scholarly journals Structure and Sequence Determinants Governing the Interactions of RNAs with Influenza A Virus Non-Structural Protein NS1

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 947
Author(s):  
Alan Wacquiez ◽  
Franck Coste ◽  
Emmanuel Kut ◽  
Virginie Gaudon ◽  
Sascha Trapp ◽  
...  
Keyword(s):  
Influenza A ◽  
Rna Binding ◽  
3D Structure ◽  
Genetically Engineered ◽  
Phenotypic Characterization ◽  
Sequence Motifs ◽  
Structural Protein ◽  
Specific Sequence ◽  
Rna Interaction

The non-structural protein NS1 of influenza A viruses is an RNA-binding protein of which its activities in the infected cell contribute to the success of the viral cycle, notably through interferon antagonism. We have previously shown that NS1 strongly binds RNA aptamers harbouring virus-specific sequence motifs (Marc et al., Nucleic Acids Res. 41, 434–449). Here, we started out investigating the putative role of one particular virus-specific motif through the phenotypic characterization of mutant viruses that were genetically engineered from the parental strain WSN. Unexpectedly, our data did not evidence biological importance of the putative binding of NS1 to this specific motif (UGAUUGAAG) in the 3′-untranslated region of its own mRNA. Next, we sought to identify specificity determinants in the NS1-RNA interaction through interaction assays in vitro with several RNA ligands and through solving by X-ray diffraction the 3D structure of several complexes associating NS1′s RBD with RNAs of various affinities. Our data show that the RBD binds the GUAAC motif within double-stranded RNA helices with an apparent specificity that may rely on the sequence-encoded ability of the RNA to bend its axis. On the other hand, we showed that the RBD binds to the virus-specific AGCAAAAG motif when it is exposed in the apical loop of a high-affinity RNA aptamer, probably through a distinct mode of interaction that still requires structural characterization. Our data are consistent with more than one mode of interaction of NS1′s RBD with RNAs, recognizing both structure and sequence determinants.

scholarly journals Shortening the unstructured, interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens

2014 ◽  
Vol 95 (6) ◽  
pp. 1233-1243 ◽  
Author(s):  
Sascha Trapp ◽  
Denis Soubieux ◽  
Hélène Marty ◽  
Evelyne Esnault ◽  
Thomas W. Hoffmann ◽  
...  
Keyword(s):  
Rna Binding ◽  
H1n1 Influenza ◽  
Genetically Engineered ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
General Effect ◽  
Structural Protein ◽  
H5n1 Influenza

Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in 2000 that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbour a deletion of aa 80–84 in the unstructured region of the non-structural (NS) protein NS1 linking its RNA-binding domain to its effector domain. NS segments harbouring this mutation have since been found in non-H5N1 viruses and we asked whether this 5 aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the WT and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrated that the increased replicative potential conferred by this small deletion was a general feature not restricted to NS1 from H5N1 viruses and suggested that viruses acquiring this mutation may be selected positively in the future.

DNA Binding Activity of Marine Shrimp LvProfilin

2021 ◽  
Author(s):  
Yanisa Laoong-u-thai ◽  
Warapond Wanna ◽  
Autaipohn Kaikaew
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
3D Structure ◽  
Binding Activity ◽  
Shrimp Farming ◽  
Binding Prediction ◽  
3D Protein Structure ◽  
Dna Binding Activity ◽  
Marine Shrimp

Shrimp farming is an important business in Thailand and worldwide. The study of molecular biology and biochemical pathway of the key molecules controlling muscle growth is an essential to improve shrimp livestock. Profilin is a pivotal protein in muscle formation, especially actin protein. Its nuclear function has been reported in many species for gene regulation. Here in this work, we characterized the function of LvProfilin, a marine shrimp profilin from Litopenaeus vannamei, both in silico and in vitro. The phylogenetic tree of LvProfilin among organisms and its 3D protein structure showed that LvProfilin was highly conserved among shrimp and arthropods. The homology modeling of its 3D structure revealed 3 alpha-helices and 6 beta-strands similar to most eukaryotic profilins. To interpret its possible function, the gene expression of LvProfilin in various tissues was performed. We found that this gene was expressed in various tissues. This result may imply that LvProfilin could share a common function in all tissues. Nuclear activity has been a promising function of LvProfilin. We performed a DNA/RNA binding prediction analysis using DRNApred. The result indicated that Lysine-90 and Threonine-91 were the putative DNA-binding sites with the probability of 63.12% and 54.16%, respectively. Its binding activity was confirmed in vitro which bound stronger to single strand DNA than double strand DNA. To our best knowledge, this is the first report of DNA binding activity of profilin in invertebrates.

scholarly journals A deep learning framework to predict binding preference of RNA constituents on protein surface

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jordy Homing Lam ◽  
Yu Li ◽  
Lizhe Zhu ◽  
Ramzan Umarov ◽  
Hanlun Jiang ◽  
...  
Keyword(s):  
Deep Learning ◽  
Protein Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Preference ◽  
Rna Backbone ◽  
Binding Pockets ◽  
Rna Interaction

Abstract Protein-RNA interaction plays important roles in post-transcriptional regulation. However, the task of predicting these interactions given a protein structure is difficult. Here we show that, by leveraging a deep learning model NucleicNet, attributes such as binding preference of RNA backbone constituents and different bases can be predicted from local physicochemical characteristics of protein structure surface. On a diverse set of challenging RNA-binding proteins, including Fem-3-binding-factor 2, Argonaute 2 and Ribonuclease III, NucleicNet can accurately recover interaction modes discovered by structural biology experiments. Furthermore, we show that, without seeing any in vitro or in vivo assay data, NucleicNet can still achieve consistency with experiments, including RNAcompete, Immunoprecipitation Assay, and siRNA Knockdown Benchmark. NucleicNet can thus serve to provide quantitative fitness of RNA sequences for given binding pockets or to predict potential binding pockets and binding RNAs for previously unknown RNA binding proteins.

scholarly journals Homozygous Inactivating Mutation inNANOS3in Two Sisters with Primary Ovarian Insufficiency

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Mariza G. Santos ◽  
Aline Z. Machado ◽  
Conceição N. Martins ◽  
Sorahia Domenice ◽  
Elaine M. F. Costa ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Primordial Germ Cells ◽  
Embryonic Cell ◽  
Primary Ovarian Insufficiency ◽  
Primary Amenorrhea ◽  
Female Reproduction ◽  
Ovarian Insufficiency ◽  
Rna Interaction

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis ofNANOS3in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation inNANOS3was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 andin silicomolecular modelling suggests destabilization of protein-RNA interaction.In vitroanalyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation inNANOS3suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

scholarly journals Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process

2000 ◽  
Vol 74 (18) ◽  
pp. 8252-8261 ◽  
Author(s):  
Hui Zhang ◽  
Roger J. Pomerantz ◽  
Geethanjali Dornadula ◽  
Yong Sun
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Binding ◽  
Viral Rna ◽  
Immunodeficiency Virus ◽  
Mrnp Complex ◽  
Rna Interaction ◽  
Vif Protein ◽  
Hiv 1

ABSTRACT Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not supportvif-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.

scholarly journals A NS1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza A virus

2019 ◽  
Vol 77 (1) ◽  
Author(s):  
Su Hui Catherine Teo ◽  
Jian-Ping Wu ◽  
Chee-Keng Mok ◽  
Yee-Joo Tan
Keyword(s):  
Monoclonal Antibody ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Structural Protein ◽  
Multifunctional Protein ◽  
Good Target ◽  
Conformational Plasticity

Abstract The non-structural protein 1 (NS1) of influenza A virus (IAV) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. As such, it serves as a good target for research and diagnostic assay development. In this study, we have generated a novel monoclonal antibody (mAb) 19H9 and epitope mapping revealed that two residues, P85 and Y89, of NS1 are essential for interacting with this mAb. Furthermore, residues P85 and Y89 are found to be highly conserved across different IAV subtypes, namely seasonal H1N1 and H3N2, as well as the highly pathogenic H5N1 and H5N6 avian strains. Indeed, mAb 19H9 exhibits broad cross-reactivity with IAV strains of different subtypes. The binding of mAb 19H9 to residue Y89 was further confirmed by the abrogation of interaction between NS1 and p85β. Additionally, mAb 19H9 also detected NS1 proteins expressed in IAV-infected cells, showing NS1 intracellular localization in the cytoplasm and nucleolus. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N-terminal RNA-binding domain and C-terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.

scholarly journals Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides

2020 ◽  
Vol 21 (17) ◽  
pp. 6341
Author(s):  
Maria Sendino ◽  
Miren Josu Omaetxebarria ◽  
Gorka Prieto ◽  
Jose Antonio Rodriguez
Keyword(s):  
Nuclear Export ◽  
Relevant Information ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Cellular Assay ◽  
Small Proteins ◽  
Binding Groove ◽  
Related Proteins ◽  
Insight Into

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.

scholarly journals Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

2005 ◽  
Vol 79 (11) ◽  
pp. 6631-6643 ◽  
Author(s):  
Diego E. Alvarez ◽  
María F. Lodeiro ◽  
Silvio J. Ludueña ◽  
Lía I. Pietrasanta ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Virus Genome ◽  
Viral Rna ◽  
Physical Interaction ◽  
Virus Rna ◽  
Rna Elements ◽  
Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

scholarly journals Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

2019 ◽  
Author(s):  
Linjiang Yang ◽  
Mingshu Wang ◽  
Chunhui Zeng ◽  
Yong Shi ◽  
Anchun Cheng ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Rna Binding ◽  
Gene Interaction ◽  
Duck Enteritis Virus ◽  
Late Gene ◽  
Structural Protein ◽  
Virus Transport ◽  
Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Methods In our study, recombinant pUL21 was expressed using an pET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody used for the indirect immunofluorescence (IFA) and western blotting (WB) analysis were prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. Keywords: Duck enteritis virus, UL21, UL16, late gene, interaction

scholarly journals The Nucleoside Triphosphatase and Helicase Activities of Vaccinia Virus NPH-II Are Essential for Virus Replication

1998 ◽  
Vol 72 (6) ◽  
pp. 4729-4736 ◽  
Author(s):  
Christian H. Gross ◽  
Stewart Shuman
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Rna Binding ◽  
Rna Helicase ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Sequence Motifs ◽  
Rna Unwinding

ABSTRACT Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-tsviruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.

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