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Journal articles on the topic "Nonstructural Protein 4"
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Hagemeijer, M. C., M. Ulasli, A. M. Vonk, F. Reggiori, P. J. M. Rottier, and C. A. M. de Haan. "Mobility and Interactions of Coronavirus Nonstructural Protein 4." Journal of Virology 85, no. 9 (February 23, 2011): 4572–77. http://dx.doi.org/10.1128/jvi.00042-11.
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Guix, Susana, Santiago Caballero, Albert Bosch, and Rosa M. Pintó. "C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA." Journal of Virology 78, no. 24 (December 15, 2004): 13627–36. http://dx.doi.org/10.1128/jvi.78.24.13627-13636.2004.
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ABSTRACT Computational and biological approaches were undertaken to characterize the role of the human astrovirus nonstructural protein nsP1a/4, located at the C-terminal fragment of nsP1a. Computer analysis reveals sequence similarities to other nonstructural viral proteins involved in RNA replication and/or transcription and allows the identification of a glutamine- and proline-rich region, the prediction of many phosphorylation and O-glycosylation sites, and the occurrence of a KKXX-like endoplasmic reticulum retention signal. Immunoprecipitation analysis with an antibody against a synthetic peptide of the nsP1a/4 sequence detected polyprotein precursors of 160, 75, and 38 to 40 kDa as well as five smaller proteins in the range of 21 to 27 kDa. Immunofluorescence labeling showed that the nsP1a/4 protein is accumulated at the perinuclear region, in association with the endoplasmic reticulum and the viral RNA. These results suggest the involvement of nsP1a/4 protein in the RNA replication process in endoplasmic reticulum-derived intracellular membranes.
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Doyle, Nicole, Benjamin Neuman, Jennifer Simpson, Philippa Hawes, Judith Mantell, Paul Verkade, Hasan Alrashedi, and Helena Maier. "Infectious Bronchitis Virus Nonstructural Protein 4 Alone Induces Membrane Pairing." Viruses 10, no. 9 (September 6, 2018): 477. http://dx.doi.org/10.3390/v10090477.
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Positive-strand RNA viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral RNA synthesis. Infectious bronchitis virus (IBV), a pathogenic avian Gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (DMVs), zippered endoplasmic reticulum (zER) and tethered vesicles, known as spherules. These membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. Three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. This study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family.
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Zou, Wei, Fang Cheng, Weiran Shen, John F. Engelhardt, Ziying Yan, and Jianming Qiu. "Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins." Journal of Virology 90, no. 9 (February 24, 2016): 4658–69. http://dx.doi.org/10.1128/jvi.02964-15.
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ABSTRACTA novel chimeric parvoviral vector, rAAV2/HBoV1, in which the recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged by the human bocavirus 1 (HBoV1) capsid, has been shown to be highly efficient in gene delivery to human airway epithelia (Z. Yan et al., Mol Ther 21:2181–2194, 2013,http://dx.doi.org/10.1038/mt.2013.92). In this vector production system, we used an HBoV1 packaging plasmid, pHBoV1NSCap, that harbors HBoV1 nonstructural protein (NS) and capsid protein (Cap) genes. In order to simplify this packaging plasmid, we investigated the involvement of the HBoV1 NS proteins in capsid protein expression. We found that NP1, a small NS protein encoded by the middle open reading frame, is required for the expression of the viral capsid proteins (VP1, VP2, and VP3). We also found that the other NS proteins (NS1, NS2, NS3, and NS4) are not required for the expression of VP proteins. We performed systematic analyses of the HBoV1 mRNAs transcribed from the pHBoV1NSCap packaging plasmid and its derivatives in HEK 293 cells. Mechanistically, we found that NP1 is required for both the splicing and the read-through of the proximal polyadenylation site of the HBoV1 precursor mRNA, essential functions for the maturation of capsid protein-encoding mRNA. Thus, our study provides a unique example of how a small viral nonstructural protein facilitates the multifaceted regulation of capsid gene expression.IMPORTANCEA novel chimeric parvoviral vector, rAAV2/HBoV1, expressing a full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene, is capable of correcting CFTR-dependent chloride transport in cystic fibrosis human airway epithelium. Previously, an HBoV1 nonstructural and capsid protein-expressing plasmid, pHBoV1NSCap, was used to package the rAAV2/HBoV1 vector, but yields remained low. In this study, we demonstrated that the nonstructural protein NP1 is required for the expression of capsid proteins. However, we found that the other four nonstructural proteins (NS1 to -4) are not required for expression of capsid proteins. By mutating theciselements that function as internal polyadenylation signals in the capsid protein-expressing mRNA, we constructed a simple HBoV1 capsid protein-expressing gene that expresses capsid proteins as efficiently as pHBoV1NSCap does, and at similar ratios, but independently of NP1. Our study provides a foundation to develop a better packaging system for rAAV2/HBoV1 vector production.
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Khamrin, Pattara, Shoko Okitsu, Hiroshi Ushijima, and Niwat Maneekarn. "Novel Nonstructural Protein 4 Genetic Group in Rotavirus of Porcine Origin." Emerging Infectious Diseases 14, no. 4 (April 2008): 686–88. http://dx.doi.org/10.3201/eid1404.07111.
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Khamrin, Pattara, Shoko Okitsu, Hiroshi Ushijima, and Niwat Maneekarn. "Novel Nonstructural Protein 4 Genetic Group in Rotavirus of Porcine Origin." Emerging Infectious Diseases 14, no. 4 (April 2008): 686–88. http://dx.doi.org/10.3201/eid1404.071111.
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Ding, Chuantian, Masashi Urabe, Max Bergoin, and Robert M. Kotin. "Biochemical Characterization of Junonia coenia Densovirus Nonstructural Protein NS-1." Journal of Virology 76, no. 1 (January 1, 2002): 338–45. http://dx.doi.org/10.1128/jvi.76.1.338-345.2002.
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ABSTRACT Junonia coenia densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly, Junonia coenia. Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF). The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3. These proteins are likely to be involved in viral DNA replication, among other functions. In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been characterized. Here, we describe biochemical properties of the NS-1 protein of JcDNV. The NS-1 ORF was cloned in frame with the Escherichia coli malE gene, which encodes the bacterial maltose binding protein (MBP). Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein. The site consists of (GAC)4 and is located on the A-A′ region of the terminal palindrome. In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5′-G*TAT*TG-3′. One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number A12984 . Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis. Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs. These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity. These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents.
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Mukherjee, Arpita, Upayan Patra, Rahul Bhowmick, and Mamta Chawla-Sarkar. "Rotaviral nonstructural protein 4 triggers dynamin-related protein 1-dependent mitochondrial fragmentation during infection." Cellular Microbiology 20, no. 6 (February 28, 2018): e12831. http://dx.doi.org/10.1111/cmi.12831.
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Dahal, Bibha, Caitlin W. Lehman, Ivan Akhrymuk, Nicole R. Bracci, Lauren Panny, Michael D. Barrera, Nishank Bhalla, Jonathan L. Jacobs, Jonathan D. Dinman, and Kylene Kehn-Hall. "PERK Is Critical for Alphavirus Nonstructural Protein Translation." Viruses 13, no. 5 (May 12, 2021): 892. http://dx.doi.org/10.3390/v13050892.
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Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4–5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.
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Bhowmick, Rahul, Umesh Chandra Halder, Shiladitya Chattopadhyay, Shampa Chanda, Satabdi Nandi, Parikshit Bagchi, Mukti Kant Nayak, Oishee Chakrabarti, Nobumichi Kobayashi, and Mamta Chawla-Sarkar. "Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection." Journal of Biological Chemistry 287, no. 42 (August 10, 2012): 35004–20. http://dx.doi.org/10.1074/jbc.m112.369595.
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Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca2+ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.
Dissertations / Theses on the topic "Nonstructural Protein 4"
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Lindström, Hannah Kim. "Molecular studies of the hepatitis C virus : the role of IRES activity for therapy response, and the impact of the non-structural protein NS4B on the viral proliferation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-875-4/.
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Mir, Kiran D. "The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1." Texas A&M University, 2003. http://hdl.handle.net/1969.1/3976.
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Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants
worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first
described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular
calcium through multiple mechanisms that promote abnormal ion transport and
subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially
interact with model membranes mimicking caveolae in lipid composition and radius of
curvature. Our laboratory has recently reported the colocalization and
coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae.
Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic
peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one
terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed
in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least
one terminus were capable of binding the NSP4 bait. An in vitro binding assay
confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1
residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates
NSP4 signaling and/or intracellular trafficking.
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Palla, Narayan Sastri. "Studies On The Structural And Biological Properties Of Rotavirus Enterotoxigenic Non-structural Protein 4 (NSP4)." Thesis, 2011. http://etd.iisc.ernet.in/handle/2005/1958.
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Rotavirus is the major cause of infantile gastroenteritis. Each year more than 600,000 young children are estimated to die in developing countries throughout the world. Rotavirus infection can be either symptomatic or asymptomatic. But the genetic or molecular basis for rotavirus virulence is not yet clearly understood. NSP4, encoded by genome segment 10, is a multifunctional protein. It is identified as the first viral enterotoxin and is essential for virus morphogenesis and pathogenesis. Analysis of NSP4 from more than 175 strains failed to reveal any sequence motif or amino acid that segregated with the virulence phenotype of the virus. Further, a few studies indicated a lack of consistent correlation between virus virulence and diarrhea inducing ability of the cognate NSP4.
To understand the basis for the inconsistency in the enterotoxigenic activity of a few NSP4s reported in a limited number of studies, comparative analysis of the biophysical, biochemical, and biological properties of NSP4ΔN72, which from SA11 and Hg18 was earlier shown to be highly diarrheagenic, from 17 different symptomatic and asymptomatic strains was carried out. To study structure-function relationship we used Thioflavin T fluorescence assay, gel filtration, CD spectroscopy, trypsin susceptibility and enterotoxin assay in newborn mice for all the proteins. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thoflavin T binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. Since earlier studies showed that a secreted peptide (ΔN112) of SA11-NSP4 also induced diarrhea in newborn mice pups, we have generated NSP4ΔN112 deletions from six different strains and tested for their diarrhea inducing ability. The patterns of DD50 values of the ΔN112 peptides was similar to that for ΔN72 peptides, but were 1000-1200-fold less efficient than that of SA11ΔN72.
NSP4 exists in multiple forms in the infected cells- as oligomers, higher molecular weight complexes and ER- and cytoplasmic membrane anchored forms. Previous studies suggest that the N-terminal boundary of the oligomerization domain could lie downstream to residue 94 from the N-terminus. A peptide from residue 112-175, secreted from rotavirus infected cells, was reported to induce dose-dependent diarrhea in suckling mice, suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, the precise N-terminal boundaries in NSP4 for oligomerization and diarrhea induction have not been identified. To address this question, a large number of deletion mutants C-terminal to residue 94 were generated and tested for their ability to induce diarrhea in newborn mouse pups. Our data suggest that while the deletions ∆N121 to ∆N131 failed to induce diarrhea, ΔN118 was diarrheagenic suggesting that the N-terminal boundary of the minimal diarrhea inducing domain lies between aa 118 and 121. Size exclusion chromatography revealed that residues 95 to 98 are critical and sufficient for oligomerization. Studies on oligomerization further revealed that NSP4ΔN94 exists in pentamers, tetramers and dimers, while deletion mutants C-terminal to aa 94 exist only as dimers. Our studies demonstrate for the first time that not only tetramers but pentamers as well as dimers possess enterotoxigenic properties.
Most human rotavirus infections are caused by group A rotaviruses. Within this group, rotaviruses are further classified into subgroups based on the antigenic specificity associated with the protein product of the sixth gene, VP6. Previous studies have mapped SG I specificity to aa position 305 and the region between 296 and 299, and SG II specificity to residue 315 on VP6. However, the subgroup specific determinants on NSP4 have not been identified till date. In this study, we generated several amino acid substitution mutants in the SG I-specific SA11 NSP4∆N72 protein as in previous studies ∆N72 was found to efficiently bind DLPs. Using an enzyme linked immunosorbent assay method, the effect of the mutations in the C-terminal and N-terminal regions in ∆N72 on their binding ability to SG I and SG II DLPs was assayed. Residues at positions 85, 169, 174 and 175 and in the ISVD appear to collectively determine the specificity of binding to DLPs. While the conserved proline and glycines at positions 165, 168 and 162, respectively, are important for maintaining the required conformation for general recognition of DLP. The present study provides important insights towards understanding the determinants in NSP4 for SG-specific DLP interaction.
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Jagannath, M. R. "Characterization Of A Bovine Rotavirus From Humans And Studies On The Structural And Biological Properties Of Rotaviral Enterotoxigenic Nonstructural Protein 4." Thesis, 2005. http://etd.iisc.ernet.in/handle/2005/2202.
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Kumar, Sushant. "Structural Studies on DNA Damage Inducible Protein 1 (Ddi1) of Leishmania and the Rotavirus Nonstructural Protein NSP4." Thesis, 2016. http://hdl.handle.net/2005/3018.
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Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66.
Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2.
Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds.
Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.
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Deepa, R. "Diversity In Indian Equine Rotaviruses And Structure And Function Of Rotavirus Non Structural Protein 4 (NSP4)." Thesis, 2006. http://hdl.handle.net/2005/422.
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Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering
economic burden worldwide. Most of these deaths occur in the developing countries
and India is estimated to account for about a quarter of these deaths. Extensive
molecular epidemiology studies carried out by our laboratory have revealed many
interesting aspects about rotavirus diversity in this country.
Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two
organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16.
Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are
dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids
(aa) from the C-terminus is predicted to be unstructured and highly susceptible to
trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18
NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion
and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas,
proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to
DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4.
Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were
unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa
95-135 has been reported. Our recent studies indicate that the interspecies variable
regions from aa 135-141 as well as the extreme C-terminus are critical determinants
of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the
stretch of 9 residues from the C-terminus could not be seen suggesting its highly
flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization
under the experimental conditions. The present structure revealed significant
differences from that of the synthetic peptide in the conformation of amino acids at
the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational
differences in this critical region effected by the presence or absence of proline or
glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from
different strains in newborn mouse pups. Although symptomatic and asymptomatic
strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity.
In conclusion, we demonstrate that the predicted unstructured C-terminal region is
indeed highly flexible and is an important determinant of biological functions of the
NSP4, mutations in which probably correlates with the virulence properties of the virus.
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Williams, Cecelia V. "Mapping of the rotavirus nonstructural protein-4-caveolin-1 binding site to three hydrophobic residues within the extended, c-terminal amphipathic alpha helix." 2008. http://hdl.handle.net/1969.1/ETD-TAMU-3258.
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Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma
membrane of infected cells, possibly through interaction with caveolin-1. A direct
interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown
by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4
binding domain to caveolin-1, mutants were prepared by altering either the charged or
hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for
binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala)
disrupted the charged face, while the hydrophobic face was disrupted by replacing
selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast
two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity,
whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal
truncated clone (NSP446-175), which removed the hydrophobic domains and
aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and
NSP446-175 is sufficient for binding.
Seven additional mutants were prepared from NSP4HydroMut in which individually
charged residues were reverted to the original hydrophobic aa or were replaced with
alanine. Analyses of the interaction of these revertants with caveolin-1 localized the
NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional
aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound
caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to
a centrally located peptide. These data suggest conformational and hydrophobic
constraints play a role in the NSP4-caveolin-1 association.
The mutant NSP4 molecules also were evaluated for transport to the plasma
membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and
NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western
blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were
exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut
remained intracellular. Further, NSP4-transfected cells displayed an intracellular
association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data
indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.
Book chapters on the topic "Nonstructural Protein 4"
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Kato, Naoya, Keng-Hsin Lan, Suzane Kioko Ono-Nita, Hideo Yoshida, Yasushi Shiratori, and Masao Omata. "Hepatitis C Virus Nonstructural Region 5A Protein: A Potent Transcriptional Activator." In HCV and Related Liver Diseases, 45–58. Tokyo: Springer Japan, 1999. http://dx.doi.org/10.1007/978-4-431-68488-6_4.
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Suzuki, Tetsuro, and Ryosuke Suzuki. "Role of Nonstructural Proteins in HCV Replication." In Hepatitis C Virus I, 129–48. Tokyo: Springer Japan, 2016. http://dx.doi.org/10.1007/978-4-431-56098-2_7.
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Hijikata, Makoto, Hiroto Mizushima, Yasunori Tanji, Yasumasa Komoda, Yuji Hirowatari, Tsuyoshi Akagi, Nobuyuki Kato, Torahiko Tanaka, Koichi Kimura, and Kunitada Shimotohno. "Processing Mechanisms of Nonstructural Proteins of Hepatitis C Virus." In Viral Hepatitis and Liver Disease, 140–43. Tokyo: Springer Japan, 1994. http://dx.doi.org/10.1007/978-4-431-68255-4_37.
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Lai, C. J., M. Pethel, L. R. Jan, H. Kawano, A. Cahour, and B. Falgout. "Processing of dengue type 4 and other flavivirus nonstructural proteins." In Positive-Strand RNA Viruses, 359–68. Vienna: Springer Vienna, 1994. http://dx.doi.org/10.1007/978-3-7091-9326-6_36.
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Hong, Zhi, Eric B. Ferrari, Angela Skelton, Jacquelyn Wright-Minogue, Weidong Zhong, and Charles A. Lesburg. "Effects of genotypic variations on hepatitis C virus nonstructural protein 5B structure and activity." In Frontiers in Viral Hepatitis, 109–21. Elsevier, 2003. http://dx.doi.org/10.1016/b978-044450986-4/50061-8.
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Sastri, N. P., S. E. Crawford, and M. K. Estes. "Pleiotropic Properties of Rotavirus Nonstructural Protein 4 (NSP4) and Their Effects on Viral Replication and Pathogenesis." In Viral Gastroenteritis, 145–74. Elsevier, 2016. http://dx.doi.org/10.1016/b978-0-12-802241-2.00008-0.
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Puerta-Guardo, Henry, Scott B. Biering, Eva Harris, Norma Pavia-Ruz, Gonzalo Vázquez-Prokopec, Guadalupe Ayora-Talavera, and Pablo Manrique-Saide. "Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease: Part I - Dengue Virus Tropism, Host Innate Immune Responses, and Subversion of Antiviral Responses." In Dengue Fever in a One Health Perspective. IntechOpen, 2020. http://dx.doi.org/10.5772/intechopen.93140.
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Dengue is the most prevalent emerging mosquito-borne viral disease, affecting more than 40% of the human population worldwide. Many symptomatic dengue virus (DENV) infections result in a relatively benign disease course known as dengue fever (DF). However, a small proportion of patients develop severe clinical manifestations, englobed in two main categories known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Secondary infection with any of the four dengue virus serotypes (DENV1, -2, -3, and -4) is a risk factor to develop severe forms of dengue disease. DSS is primarily characterized by sudden and abrupt endothelial dysfunction, resulting in vascular leak and organ impairment, which may progress to hypovolemic shock and death. Severe DENV disease (DHF/DSS) is thought to follow a complex relationship between distinct immunopathogenic processes involving host and viral factors, such as the serotype cross-reactive antibody-dependent enhancement (ADE), the activation of T cells and complement pathways, the phenomenon of the cytokine storm, and the newly described viral toxin activity of the nonstructural protein 1 (NS1), which together play critical roles in inducing vascular leak and virus pathogenesis. In this chapter that is divided in two parts, we will outline the recent advances in our understanding of DENV pathogenesis, highlighting key viral-host interactions and discussing how these interactions may contribute to DENV immunopathology and the development of vascular leak, a hallmark of severe dengue. Part I will address the general features of the DENV complex, including the virus structure and genome, epidemiology, and clinical outcomes, followed by an updated review of the literature describing the host innate immune strategies as well as the viral mechanisms acting against and in favor of the DENV replication cycle and infection.
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Shahid, Imran, and Qaiser Jabeen. "HCV-Host Interactions: Interplay Part 2: Host Related Determinants and Intracellular Signaling." In Hepatitis C Virus-Host Interactions and Therapeutics: Current Insights and Future Perspectives, 26–53. BENTHAM SCIENCE PUBLISHERS, 2023. http://dx.doi.org/10.2174/9789815123432123010005.
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The progression of acute HCV infection to chronic disease and subsequent extrahepatic comorbidities involve both viruses and host cellular proteins interactions as well as insurrection or subjection of cell signaling and metabolic pathways in infected cells. This interaction between host-specific factors and the hepatitis C genome also weakens or impairs other physiological or metabolic regulatory roles of the hepatocytes. Several host cell proteins promote hepatitis C infection through binding to HCV nonstructural proteins (e.g., PPP2R5D). Some studies also found cytokine (e.g., IL-10, IL-6, TNF-α, and TGF-β1) gene polymorphisms to be highly associated with chronic hepatitis C (CHC) infection progression, whereas, polymorphism in some host genes (e.g., PNPLA3, ADAR-1, and IFIH1) are found to be actively involved in the induction of advanced liver fibrosis in patients co-infected with HIV-1/HCV. Host lipid metabolism reprogramming through host lipid regulators (e.g., ANGPTL-3 and 4) is also considered essential for CHC progression to severe liver disease (e.g., cirrhosis and HCC). Several microRNAs (e.g., miR-122, miR135a) are supposed to be key mediators of HCV infection progression and development of HCC in infected individuals and associated hepatic comorbidities. In chapter 1, we have illustrated the potential roles of virus-specific proteins in HCV molecular pathogenesis. Herein, we will elucidate the host-specific culprits that subvert, impede or disrupt host cells' communications, cell signaling, and metabolic pathways to propagate HCV infection. We will also elaborate that how the subversion of infected host-cell signaling and metabolic pathways disrupt cellular networks to evolve advanced fibrosis and hepatocarcinogenesis in HCV-infected individuals.
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Tijjani, Habibu, Ahmed Olatunde, Adegbenro Peter Adegunloye, and Ahmed Adebayo Ishola. "In silico insight into the interaction of 4-aminoquinolines with selected SARS-CoV-2 structural and nonstructural proteins." In Coronavirus Drug Discovery, 313–33. Elsevier, 2022. http://dx.doi.org/10.1016/b978-0-323-95578-2.00001-7.
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