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.

scholarly journals Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors

2021 ◽  
pp. 247255522110262
Author(s):  
Kanchan Devkota ◽  
Matthieu Schapira ◽  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Fengling Li ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Competitive Inhibitor ◽  
Human Immune System ◽  
Mtase Activity ◽  
Competitive Inhibitors ◽  
Human Immune ◽  
Rna Capping ◽  
Socioeconomic Consequences ◽  
Bisubstrate Inhibitors

The COVID-19 pandemic has clearly brought the healthcare systems worldwide 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. Nonstructural 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 (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure–activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

scholarly journals An in vitro technique to identify the RNA binding-site sequences for RNA-binding proteins

BioTechniques ◽  
2017 ◽  
Vol 63 (1) ◽  
Author(s):  
SunKyung Choi ◽  
Chungoo Park ◽  
Kyoon Eon Kim ◽  
Kee K. Kim
Keyword(s):  
Binding Site ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
In Vitro Technique

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 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 An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

1991 ◽  
Vol 11 (10) ◽  
pp. 5312-5320 ◽  
Author(s):  
Z W Qian ◽  
J Wilusz
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Band Shift

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

scholarly journals Processivity of the Saccharomyces cerevisiae Poly(A) Polymerase Requires Interactions at the Carboxyl-Terminal RNA Binding Domain

1998 ◽  
Vol 18 (10) ◽  
pp. 5942-5951 ◽  
Author(s):  
Alexander Zhelkovsky ◽  
Steffen Helmling ◽  
Claire Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Rna Binding ◽  
Atp Binding Site ◽  
Nucleotide Binding Sites ◽  
Carboxyl Terminal ◽  
Unique Specificity ◽  
Carboxy Terminal

ABSTRACT The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action. Partial truncation of this RBS has the same effect, indicating that this site is required for processivity. A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3′ end of the primer. This site discriminates against deoxyribonucleotides at the 3′ end, and interactions at this site are not affected by Fip1. Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3′ end of the primer and at another contact site 14 nucleotides upstream of the 3′ end. These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites.

scholarly journals The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs.

1989 ◽  
Vol 9 (7) ◽  
pp. 2975-2982 ◽  
Author(s):  
C Lutz-Freyermuth ◽  
J D Keene ◽  
C Lutz-Reyermuth
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Vitro Binding ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

The site of interaction between human U1 RNA and one of its uniquely associated proteins, A, was examined with in vitro binding assays. The A protein bound directly to stem-loop II of U1 RNA in a region which exhibits sequence similarity to U2 RNA. The similarity with U2 RNA was in a region that has been shown to interact with a U2 RNA-associated protein. The A protein-binding site on U1 RNA overlapped a previously described epitope for an RNA-specific human autoantibody (S. L. Deutscher and J. D. Keene, Proc. Natl. Acad. Sci. USA 85:3299-3303, 1988), supporting the hypothesis that the anti-RNA antibody originated as an anti-idiotypic response to A protein-specific autoantibodies.

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 RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

2020 ◽  
Author(s):  
Kevin Haubrich ◽  
Sandra Augsten ◽  
Bernd Simon ◽  
Pawel Masiewicz ◽  
Kathryn Perez ◽  
...  
Keyword(s):  
Binding Site ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
E3 Ligase ◽  
Cell Fate Decisions ◽  
Ubiquitin E3 Ligase

ABSTRACTTRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25’s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA-binding have been described, the precise RNA binding site has not been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. Mutational analysis shows that RNA binding affects ubiquitination of RIG-I in mammalian cells. In addition, we present a simple model system for RNA-induced liquid-liquid phase separation of TRIM25 in vitro, resembling previously observed cellular RNA granules, that facilitates the recruitment of RIG-I.

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