scholarly journals Conserved Surface Features Form the Double-stranded RNA Binding Site of Non-structural Protein 1 (NS1) from Influenza A and B Viruses

2007 ◽  
Vol 282 (28) ◽  
pp. 20584-20592 ◽  
Author(s):  
Cuifeng Yin ◽  
Javed A. Khan ◽  
G. V. T. Swapna ◽  
Asli Ertekin ◽  
Robert M. Krug ◽  
...  
Keyword(s):  
Binding Site ◽  
Influenza A ◽  
Rna Binding ◽  
Structural Protein ◽  
Double Stranded Rna ◽  
Surface Features

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 Interactions between double-stranded RNA regulators and the protein kinase DAI.

1992 ◽  
Vol 12 (11) ◽  
pp. 5238-5248 ◽  
Author(s):  
L Manche ◽  
S R Green ◽  
C Schmedt ◽  
M B Mathews
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Rna Binding ◽  
Polypeptide Chain ◽  
Initiation Factor ◽  
Double Stranded Rna ◽  
Rna Duplexes ◽  
High Concentrations ◽  
Helical Turn ◽  
Maximal Packing

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

Antiviral activity of double‐stranded RNA‐binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase

2018 ◽  
Vol 32 (8) ◽  
pp. 4380-4393 ◽  
Author(s):  
Chi‐Ping Chan ◽  
Chun‐Kit Yuen ◽  
Pak‐Hin Hinson Cheung ◽  
Sin‐Yee Fung ◽  
Pak‐Yin Lui ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Viral Rna ◽  
Double Stranded Rna

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 Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells

2011 ◽  
Vol 92 (9) ◽  
pp. 2111-2121 ◽  
Author(s):  
Muhammad Munir ◽  
Siamak Zohari ◽  
Giorgi Metreveli ◽  
Claudia Baule ◽  
Sándor Belák ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Influenza A ◽  
Rna Binding ◽  
Type I Interferons ◽  
Lung Cells ◽  
Type I ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Promoter Activation ◽  
Influenza A Viruses

Non-structural protein 1 (NS1) counteracts the production of host type I interferons (IFN-α/β) for the efficient replication and pathogenicity of influenza A viruses. Here, we reveal another dimension of the NS1 protein of avian influenza A viruses in suppressing IFN-β production in cultured cell lines. We found that allele A NS1 proteins of H6N8 and H4N6 have a strong capacity to inhibit the activation of IFN-β production, compared with allele B from corresponding subtypes, as measured by IFN stimulatory response element (ISRE) promoter activation, IFN-β mRNA transcription and IFN-β protein expression. Furthermore, the ability to suppress IFN-β promoter activation was mapped to the C-terminal effector domain (ED), while the RNA-binding domain (RBD) alone was unable to suppress IFN-β promoter activation. Chimeric studies indicated that when the RBD of allele A was fused to the ED of allele B, it was a strong inhibitor of IFN-β promoter activity. This shows that well-matched ED and RBD are crucial for the function of the NS1 protein and that the RBD could be one possible cause for this differential IFN-β inhibition. Notably, mutagenesis studies indicated that the F103Y and Y103F substitutions in alleles A and B, respectively, do not influence the ISRE promoter activation. Apart from dsRNA signalling, differences were observed in the expression pattern of NS1 in transfected human and mink lung cells. This study therefore expands the versatile nature of the NS1 protein in inhibiting IFN responses at multiple levels, by demonstrating for the first time that it occurs in a manner dependent on allele type.

scholarly journals Molecular Dynamics Simulations of Influenza A Virus NS1 Reveal a Remarkably Stable RNA-Binding Domain Harboring Promising Druggable Pockets

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 537
Author(s):  
Hiba Abi Hussein ◽  
Colette Geneix ◽  
Camille Cauvin ◽  
Daniel Marc ◽  
Delphine Flatters ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Influenza A ◽  
Rna Binding ◽  
Biological Activities ◽  
Binding Domain ◽  
Structural Protein ◽  
Binding Interface ◽  
Rna Binding Domain ◽  
Dynamics Simulations

The non-structural protein NS1 of influenza A viruses is considered to be the major antagonist of the interferon system and antiviral defenses of the cell. It could therefore represent a suitable target for novel antiviral strategies. As a first step towards the identification of small compounds targeting NS1, we here investigated the druggable potential of its RNA-binding domain since this domain is essential to the biological activities of NS1. We explored the flexibility of the full-length protein by running molecular dynamics simulations on one of its published crystal structures. While the RNA-binding domain structure was remarkably stable along the simulations, we identified a flexible site at the two extremities of the “groove” that is delimited by the antiparallel α-helices that make up its RNA-binding interface. This groove region is able to form potential binding pockets, which, in 60% of the conformations, meet the druggability criteria. We characterized these pockets and identified the residues that contribute to their druggability. All the residues involved in the druggable pockets are essential at the same time to the stability of the RNA-binding domain and to the biological activities of NS1. They are also strictly conserved across the large sequence diversity of NS1, emphasizing the robustness of this search towards the identification of broadly active NS1-targeting compounds.

Interactions between double-stranded RNA regulators and the protein kinase DAI

1992 ◽  
Vol 12 (11) ◽  
pp. 5238-5248
Author(s):  
L Manche ◽  
S R Green ◽  
C Schmedt ◽  
M B Mathews
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Rna Binding ◽  
Polypeptide Chain ◽  
Initiation Factor ◽  
Double Stranded Rna ◽  
Rna Duplexes ◽  
High Concentrations ◽  
Helical Turn ◽  
Maximal Packing

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

scholarly journals Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

2021 ◽  
Author(s):  
Kanchan Devkota ◽  
Matthieu Schapira ◽  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Fengling Li ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Phenyl Group ◽  
Competitive Inhibitor ◽  
Structural Protein ◽  
Human Immune System ◽  
Rna Capping ◽  
Protein Methyltransferases ◽  
Socioeconomic Consequences

AbstractThe COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 ± 0.2 μM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

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