, no. 8 (August 1, 2006): 2253–62. http://dx.doi.org/10.1099/vir.0.81849-0.
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Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-γ responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-γ responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.
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Lundin, Marika, Hannah Lindström, Caroline Grönwall, and Mats A. A. Persson. "Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein." Journal of General Virology 87, no. 11 (November 1, 2006): 3263–72. http://dx.doi.org/10.1099/vir.0.82211-0.
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Among the least-known hepatitis C virus proteins is the non-structural protein 4B (NS4B). It localizes to the endoplasmic reticulum (ER) membrane and induces membrane changes, resulting in a membranous web that is reported to be the locale for virus replication. A model was presented previously for the topology of recombinant HCV NS4B of the 1a genotype based on in vitro data. In this model, the N-terminal tail of a considerable fraction of the NS4B molecules was translocated into the ER lumen via a post-translational process, giving the protein a dual transmembrane topology. It is now reported that translocation of the N terminus also occurs for processed NS4B expressed in cells in the context of the polyprotein. In the presence of NS5A, however, a lower degree of translocation was observed, which may indicate that NS5A influences the topology of NS4B. In vitro expression studies of NS4B from all major genotypes demonstrated that translocation of the N terminus to the ER lumen is conserved across genotypes. This clearly suggests an important function for this feature. Furthermore, when disrupting a previously reported amphipathic helix (AH) in the N terminus of NS4B, translocation was inhibited. As a disrupted AH also abolished the ability of NS4B to rearrange membranes, these data indicate for the first time an association between translocation of the N terminus and membrane rearrangement. Finally, the present experiments also confirm the predicted location of the first luminal loop to be around aa 112.
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Kong, Lingbao, Akira Fujimoto, Mariko Nakamura, Haruyo Aoyagi, Mami Matsuda, Koichi Watashi, Ryosuke Suzuki, et al. "Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B." Journal of Virology 90, no. 6 (January 6, 2016): 3093–111. http://dx.doi.org/10.1128/jvi.01540-15.
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ABSTRACTIt has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infectionin vitroandin vivo. Our findings provide new insights into HCV host cofactors.IMPORTANCEThe hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors.
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Gao, Lu, Hideki Aizaki, Jian-Wen He, and Michael M. C. Lai. "Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft." Journal of Virology 78, no. 7 (April 1, 2004): 3480–88. http://dx.doi.org/10.1128/jvi.78.7.3480-3488.2004.
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ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.
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Bashir, Shahbaz, Andrey Kossarev, Violeta Cascon Martin, and Jan Paeshuyse. "Deciphering the Role of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Viral Pathogenesis." Veterinary Sciences 7, no. 4 (October 31, 2020): 169. http://dx.doi.org/10.3390/vetsci7040169.
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Bovine viral diarrhea virus (BVDV) is a (+) ssRNA virus that belongs to the family Flaviviridae. BVDV is a significant animal pathogen causing substantial economic losses to the cattle industry worldwide through respiratory and gastrointestinal infections and abortion or birth of persistently infected calves. While the immunogenic profile of some of the BVDV proteins (i.e., Erns, E2 and NS3) is well established during viral pathogenesis, very little information is available about most of BVDV’s non-structural proteins in this regard. In recent times, the NS4B protein has emerged as an interesting target of diagnostic, vaccination and therapeutic value in viral infections of other members of the family Flaviviridae due to its key scaffold-like contribution in the viral replication complex. Although, BVDV-NS4B has a membrane topology alongside its role in induction of autophagosomes in vitro. However, information on its immunogenicity during BVDV pathogenesis and vaccination is scarce. To characterize the immunogenic profile of the NS4B, five cows were vaccinated with the live attenuated BVDV vaccine Bovela® and blood samples were taken pre- and post-immunization for serum isolation. Virus neutralization assay (VNA) confirmed the presence of anti-BVDV antibodies in the sera of vaccinated cows. VNA also revealed pre-existing antibodies against BVDV in the pre-immunization sera of two cows. To identify BVDV-NS4B specific antibodies, the NS4B protein was expressed in mammalian cells by using the pCI-neo vector system. The sera from BVDV vaccinated cows were evaluated for the presence of BVDV-NS4B specific antibodies through western blot and indirect ELISA. Interestingly, t sera from cows with pre-existing immunity against BVDV were able to detect NS4B in western blot and ELISA, suggesting the presence of NS4B-specific antibodies. The obtained results provide the first indication of the immunogenic nature of BVDV-NS4B protein in sero-converted animals. These findings are consistent with the observation made for NS4B in other Flaviviridae members and confirm this protein as an interesting target with diagnostic, vaccination and therapeutic value.
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Zou, Jing, Le Tian Lee, Qing Yin Wang, Xuping Xie, Siyan Lu, Yin Hoe Yau, Zhiming Yuan, et al. "Mapping the Interactions between the NS4B and NS3 Proteins of Dengue Virus." Journal of Virology 89, no. 7 (January 14, 2015): 3471–83. http://dx.doi.org/10.1128/jvi.03454-14.
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ABSTRACTFlavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and anin situproximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn α-helix and two short β-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy.IMPORTANCENS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details of how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3-NS4B interaction for antiviral drug discovery.
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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.
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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.
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Condotta, Stephanie A., Morgan M. Martin, Martine Boutin, and François Jean. "Detection and in-cell selectivity profiling of the full-length West Nile virus NS2B/NS3 serine protease using membrane-anchored fluorescent substrates." Biological Chemistry 391, no. 5 (May 1, 2010): 549–59. http://dx.doi.org/10.1515/bc.2010.051.
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AbstractFlaviviral NS2B/NS3 heterocomplex serine proteases are a primary target for anti-flavivirus drug discovery. To gain insights into the enzymatic properties and molecular determinants of flaviviral NS2B/NS3 protease substrate specificity in host cells, we developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 endoproteolytic activity in human cells. The substrate consists of a fluorescent reporter group (DsRed) tethered to the endoplasmic reticulum membrane by a membrane-anchoring domain. Between the two domains is a specific peptide linker that corresponds to the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions within the WNV polyprotein precursor. When the protease cleaves the peptide linker, the DsRed reporter group is released, changing its localization in the cell from membrane-bound punctate perinuclear to diffuse cytoplasmic. This change in protein location can be monitored by fluorescent microscopy, and cleavage products can be quantified by Western blotting. Our data demonstrate the robustness of ourtrans-cleavage fluorescence assay to capture single-cell imaging of membrane-associated WNV NS2B/NS3 endoproteolytic activity and to perform in-cell selectivity profiling of the NS2B/NS3 protease. Our study is the first to provide cellular insights into the biological and enzymatic properties of a prime target for inhibitors of WNV replication.
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Naik, Nenavath Gopal, and Huey-Nan Wu. "Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication." Journal of Virology 89, no. 13 (April 15, 2015): 6746–60. http://dx.doi.org/10.1128/jvi.00423-15.
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ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.
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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.
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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.
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Blight, Keril J. "Allelic Variation in the Hepatitis C Virus NS4B Protein Dramatically Influences RNA Replication." Journal of Virology 81, no. 11 (March 14, 2007): 5724–36. http://dx.doi.org/10.1128/jvi.02481-06.
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ABSTRACT In the Huh-7.5 hepatoma cell line, replication of the genotype 1a H77 strain of hepatitis C virus (HCV) is attenuated compared to that of the genotype 1b Con1 strain. This study identifies the poorly characterized integral membrane protein, NS4B, as a major determinant for this replication difference. Chimeric H77 subgenomic replicons containing the entire NS4B gene from Con1 in place of the H77 NS4B sequence replicated approximately 10-fold better than the H77 parent and to levels similar to that of the adapted Con1 replicon. An intermediate level of replication enhancement was conferred by H77 chimeras containing the poorly conserved N-terminal 47 residues or the remaining less-divergent C terminus of Con1 NS4B. The replication-enhancing activity within the N terminus of NS4B was further mapped to two Con1-specific amino acids. Experiments to elucidate the mechanism of enhanced H77 replication revealed that Con1 NS4B primarily increased H77 RNA synthesis on a per cell basis, as indicated by the similar capacities of chimeric and parental replicons to establish replication in Huh-7.5 cells and the higher levels of both positive- and negative-strand RNAs for the chimeras than for the H77 parent. Additionally, enhanced H77 replication was not the result of Con1 NS4B-mediated effects on HCV translation efficiency or alterations in polyprotein processing. Expression of Con1 NS4B in trans did not improve the replication of the H77 parental replicon, suggesting a cis-dominant role for NS4B in HCV replication. These results provide the first evidence that allelic variation in the NS4B sequence between closely related isolates significantly impacts HCV replication in cell culture.
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Jones, Daniel M., Arvind H. Patel, Paul Targett-Adams, and John McLauchlan. "The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus." Journal of Virology 83, no. 5 (December 10, 2008): 2163–77. http://dx.doi.org/10.1128/jvi.01885-08.
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ABSTRACT Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.
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Chatel-Chaix, Laurent, Wolfgang Fischl, Pietro Scaturro, Mirko Cortese, Stephanie Kallis, Marie Bartenschlager, Bernd Fischer, and Ralf Bartenschlager. "A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication." Journal of Virology 89, no. 14 (April 29, 2015): 7170–86. http://dx.doi.org/10.1128/jvi.00867-15.
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ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.
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Van Slyke, Greta A., Yongqing Jia, Melissa C. Whiteman, Jason A. Wicker, Alan D. T. Barrett, and Laura D. Kramer. "Vertebrate attenuated West Nile virus mutants have differing effects on vector competence in Culex tarsalis mosquitoes." Journal of General Virology 94, no. 5 (May 1, 2013): 1069–72. http://dx.doi.org/10.1099/vir.0.049833-0.
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Previous mutational analyses of naturally occurring West Nile virus (WNV) strains and engineered mutant WNV strains have identified locations in the viral genome that can have profound phenotypic effect on viral infectivity, temperature sensitivity and neuroinvasiveness. We chose six mutant WNV strains to evaluate for vector competence in the natural WNV vector Culex tarsalis, two of which contain multiple ablations of glycosylation sites in the envelope and NS1 proteins; three of which contain mutations in the NS4B protein and an attenuated natural bird isolate (Bird 1153) harbouring an NS4B mutation. Despite vertebrate attenuation, all NS4B mutant viruses displayed enhanced vector competence by Cx. tarsalis. Non-glycosylated mutant viruses displayed decreased vector competence in Cx. tarsalis mosquitoes, particularly when all three NS1 glycosylation sites were abolished. These results indicate the importance of both the NS4B protein and NS1 glycosylation in the transmission of WNV by a significant mosquito vector.
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Fanunza, Elisa, Nicole Grandi, Marina Quartu, Fabrizio Carletti, Laura Ermellino, Jessica Milia, Angela Corona, Maria Rosaria Capobianchi, Giuseppe Ippolito, and Enzo Tramontano. "INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation." Viruses 13, no. 12 (December 6, 2021): 2448. http://dx.doi.org/10.3390/v13122448.
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The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.
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Lin, C., J. W. Wu, K. Hsiao, and M. S. Su. "The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins." Journal of virology 71, no. 9 (1997): 6465–71. http://dx.doi.org/10.1128/jvi.71.9.6465-6471.1997.
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Zou, J., X. Xie, L. T. Lee, R. Chandrasekaran, A. Reynaud, L. Yap, Q. Y. Wang, et al. "Dimerization of Flavivirus NS4B Protein." Journal of Virology 88, no. 6 (January 3, 2014): 3379–91. http://dx.doi.org/10.1128/jvi.02782-13.
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Wölk, Benno, Domenico Sansonno, Hans-Georg Kräusslich, Franco Dammacco, Charles M. Rice, Hubert E. Blum, and Darius Moradpour. "Subcellular Localization, Stability, andtrans-Cleavage Competence of the Hepatitis C Virus NS3-NS4A Complex Expressed in Tetracycline-Regulated Cell Lines." Journal of Virology 74, no. 5 (March 1, 2000): 2293–304. http://dx.doi.org/10.1128/jvi.74.5.2293-2304.2000.
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ABSTRACT A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3′-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.
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Suda, Yuto, Daisuke Yamane, Muhammad Atif Zahoor, Yassir Mahgoub Mohamed, Shin Murakami, Kentaro Kato, Taisuke Horimoto, and Hiroomi Akashi. "Unique Localization of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Infected Cells." JOURNAL OF ADVANCES IN AGRICULTURE 6, no. 1 (July 20, 2016): 914–21. http://dx.doi.org/10.24297/jaa.v6i1.5396.
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Bovine viral diarrhea virus (BVDV), an important pathogen infecting ruminants, has 2 biotypes: cytopathic (cp) and noncytopathic (ncp), which are related to the onset of disease. The viral replication complex is composed of viral non-structural (NS) proteins, raising the possibility that NS protein(s) play a role in viral biotypes. To gain insight into this possible role, we analysed the intracellular localization of each NS protein in both cp and ncp virus-infected cells, and found that NS4B protein, a possible anchor protein of the viral replication complex, showed a unique dotted localization pattern that markedly merged with an endoplasmic reticulum marker, unlike other NS proteins, although there was no difference in the localization of NS4B protein between the 2 biotypes.Â
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Pascut, Devis, Minh Hoang, Nhu N. Q. Nguyen, Muhammad Yogi Pratama, and Claudio Tiribelli. "HCV Proteins Modulate the Host Cell miRNA Expression Contributing to Hepatitis C Pathogenesis and Hepatocellular Carcinoma Development." Cancers 13, no. 10 (May 19, 2021): 2485. http://dx.doi.org/10.3390/cancers13102485.
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Hepatitis C virus (HCV) genome encodes for one long polyprotein that is processed by cellular and viral proteases to generate 10 polypeptides. The viral structural proteins include the core protein, and the envelope glycoproteins E1 and E2, present at the surface of HCV particles. Non-structural (NS) proteins consist of NS1, NS2, NS3, NS4A, NS4B, NS5a, and NS5b and have a variable function in HCV RNA replication and particle assembly. Recent findings evidenced the capacity of HCV virus to modulate host cell factors to create a favorable environment for replication. Indeed, increasing evidence has indicated that the presence of HCV is significantly associated with aberrant miRNA expression in host cells, and HCV structural and non-structural proteins may be responsible for these alterations. In this review, we summarize the recent findings on the role of HCV structural and non-structural proteins in the modulation of host cell miRNAs, with a focus on the molecular mechanisms responsible for the cell re-programming involved in viral replication, immune system escape, as well as the oncogenic process. In this regard, structural and non-structural proteins have been shown to modulate the expression of several onco-miRNAs or tumor suppressor miRNAs.
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Pouliot, Jeffrey J., Michael Thomson, Mi Xie, Joseph Horton, John Johnson, David Krull, Amanda Mathis, et al. "Preclinical Characterization andIn VivoEfficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein." Antimicrobial Agents and Chemotherapy 59, no. 10 (August 10, 2015): 6539–50. http://dx.doi.org/10.1128/aac.00813-15.
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ABSTRACTThe hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibitin vivoefficacy. We describe here thein vitroandin vivoantiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developedin vitroresistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-dayin vitrotreatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates.In vivoefficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end ofin vivotreatment revealed resistance mutations encoding amino acid changes that had not been identified byin vitrostudies, including NS4B N56I and N99H. Our findings provide anin vivoproof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.
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Gouttenoire, Jérôme, Philippe Roingeard, François Penin, and Darius Moradpour. "Amphipathic α-Helix AH2 Is a Major Determinant for the Oligomerization of Hepatitis C Virus Nonstructural Protein 4B." Journal of Virology 84, no. 24 (October 6, 2010): 12529–37. http://dx.doi.org/10.1128/jvi.01798-10.
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ABSTRACT Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic α-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.
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Gretton, Sarah N., Annette I. Taylor, and John McLauchlan. "Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci." Journal of General Virology 86, no. 5 (May 1, 2005): 1415–21. http://dx.doi.org/10.1099/vir.0.80768-0.
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The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP–NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.
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Gu, Zhengxian, Jason D. Graci, Frederick C. Lahser, Jamie J. Breslin, Stephen P. Jung, James H. Crona, Patricia McMonagle, et al. "Identification of PTC725, an Orally Bioavailable Small Molecule That Selectively Targets the Hepatitis C Virus NS4B Protein." Antimicrobial Agents and Chemotherapy 57, no. 7 (April 29, 2013): 3250–61. http://dx.doi.org/10.1128/aac.00527-13.
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ABSTRACTWhile new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.
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Luo, Huanle, Guorui Xie, Michael Diamond, Michael Gale, Alan Barrett, and Tian Wang. "A West Nile virus NS4B P38G mutant induces MAVS-dependent type 1 interferon and the host anti-viral responses (INC1P.353)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 54.10. http://dx.doi.org/10.4049/jimmunol.194.supp.54.10.
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Abstract The nonstructural (NS) proteins of West Nile virus (WNV) are important determinants of viral pathogenesis. WNV NS4B-P38G mutant, which has a P38G substitution in the NS4B protein is highly attenuated in mice, but induces stronger type 1 interferon (IFN) and T cell responses than the wild-type WNV. Here, we investigated mice lacking the RIG-I-like receptor adaptor gene Mavs, and found increased viremia and lethality following WNV NS4B-P38G infection. MAVS-/- mice had a reduced IFNβ production and impaired primary T cell response after infection. WNV NS4B-P38G also induced lower levels of type 1 IFNs and cell surface expression of MHC class II and co-stimulatory molecules on MAVS-/- dendritic cell (DC)s. IRF-3, -5, and -7 are the key transcription factors responsible for mediating the type I IFN and ISG response in mDCs during wild-type WNV infection. PCR array analysis showed a significantly decreased expression of IFNβ and most IFN stimulated gene (ISG)s, except IRF3 and IRF5 in WNV NS4B-P38G-infected MAVS-/- DCs compared to the wild-type group. Moreover, DCs of IRF3-/- x IRF7-/- double knockout or Irf3-/-×Irf5-/- ×Irf7-/- triple knockout mice had either unaffected or a higher IFNβ expression after WNV NS4B-P38G infection. Collectively, our results suggest that MAVS signaling is essential for the induction of type 1 IFN and T cell responses upon WNV NS4B-P38G mutant infection and this could occur through an IRF-3 and IRF-5-independent pathway.
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Svitkin, Yuri V., Arnim Pause, Marcelo Lopez-Lastra, Sandra Perreault, and Nahum Sonenberg. "Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3." Journal of Virology 79, no. 11 (June 1, 2005): 6868–81. http://dx.doi.org/10.1128/jvi.79.11.6868-6881.2005.
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ABSTRACT We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNAMet was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.
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Fernandez-Sainz, I., D. P. Gladue, L. G. Holinka, V. O'Donnell, I. Gudmundsdottir, M. V. Prarat, J. R. Patch, et al. "Mutations in Classical Swine Fever Virus NS4B Affect Virulence in Swine." Journal of Virology 84, no. 3 (November 18, 2009): 1536–49. http://dx.doi.org/10.1128/jvi.02050-09.
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ABSTRACT NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.
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Florese, Ruth H., Motoko Nagano-Fujii, Yasuhiro Iwanaga, Rachmat Hidajat, and Hak Hotta. "Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus." Virus Research 90, no. 1-2 (December 2002): 119–31. http://dx.doi.org/10.1016/s0168-1702(02)00146-6.
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Casseb, Samir Mansour Moraes, Karla Fabiane Lopes de Melo, Carlos Alberto Marques de Carvalho, Carolina Ramos dos Santos, Edna Cristina Santos Franco, and Pedro Fernando da Costa Vasconcelos. "Experimental Dengue Virus Type 4 Infection Increases the Expression of MicroRNAs-15/16, Triggering a Caspase-Induced Apoptosis Pathway." Current Issues in Molecular Biology 45, no. 6 (May 26, 2023): 4589–99. http://dx.doi.org/10.3390/cimb45060291.
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The World Health Organization has estimated the annual occurrence of approximately 392 million dengue virus (DENV) infections in more than 100 countries where the virus is endemic, which represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how the NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different durations of infection. This study demonstrated that miRNAs-15/16 were overexpressed during the infection of HepG2 and Huh 7.5 cells with DENV-4 and had a relationship with NS1 protein expression, viral load, and the activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.
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Gallinari, Paola, Debra Brennan, Chiara Nardi, Mirko Brunetti, Licia Tomei, Christian Steinkühler, and Raffaele De Francesco. "Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus." Journal of Virology 72, no. 8 (August 1, 1998): 6758–69. http://dx.doi.org/10.1128/jvi.72.8.6758-6769.1998.
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ABSTRACT The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.
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Fernandez, Stefan, Emily D. Cisney, Alexander P. Tikhonov, Barry Schweitzer, Robert J. Putnak, Monika Simmons, and Robert G. Ulrich. "Antibody Recognition of the Dengue Virus Proteome and Implications for Development of Vaccines." Clinical and Vaccine Immunology 18, no. 4 (January 26, 2011): 523–32. http://dx.doi.org/10.1128/cvi.00016-11.
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ABSTRACTDengue is a mosquito-borne infection caused by four distinct serotypes of dengue virus, each appearing cyclically in the tropics and subtropics along the equator. Although vaccines are currently under development, none are available to the general population. One of the main impediments to the successful advancement of these vaccines is the lack of well-defined immune correlates of protection. Here, we describe a protein microarray approach for measuring antibody responses to the complete viral proteome comprised of the structural (capsid, membrane, and envelope) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) components of all four dengue virus serotypes (1 to 4). We examined rhesus macaques vaccinated with tetravalent vaccines consisting of live-attenuated virus (LAV) or purified inactivated virus (PIV), followed by boosting with LAV and challenging with wild-type dengue virus. We detected temporal increases in antibodies against envelope proteins in response to either vaccine, while only the PIV/LAV vaccination strategy resulted in anticapsid antibodies. In contrast to results from vaccination, naïve macaques challenged with wild-type viruses of each serotype demonstrated a balanced response to nonstructural and structural components, including responses against the membrane protein. Our results demonstrate discriminating details concerning the nature of antibody responses to dengue virus at the proteomic level and suggest the usefulness of this information for vaccine development.
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Oliver Koch, Jan, and Ralf Bartenschlager. "Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B." Journal of Virology 73, no. 9 (September 1, 1999): 7138–46. http://dx.doi.org/10.1128/jvi.73.9.7138-7146.1999.
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ABSTRACT NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320–326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities.
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Egger, Denise, Benno Wölk, Rainer Gosert, Leonardo Bianchi, Hubert E. Blum, Darius Moradpour, and Kurt Bienz. "Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex." Journal of Virology 76, no. 12 (June 15, 2002): 5974–84. http://dx.doi.org/10.1128/jvi.76.12.5974-5984.2002.
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ABSTRACT Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.
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To, Janet, and Jaume Torres. "Trimerization of the N-Terminal Tail of Zika Virus NS4A Protein: A Potential In Vitro Antiviral Screening Assay." Membranes 11, no. 5 (April 30, 2021): 335. http://dx.doi.org/10.3390/membranes11050335.
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The nonstructural (NS) protein NS4A in flaviviruses is a membrane protein that is critical for virulence, and, among other roles, it participates in membrane morphogenesis. In dengue virus (DENV), the NS4A hydrophilic N–terminal tail, together with the first transmembrane domain, is involved in both homo-oligomerization and hetero–oligomerization with NS4B. In both DENV and Zika virus (ZIKV), this N-terminal tail (residues 1–48) forms a random coil in solution but becomes mostly α-helical upon interaction with detergents or lipid membranes. Herein, we show that a peptide from ZIKV NS4A that spans residues 4–58, which includes most of the N–terminal tail and a third of its first transmembrane domain, forms homotrimers in the absence of detergents or liposomes. After interaction with the latter, α–helical content increases, consistent with binding. The oligomeric size of NS4A is not known, as it has only been reported in SDS gels. Therefore, we propose that full-length NS4A forms homotrimers mediated by this region, and that disruption of the oligomerization of peptide ZIKV NS4A 4–58 in solution can potentially constitute the basis for an in vitro assay to discover antivirals.