Journal articles: 'Nonstructural Protein 4'

1

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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7

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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9

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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10

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.

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11

Cao, Shengliang, Xinna Cai, Min Tan, Fachao Sun, Yuchao Wang, Yingli Shang, Sidang Liu, and Yihong Xiao. "Serodiagnosis, targeting nonstructural protein 4, of porcine reproductive and respiratory syndrome virus." Archives of Virology 163, no. 2 (November 2, 2017): 411–18. http://dx.doi.org/10.1007/s00705-017-3625-5.

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12

Attallah, Abdelfattah M., Mohamed El-Far, Mohamed A. Abdelrazek, Mohamed M. Omran, Ashraf Z. Mahmoud, Hager S. Khalifa, Mohamed M. Ahmed, and Ibrahim El-Dosoky. "HCV nonstructural protein 4 is associated with aggressiveness features of breast cancer." Breast Cancer 25, no. 3 (December 28, 2017): 297–302. http://dx.doi.org/10.1007/s12282-017-0829-1.

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13

Enouf, Vincent, Philippe Langella, Jacqueline Commissaire, Jean Cohen, and Gérard Corthier. "Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic." Applied and Environmental Microbiology 67, no. 4 (April 1, 2001): 1423–28. http://dx.doi.org/10.1128/aem.67.4.1423-1428.2001.

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ABSTRACT Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but thensp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactisis able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.

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14

Zhang, Minze, Zhenpeng Cao, Jiexiong Xie, Wanjun Zhu, Pei Zhou, Honglang Gu, Long Sun, Shuo Su, and Guihong Zhang. "Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7." Virus Genes 47, no. 3 (July 28, 2013): 467–77. http://dx.doi.org/10.1007/s11262-013-0957-4.

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15

Bhowmick, Rahul, Umesh Chandra Halder, Shiladitya Chattopadhyay, Shampa Chanda, Satabdi Nandi, Parikshit Bagchi, Mukti Kant Nayak, Oishee Chakrabarti, Nobumichi Kobayashi, and Mamta Chawla-Sarkar. "Withdrawal: Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection." Journal of Biological Chemistry 295, no. 40 (October 2, 2020): 13956. http://dx.doi.org/10.1074/jbc.w120.015901.

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16

HaiXiu, Wang, Sun EnCheng, Xu QingYuan, Yang Tao, Zhang Qin, Feng YuFei, Li JunPing, et al. "Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1." Applied Microbiology and Biotechnology 99, no. 3 (October 26, 2014): 1309–21. http://dx.doi.org/10.1007/s00253-014-6150-4.

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17

Oostra, M., E. G. te Lintelo, M. Deijs, M. H. Verheije, P. J. M. Rottier, and C. A. M. de Haan. "Localization and Membrane Topology of Coronavirus Nonstructural Protein 4: Involvement of the Early Secretory Pathway in Replication." Journal of Virology 81, no. 22 (September 12, 2007): 12323–36. http://dx.doi.org/10.1128/jvi.01506-07.

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ABSTRACT The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].

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18

Yang, Chee-Hing, Che-Fang Hsu, Xiang-Qing Lai, Yu-Ru Chan, Hui-Chun Li, and Shih-Yen Lo. "Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein." Viruses 14, no. 10 (October 17, 2022): 2277. http://dx.doi.org/10.3390/v14102277.

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The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.

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Wang, Chunfu, Lingling Jia, Haichang Huang, Dike Qiu, Lourdes Valera, Xin Huang, Jin-Hua Sun, et al. "In VitroActivity of BMS-790052 on Hepatitis C Virus Genotype 4 NS5A." Antimicrobial Agents and Chemotherapy 56, no. 3 (December 27, 2011): 1588–90. http://dx.doi.org/10.1128/aac.06169-11.

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ABSTRACTThe antiviral profile of BMS-790052, a potent hepatitis C virus (HCV) replication complex inhibitor targeting nonstructural protein NS5A, is well characterized for HCV genotype-1. Here, we report that BMS-790052 inhibits hybrid replicons containing HCV genotype-4 NS5A genes with 50% effective concentrations (EC50s) ranging from 7 to 13 pM. NS5A residue 30 was an important site for BMS-790052-selected resistance in the hybrid replicons. Our results support the potential of BMS-790052 as a valuable component of combination therapy for HCV genotype-4 chronic infection.

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20

Gadlage, Mark J., Jennifer S. Sparks, Dia C. Beachboard, Reagan G. Cox, Joshua D. Doyle, Christopher C. Stobart, and Mark R. Denison. "Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function." Journal of Virology 84, no. 1 (October 21, 2009): 280–90. http://dx.doi.org/10.1128/jvi.01772-09.

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ABSTRACT Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

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Borgan, Mohammed Ali, Yoshio Mori, Naoto Ito, Makoto Sugiyama, and Nobuyuki Minamoto. "Antigenic Analysis of Nonstructural Protein (NSP) 4 of Group A Avian Rotavirus Strain PO-13." Microbiology and Immunology 47, no. 9 (September 2003): 661–68. http://dx.doi.org/10.1111/j.1348-0421.2003.tb03429.x.

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22

Wada, Yuji, Yasuko Orba, Michihito Sasaki, Shintaro Kobayashi, Michael J. Carr, Haruaki Nobori, Akihiko Sato, William W. Hall, and Hirofumi Sawa. "Discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsP4) of chikungunya virus." Virology 505 (May 2017): 102–12. http://dx.doi.org/10.1016/j.virol.2017.02.014.

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23

Quadt, R., H. J. M. Verbeek, and E. M. J. Jaspars. "Involvement of a nonstructural protein in the RNA synthesis of brome mosaic virus." Virology 165, no. 1 (July 1988): 256–61. http://dx.doi.org/10.1016/0042-6822(88)90679-4.

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Go, Yun Young, Eric J. Snijder, Peter J. Timoney, and Udeni B. R. Balasuriya. "Characterization of Equine Humoral Antibody Response to the Nonstructural Proteins of Equine Arteritis Virus." Clinical and Vaccine Immunology 18, no. 2 (December 8, 2010): 268–79. http://dx.doi.org/10.1128/cvi.00444-10.

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ABSTRACTEquine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n= 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n= 4) that were persistently infected with EAV, and from horses (n= 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.

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Winkler, Gunther, Valerie B. Randolph, Graham R. Cleaves, Terence E. Ryan, and Victor Stollar. "Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer." Virology 162, no. 1 (January 1988): 187–96. http://dx.doi.org/10.1016/0042-6822(88)90408-4.

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Miknis, Zachary J., Eric F. Donaldson, Timothy C. Umland, Ryan A. Rimmer, Ralph S. Baric, and L. Wayne Schultz. "Severe Acute Respiratory Syndrome Coronavirus nsp9 Dimerization Is Essential for Efficient Viral Growth." Journal of Virology 83, no. 7 (January 19, 2009): 3007–18. http://dx.doi.org/10.1128/jvi.01505-08.

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ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel α-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-Å resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication.

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Gao, Xiujie, Yingfen Wen, Chunlin Li, Jian Wang, Fuchun Zhang, and Lei Yu. "High specificity of human antibody response to nonstructural protein NS1 elicited by Zika virus infection." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 125.29. http://dx.doi.org/10.4049/jimmunol.198.supp.125.29.

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Abstract The newly emerged Zika virus (ZIKV) belonging to the flavivirus family, may appear in the area dengue virus (DENV) infection occurred, raising the issue of diagnosis. The secreted NS1 protein from infected cell is not only an important diagnostic marker but also highly immunogenic. Here we investigated the cross-reaction of NS1 Ab responses between the two close related viruses. First we studied the binding of sera to the recombinant full length NS1 from three ZIKV infected patients (Samples collected at day 4, 7, 15, 106, 188 and 321 for patient 1; day 6, 12, and 66 for patient 2; day 2 for patient 3) by capture ELISA. ZIKV NS1 binding IgG markedly increased in convalescent sera from patient 1 and patient 2 with less cross-reaction to DENV 1–4 NS1. The serum from patient 3 did not bind to ZIKV NS1 and that may be explained by the early time point of serum collected on day 2 from the symptom onset. Specificity of ZIKV NS1 Ab response was evidenced by lack of reactivity to DENV 1–4 NS1proteins of memory B cell derived mAbs (4/5) isolated from the two ZIKV-infected. Only one mAb (1/5) cross-reacted to DENV 2 and DENV 4 NS1. Strikingly NS1 Ab responses of DENV1 infected (six samples) which serotype frequently circulates in our area, Guangdong, China with outbreak in 2014 showed strong cross-reactivity between DENV serotypes with mild or strong reaction to ZIKV NS1. Our data indicated that NS1 Ab response elicited by Zika virus infection was ZIKV-specific and the ZIKV specific mAb can be used for diagnostic tool.

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Brown, Corrie C., Richard F. Meyer, and Marvin J. Grubman. "Identification of African Horse Sickness Virus in Cell Culture using a Digoxigenin-Labeled RNA Probe." Journal of Veterinary Diagnostic Investigation 6, no. 2 (April 1994): 153–55. http://dx.doi.org/10.1177/104063879400600204.

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A digoxigenin-labeled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1. In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV. There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus.

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Miyoshi, A., I. Poquet, V. Azevedo, J. Commissaire, L. Bermudez-Humaran, E. Domakova, Y. Le Loir, S. C. Oliveira, A. Gruss, and P. Langella. "Controlled Production of Stable Heterologous Proteins in Lactococcus lactis." Applied and Environmental Microbiology 68, no. 6 (June 2002): 3141–46. http://dx.doi.org/10.1128/aem.68.6.3141-3146.2002.

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ABSTRACT The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

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Lulla, Aleksei, Valeria Lulla, Kairit Tints, Tero Ahola, and Andres Merits. "Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease." Journal of Virology 80, no. 11 (June 1, 2006): 5413–22. http://dx.doi.org/10.1128/jvi.00229-06.

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ABSTRACT The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.

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Montgomery, Stephanie A., and Robert E. Johnston. "Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2." Journal of Virology 81, no. 19 (July 25, 2007): 10268–79. http://dx.doi.org/10.1128/jvi.00371-07.

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ABSTRACT Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-α1 but not with karyopherin-α2, -3, or -4, suggesting that karyopherin-α1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.

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Carvalho, Denise Maciel, Fernanda Gonçalves Garcia, Ana Paula Sarreta Terra, Ana Cristina Lopes Tosta, Luciana de Almeida Silva, Lúcio Roberto Castellano, and David Nascimento Silva Teixeira. "Elevated Dengue Virus Nonstructural Protein 1 Serum Levels and Altered Toll-Like Receptor 4 Expression, Nitric Oxide, and Tumor Necrosis Factor Alpha Production in Dengue Hemorrhagic Fever Patients." Journal of Tropical Medicine 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/901276.

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Background. During dengue virus (DV) infection, monocytes produce tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) which might be critical to immunopathogenesis. Since intensity of DV replication may determine clinical outcomes, it is important to know the effects of viral nonstructural protein 1 (NS1) on innate immune parameters of infected patients. The present study investigates the relationships between dengue virus nonstructural protein 1 (NS1) serum levels and innate immune response (TLR4 expression and TNF-α/NO production) of DV infected patients presenting different clinical outcomes.Methodology/Principal Findings. We evaluated NO, NS1 serum levels (ELISA), TNF-αproduction by peripheral blood mononuclear cells (PBMCs), and TLR4 expression on CD14+cells from 37 dengue patients and 20 healthy controls. Early in infection, increased expression of TLR4 in monocytes of patients with dengue fever (DF) was detected compared to patients with dengue hemorrhagic fever (DHF). Moreover, PBMCs of DHF patients showed higher NS1 and lower NO serum levels during the acute febrile phase and a reduced response to TLR4 stimulation by LPS (with a reduced TNF-αproduction) when compared to DF patients.Conclusions/Significance. During DV infection in humans, some innate immune parameters change, depending on the NS1 serum levels, and phase and severity of the disease which may contribute to development of different clinical outcomes.

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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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Piron, Maria, Thierry Delaunay, Jeanne Grosclaude, and Didier Poncet. "Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3." Journal of Virology 73, no. 7 (July 1, 1999): 5411–21. http://dx.doi.org/10.1128/jvi.73.7.5411-5421.1999.

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ABSTRACT The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.

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Tsai, Sun-Lung, Pei-Jer Chen, Lih-Hwa Hwang, Jia-Horng Kao, Jyh-Hsiung Huang, Tong-Hsuan Chang, and Ding-Shinn Chen. "Immune response to a hepatitis C virus nonstructural protein in chronic hepatitis C virus infection." Journal of Hepatology 21, no. 3 (January 1994): 403–11. http://dx.doi.org/10.1016/s0168-8278(05)80320-4.

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Parida, Satya, Lucy Fleming, Debi Gibson, Pip A. Hamblin, Santina Grazioli, Emiliana Brocchi, and David J. Paton. "Bovine Serum Panel for Evaluating Foot-and-Mouth Disease Virus Nonstructural Protein Antibody Tests." Journal of Veterinary Diagnostic Investigation 19, no. 5 (September 2007): 539–44. http://dx.doi.org/10.1177/104063870701900513.

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A panel of 36 sera has been assembled from experimental cattle that had been infected by inoculation or contact exposure with 4 serotypes of foot-and-mouth disease virus (FMDV) with or without prior vaccination. Virus replication and persistence had been characterized in all of the animals. The proportion of the sera scored positive by 5 tests for antibodies to the nonstructural proteins of FMDV varied, suggesting that the panel can discriminate between the sensitivity with which such tests are able to identify infected cattle. Use of this panel will help in assessment of new tests and quality control of existing methods.

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Quirino-Teixeira, Anna Cecíllia, Stephane Vicente Rozini, Giselle Barbosa-Lima, Diego Rodrigues Coelho, Pedro Henrique Carneiro, Ronaldo Mohana-Borges, Patrícia T. Bozza, and Eugenio D. Hottz. "Inflammatory signaling in dengue-infected platelets requires translation and secretion of nonstructural protein 1." Blood Advances 4, no. 9 (May 11, 2020): 2018–31. http://dx.doi.org/10.1182/bloodadvances.2019001169.

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Abstract Emerging evidence identifies major contributions of platelets to inflammatory amplification in dengue, but the mechanisms of infection-driven platelet activation are not completely understood. Dengue virus nonstructural protein-1 (DENV NS1) is a viral protein secreted by infected cells with recognized roles in dengue pathogenesis, but it remains unknown whether NS1 contributes to the inflammatory phenotype of infected platelets. This study shows that recombinant DENV NS1 activated platelets toward an inflammatory phenotype that partially reproduced DENV infection. NS1 stimulation induced translocation of α-granules and release of stored factors, but not of newly synthesized interleukin-1β (IL-1β). Even though both NS1 and DENV were able to induce pro-IL-1β synthesis, only DENV infection triggered caspase-1 activation and IL-1β release by platelets. A more complete thromboinflammatory phenotype was achieved by synergistic activation of NS1 with classic platelet agonists, enhancing α-granule translocation and inducing thromboxane A2 synthesis (thrombin and platelet-activating factor), or activating caspase-1 for IL-1β processing and secretion (adenosine triphosphate). Also, platelet activation by NS1 partially depended on toll-like receptor-4 (TLR-4), but not TLR-2/6. Finally, the platelets sustained viral genome translation and replication, but did not support the release of viral progeny to the extracellular milieu, characterizing an abortive viral infection. Although DENV infection was not productive, translation of the DENV genome led to NS1 expression and release by platelets, contributing to the activation of infected platelets through an autocrine loop. These data reveal distinct, new mechanisms for platelet activation in dengue, involving DENV genome translation and NS1-induced platelet activation via platelet TLR4.

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Tao, Ran, Liurong Fang, Dongcheng Bai, Wenting Ke, Yanrong Zhou, Dang Wang, and Shaobo Xiao. "Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Cleaves Porcine DCP1a To Attenuate Its Antiviral Activity." Journal of Immunology 201, no. 8 (August 29, 2018): 2345–53. http://dx.doi.org/10.4049/jimmunol.1701773.

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39

Tan, Yaw Bia, Laura Sandra Lello, Xin Liu, Yee-Song Law, Congbao Kang, Julien Lescar, Jie Zheng, Andres Merits, and Dahai Luo. "Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold." Nucleic Acids Research 50, no. 2 (January 17, 2022): 1000–1016. http://dx.doi.org/10.1093/nar/gkab1302.

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Abstract Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

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Chao, Chiao-Hsuan, Wei-Chueh Wu, Yen-Chung Lai, Pei-Jane Tsai, Guey-Chuen Perng, Yee-Shin Lin, and Trai-Ming Yeh. "Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage." PLOS Pathogens 15, no. 4 (April 22, 2019): e1007625. http://dx.doi.org/10.1371/journal.ppat.1007625.

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Yuan, Shuaizhen, Ning Zhang, Lei Xu, Lei Zhou, Xinna Ge, Xin Guo, and Hanchun Yang. "Induction of Apoptosis by the Nonstructural Protein 4 and 10 of Porcine Reproductive and Respiratory Syndrome Virus." PLOS ONE 11, no. 6 (June 16, 2016): e0156518. http://dx.doi.org/10.1371/journal.pone.0156518.

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42

Lim, Pei-Yin, Appanna Ramapraba, Thomas Loy, Angeline Rouers, Tun-Linn Thein, Yee-Sin Leo, Dennis R. Burton, Katja Fink, and Cheng-I. Wang. "A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue viruses." PLOS ONE 18, no. 5 (May 18, 2023): e0285878. http://dx.doi.org/10.1371/journal.pone.0285878.

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Dengue non-structural protein (NS1) is an important diagnostic marker during the acute phase of infection. Because NS1 is partially conserved across the flaviviruses, a highly specific DENV NS-1 diagnostic test is needed to differentiate dengue infection from Zika virus (ZIKV) infection. In this study, we characterized three newly isolated antibodies against NS1 (A2, D6 and D8) from a dengue-infected patient and a previously published human anti-NS1 antibody (Den3). All four antibodies recognized multimeric forms of NS1 from different serotypes. A2 bound to NS1 from DENV-1, -2, and -3, D6 bound to NS1 from DENV-1, -2, and -4, and D8 and Den3 interacted with NS1 from all four dengue serotypes. Using a competition ELISA, we found that A2 and D6 bound to overlapping epitopes on NS1 whereas D8 recognized an epitope distinct from A2 and D6. In addition, we developed a capture ELISA that specifically detected NS1 from dengue viruses, but not ZIKV, using Den3 as the capture antibody and D8 as the detecting antibody. This assay detected NS1 from all the tested dengue virus strains and dengue-infected patients. In conclusion, we established a dengue-specific capture ELISA using human antibodies against NS1. This assay has the potential to be developed as a point-of-care diagnostic tool.

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Rollier, C., E. Depla, J. A. R. Drexhage, E. J. Verschoor, B. E. Verstrepen, A. Fatmi, C. Brinster, et al. "Control of Heterologous Hepatitis C Virus Infection in Chimpanzees Is Associated with the Quality of Vaccine-Induced Peripheral T-Helper Immune Response." Journal of Virology 78, no. 1 (January 1, 2004): 187–96. http://dx.doi.org/10.1128/jvi.78.1.187-196.2004.

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ABSTRACT Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

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Hwang, Jungwook, Luyun Huang, Daniel G. Cordek, Robert Vaughan, Shelley L. Reynolds, George Kihara, Kevin D. Raney, C. Cheng Kao, and Craig E. Cameron. "Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins." Journal of Virology 84, no. 24 (October 6, 2010): 12480–91. http://dx.doi.org/10.1128/jvi.01319-10.

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ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.

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Huang, Angkana T., Henrik Salje, Ana Coello Escoto, Nayeem Chowdhury, Christian Chávez, Bernardo Garcia-Carreras, Wiriya Rutvisuttinunt, et al. "Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand." PLOS Pathogens 18, no. 5 (May 2, 2022): e1010500. http://dx.doi.org/10.1371/journal.ppat.1010500.

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Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok, Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within ±3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances from when E alone (50.5% to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (ΔRMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface.

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Belliot, Gaël, Stanislav V. Sosnovtsev, Tanaji Mitra, Carl Hammer, Mark Garfield, and Kim Y. Green. "In Vitro Proteolytic Processing of the MD145 Norovirus ORF1 Nonstructural Polyprotein Yields Stable Precursors and Products Similar to Those Detected in Calicivirus-Infected Cells." Journal of Virology 77, no. 20 (October 15, 2003): 10957–74. http://dx.doi.org/10.1128/jvi.77.20.10957-10974.2003.

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ABSTRACT The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q330/G331, Q696/G697, E875/G876, E1008/A1009, and E1189/G1190) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.

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Wang, Chunxiao, Sundy N. Y. Yang, Kate Smith, Jade K. Forwood, and David A. Jans. "Nuclear import inhibitor N -(4-hydroxyphenyl) retinamide targets Zika virus (ZIKV) nonstructural protein 5 to inhibit ZIKV infection." Biochemical and Biophysical Research Communications 493, no. 4 (December 2017): 1555–59. http://dx.doi.org/10.1016/j.bbrc.2017.10.016.

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Brown, Corrie C., Richard F. Meyer, and Marvin J. Grubman. "Use of a Digoxigenin-Labeled RNA Probe to Detect All 24 Serotypes of Bluetongue Virus in Cell Culture." Journal of Veterinary Diagnostic Investigation 5, no. 2 (April 1993): 159–62. http://dx.doi.org/10.1177/104063879300500203.

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A digoxigenin-labeled RNA probe, corresponding to the section of the bluetongue virus (BTV) serotype 17 genome coding for nonstructural protein-1 (NS1), was applied to noninfected cell cultures and cell cultures infected with 24 different serotypes of BTV, 2 serotypes of epizootic hemorrhagic disease virus, and African horse sickness virus type 4. The probe hybridized to all cell cultures infected with the various BTV serotypes but did not hybridize to noninfected cell cultures or cell cultures infected with any of the other orbiviruses.

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Cherkashchenko, Liubov, Kai Rausalu, Sanjay Basu, Luke Alphey, and Andres Merits. "Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner." Viruses 14, no. 6 (June 17, 2022): 1327. http://dx.doi.org/10.3390/v14061327.

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Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.

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Tai, Dar-Fu, Chung-Yin Lin, Tzong-Zeng Wu, Jyh-Hsiung Huang, and Pei-Yun Shu. "Artificial Receptors in Serologic Tests for the Early Diagnosis of Dengue Virus Infection." Clinical Chemistry 52, no. 8 (August 1, 2006): 1486–91. http://dx.doi.org/10.1373/clinchem.2005.064501.

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Abstract Background: Because of the range and nonspecificity of clinical presentations of dengue virus infections, we felt there was a need to create diagnostic tests. We used artificial receptors for the virus to develop serologic assays to detect dengue virus infection. Methods: We coated a quartz crystal microbalance (QCM) with molecularly imprinted polymers specific for nonstructural protein 1 of flavivirus. These artificial receptors were specifically created on a QCM chip by polymerization of monomers and were cross-linked in the presence of the epitope site of nonstructural protein 1. We tested serum samples from patients with confirmed cases of dengue reported to the Center for Disease Control in Taipei. Samples were diluted 100-fold; no other sample pretreatment was used. The QCM response was compared with results of monoclonal ELISA. Results: QCM signals were >15 Hz in 18 of 21 (86%) of dengue samples and in 0 of 16 control samples. The correlation (r2) of the QCM response and the ELISA result was 0.73. Within-run and run-to-run imprecisions (CV) were 4%–28% and 10%–32%, respectively. Conclusions: The described assay offers a serologic technique for diagnosis of early viremia. The results illustrate the potential of well-organized polymers on the highly sensitive sensor system for diagnostic and biotechnological applications.

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Journal articles on the topic 'Nonstructural Protein 4'

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