Journal: bioRxiv

Article Title: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

doi: 10.1101/2022.01.19.476693

Figure Lengend Snippet: ( a ) PLA detection of NSP2-GIGYF2 interactions visible as fluorescent punctate in HEK293T cells transfected with vectors expressing v5-tagged GIGYF2, 4EHP, or GIGYF1 together with FLAG-NSP2. 24 h post-transfection cells were fixed and subjected to PLA using FLAG and v5 antibodies. PLA signals are shown in yellow. Nucleus and actin cytoskeleton were counterstained with DAPI (blue) and phalloidin (red), respectively. Scale bar= 10 µm. ( b ) Quantification of positive PLA signals in (a). The number of PLA signals from at least 30 cells was counted in each sample. ****P<0.0001 one-way ANOVA with Bonferroni’s post hoc test; n=5 independent replicates. Data are presented as mean ± SD. ( c ) WT HEK293 cells were co-transfected with plasmids expressing either λN-v5-GIGYF2 or λN-v5 as control, along with R-Luc -5BoxB-A114-N40-HhR and F-Luc (as control), followed by dual-luciferase measurement assay. Data are presented as mean ± SD (n=3). ***p<0.001, one-way ANOVA with Bonferroni’s post hoc test. The schematic shows a graphic model of λN-v5-GIGYF2 tethering system. ( d ) Analysis of λN-v5-GIGYF2 tethering-induced silencing in GIGYF2-KO cells that overexpress FLAG-NSP2. Data are presented as mean ± SD (n=3). ns= non-significant, one-way ANOVA with Bonferroni’s post hoc test. See also Extended Data Fig. 1.

Article Snippet: Synthesized viral coding sequences were incorporated into Gateway-compatible Entry vectors; pDONR207 SARS-CoV-2 NSP1 (Addgene, 141255), pDONR223 SARS-CoV-2 NSP2 (Addgene, 141256) and expression clones with N-terminal fusion tags were produced simply by Gateway cloning (GatewayTM LR ClonaseTM II Enzyme mix, Invitrogen, 11791020).

Techniques: Transfection, Expressing, Control, Luciferase

Journal: bioRxiv

Article Title: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

doi: 10.1101/2022.01.19.476693

Figure Lengend Snippet: (a) WT cells were co-transfected with vectors expressing either λN-v5-GIGYF2 or λN-v5 control, along with R-Luc -5BoxB-A114-N40-HhR and F-Luc (as control), in combination with FLAG-4EHP or FLAG-Empty plasmids. Dual-luciferase assay was performed 24 h post-transection. Data are presented as mean ± SD (n=3). *p<0.05, one-way ANOVA with Bonferroni’s post hoc test. ( b-c ) GIGYF2-tethering assay carried out in 4EHP-KO cells in ( b ) and GIGYF2-KO cells in ( c ). Data are presented as mean ± SD (n=3). ns= non-significant, **p<0.01, ***p<0.001, one-way ANOVA with Bonferroni’s post hoc test. ( d ) Graphic illustration of the GIGYF2/4EHP-mediated induction of translational repression by NSP2. ( e ) PLA assay for detection of GIGYF2-4EHP interactions in HEK293T cells transfected with vectors expressing v5-GIGYF2 and HA-4EHP together with FLAG-Empty or FLAG-NSP2. Cells were fixed and subjected to PLA using v5 and HA antibodies 24 h post-transfection. Scale bar= 10 µm. ( f ) Quantification of positive PLA signals from (e). The number of PLA signals from at least 20 cells was counted in each sample. n=5 independent experiments. Data are presented as mean ± SD (n=5). ****P< 0.0001, one-way ANOVA with Bonferroni’s post hoc test. See also Extended Data Fig. 2 and .

Article Snippet: Synthesized viral coding sequences were incorporated into Gateway-compatible Entry vectors; pDONR207 SARS-CoV-2 NSP1 (Addgene, 141255), pDONR223 SARS-CoV-2 NSP2 (Addgene, 141256) and expression clones with N-terminal fusion tags were produced simply by Gateway cloning (GatewayTM LR ClonaseTM II Enzyme mix, Invitrogen, 11791020).

Techniques: Transfection, Expressing, Control, Luciferase

Journal: bioRxiv

Article Title: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

doi: 10.1101/2022.01.19.476693

Figure Lengend Snippet: (a) 24 h post-transfection with GFP, NSP2, NSP1, or E protein, HEK293 cells were transfected with psiCHECK2- R-Luc-Ifnb1 3′ UTR reporter or the psiCHECK2 reporter (control). R-Luc and F-Luc activities were measured 24 h after the 2 nd transfection. The R-Luc/F-Luc ratio of psiCHECK2- R-Luc-Ifnb1 3′ UTR cells was normalized to the value for the psiCHECK2 cells as a percentage. Data are presented as mean ± SD (n=3). ns= non-significant, *p< 0.05, two-way ANOVA with Bonferroni’s post hoc test. (b) WT, 4EHP-KO, and GIGYF2-KO HEK293 cells were transfected with GFP or NSP2 expression plasmid for 24 h, followed by the 2 nd transfection with psiCHECK2- R-Luc - Ifnb1 3′ UTR reporter or the psiCHECK2 control reporter. 24h post-transfection the R-Luc / F-Luc ratio was measured as described in (a). (c) ELISA measurement of IFN-β and (d) western blot analysis of poly(I:C) treated WT and GIGYF2-KO A549 cells stably expressing either empty vector (EV) or NSP2 using lentiviral vector at 8 h post transfection. Data are presented as mean ± SD (n=3). ns= non-significant, ***p< 0.001, two-way ANOVA with Bonferroni’s post hoc test was used. (e) Graphic illustration of recruitment of the GIGYF2/4EHP repressor complex by NSP2 to silence IFN-β production in response to SARS-CoV-2 infection. GIGYF2/4EHP complex enables the miRISC-induced repression of the cap-dependent mRNA translation. Binding of NSP2 to GIGYF2 enhances the interaction of GIGYF2 with 4EHP, resulting in co-stabilisation of GIGYF2 and 4EHP and augmented translational repression of Ifnb1 mRNA. See also Extended Data Fig. 6 and Fig. 7.

Article Snippet: Synthesized viral coding sequences were incorporated into Gateway-compatible Entry vectors; pDONR207 SARS-CoV-2 NSP1 (Addgene, 141255), pDONR223 SARS-CoV-2 NSP2 (Addgene, 141256) and expression clones with N-terminal fusion tags were produced simply by Gateway cloning (GatewayTM LR ClonaseTM II Enzyme mix, Invitrogen, 11791020).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Stable Transfection, Infection, Binding Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

doi: 10.1101/2022.01.19.476693

Figure Lengend Snippet: (a) Structural prediction and modeling of human GIGYF2 by Pfam and AlphaFold-2. The predicted three-dimensional structure of GIGYF2 was obtained from the AlphaFold Protein Structure Database and annotated in PyMOL: cyan – long helix region, orange – GYF domain, grey – rest of GIGYF2. The schematic of predicted domains and folding regions along the primary structure of GIGYF2 was generated by querying human GIGYF2 (accession number: Q6Y7W6) against the Pfam database. Grey – regions predicted to be disordered, blue – regions predicted to have low complexity, green – regions predicted to fold into known domains or secondary structures. (b) Cryo-EM structure of SARS-CoV-2 NSP2. The cryo-EM structure of NSP2 (4-104, 119-475, 491-505) was obtained from the Protein Data Bank (PDB code: 7MSX). Discrete folding regions within NSP2 were annotated in PyMOL: red – NSP2-R1, green – NSP2-R2, magenta – NSP2-R3, yellow – NSP2-R4. (c) Model of the interaction between GIGYF2-LHR and NSP2 by HADDOCK 2.4. The predicted model was annotated in PyMOL as follows: cyan – GIGYF2-LHR ribbon model superimposed over a GIGYF2-LHR 60% transparent surface model, magenta – NSP2 cryo-EM ribbon model superimposed over an NSP2 cryo-EM 60% transparent surface model, yellow – G262/G265 containing alpha helix in NSP2. See also Extended Data Fig. 8-10.

Article Snippet: Synthesized viral coding sequences were incorporated into Gateway-compatible Entry vectors; pDONR207 SARS-CoV-2 NSP1 (Addgene, 141255), pDONR223 SARS-CoV-2 NSP2 (Addgene, 141256) and expression clones with N-terminal fusion tags were produced simply by Gateway cloning (GatewayTM LR ClonaseTM II Enzyme mix, Invitrogen, 11791020).

Techniques: Structural Proteomics, Generated, Cryo-EM Sample Prep

Journal: Molecular medicine reports

Article Title: RTN2, a new member of circadian clock genes identified by database mining and bioinformatics prediction, is highly expressed in ovarian cancer.

doi: 10.3892/mmr.2022.12866

Figure Lengend Snippet: Figure 8. RTN2 mRNA expression level in ovarian cancer tissues and normal ovarian tissues determined by reverse transcription‑quantitative PCR. Data were presented as 2‑ΔΔCq. *P<0.05. RTN2, reticulon 2.

Article Snippet: Paraffin‐embedded normal ovarian tissue and oV tissue samples were routinely processed and then incubated with rabbit primary antibodies against RTN2 overnight at 4 ̊C (cat. no. 11168‐1‐aP; 1:200 dilution; Proteintech Group, inc.). after a 1‐h incubation with HrP‐linked anti‐rabbit secondary antibodies (cat. no. a0545; 1:4,000 dilution; MilliporeSigma) at room temperature, the daB reagent was utilized for development, followed by hematoxylin counterstaining. a leica dM 2000 microscope (leica Microsystems) was used for analysis.

Techniques: Expressing

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Expression and purification of recombinant NSP2. NSP2 expressed in E. coli with a C-terminal His tag was purified by NTA affinity chromatography, and the final eluate was dialyzed against LSB. The eluate (lane 2) and protein dialyzed in LSB (lane 1) were resolved by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. M, molecular size marker.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Expressing, Purification, Recombinant, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Marker

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: NSP2 possesses helix-destabilizing activity. (A) A schematic representation of the 32P-labeled DNA-RNA partial duplex A11-StyI–18AD. (B) From 1 to 200 pmol of NSP2 (lanes 4 to 9) was incubated with 0.1 pmol of A11-StyI–18AD for 30 min at 37°C. Afterwards, the reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. Reaction mixtures containing 0.1 pmol of the 32P-labeled 18AD DNA oligonucleotide instead of the duplex (lane 1), 0.1 pmol of the duplex and no NSP2 (lane 2), and 0.1 pmol of the duplex denatured by heating at 95°C for 2 min and containing no NSP2 (lane 3) were also analyzed.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Activity Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Autoradiography

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Directionality of the unwinding activity of NSP2. (A) A schematic representation of the possible duplexes formed by annealing the 32P-labeled DNA oligonucleotides 5′18AD and 3′22AD to the A11-SacII RNA. (B) A standard strand displacement assay was performed by incubating 0.1 pmol of the duplex mixture as shown in panel A with 1 to 100 pmol of recombinant NSP2 (rNSP2) (lanes 4 to 7). The reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. Reaction mixtures containing 32P-labeled 5′18AD (lane 1) or 5′22AD (lane 2) and no duplexes were also analyzed.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Activity Assay, Labeling, Recombinant, Nucleic Acid Electrophoresis, Autoradiography

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Helix destabilization by NSP2 is cooperative. (A) A schematic representation of the 32P-labeled DNA-RNA duplex A11-SacII–5′18AD, formed by incubating the DNA oligonucleotide, 5′18AD, with the A11-SacII RNA. (B) A set of standard strand displacement assays was performed in parallel by incubating 0.1 pmol of the duplex with 1 to 200 pmol of NSP2. The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity of NSP2 was plotted as a function of the amount of NSP2 in the reaction mixtures.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Labeling, Nucleic Acid Electrophoresis, Activity Assay

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Kinetics of helix destabilization by NSP2. The 32P-labeled DNA-RNA duplex A11-StyI–5′18AD (0.1 pmol) was incubated with 50 (●), 100 (▴), or 200 (■) pmol of NSP2 for 2, 10, 15, or 30 min at 37°C. Afterwards, the reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity of NSP2 was plotted as a function of reaction time.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Labeling, Incubation, Nucleic Acid Electrophoresis, Activity Assay

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Effect of cations on helix destabilization by NSP2. The DNA-RNA duplex, A11-StyI–18AD (0.1 pmol), was incubated with NSP2 (200 pmol) in the presence of 0 to 10 mM MgCl2 (○), CaCl2 (□), or MnCl2 (◊) (panel A) or 0 to 250 mM NaCl (●) (panel B). The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity was plotted as a function of salt concentration, with the value obtained for the reaction mixture lacking salt normalized to 100%.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Incubation, Nucleic Acid Electrophoresis, Activity Assay, Concentration Assay

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Combined effect of Mg2+ and NTP on helix destabilization by NSP2. (A) NSP2 (200 pmol) was incubated with 0.1 pmol of the DNA-RNA duplex, A11-StyI–18AD, in the absence or presence of 5 mM ATP, GTP, CTP, or UTP for 30 min at 37°C. (B) Reaction mixtures containing the same components as those of the reaction mixtures in panel A, except that these contained 5 mM MgCl2, were also incubated. (C) NSP2 (100 pmol) was incubated with 0.1 pmol of the DNA-RNA duplex, A11-StyI–18AD, in the absence or presence of 1 mM MgCl2 or in the presence of 1 mM MgCl2 and 1 mM ATP, ADP, or ATP-γ-S. The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The values were normalized to that of 100% for the assays performed in the absence of MgCl2.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Incubation, Nucleic Acid Electrophoresis, Activity Assay

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: Destabilization of an RNA-RNA duplex by NPS2. (A) A schematic representation of the 32P-labeled RNA-RNA duplex A11-StyI–14AR produced by annealing the RNAs A11-StyI and 14AR. (B) Strand displacement assays were performed by incubating 0.1 pmol of the RNA-RNA duplex with 1 to 200 pmol of NSP2 in the presence (lanes 4 to 9) or absence (lanes 10 to 15) of 5 mM MgCl2. As controls, reaction mixtures were also prepared that contained 0.1 pmol of the 14AR RNA instead of the duplex (lane 1), 0.1 pmol of A11-StyI–14AR duplex and no NSP2 (lane 2), or 0.1 pmol of A11-StyI–14AR and no NSP2, denatured by heating at 95°C for 2 min (lane 3). (C) Strand displacement assays were performed by incubating 5 to 20 fmol of the RNA-RNA duplex in the presence or absence of recombinant NSP2 (rNSP2). The reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. A PhosphorImager was used to determine the percent helix-destabilizing activity for each reaction. The values were normalized to that of 100% for the assay reaction wherein the substrate duplex was denatured by heating.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Labeling, Produced, Recombinant, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay

Journal:

Article Title: Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2

doi: 10.1128/JVI.75.10.4519-4527.2001

Figure Lengend Snippet: NSP2 destabilizes a short RNA-RNA duplex with greater efficiency. (A) A schematic representation of the 32P-labeled RNA-RNA duplex A11-StyI–10AR produced by annealing the RNAs A11-StyI and 10AR. (B) Strand displacement assays were performed by incubating 0.1 pmol of the RNA-RNA duplex with 1 to 200 pmol of NSP2 (lanes 5 to 10). As controls, reaction mixtures were also prepared that contained 0.1 pmol of the 10AR RNA instead of the duplex (lane 1), 0.1 pmol of the A11-StyI–14AR duplex and no NSP2 (lane 2 and 4), or 0.1 pmol of A11-StyI–10AR and no NSP2, denatured by heating at 95°C for 2 min (lane 3). A PhosphorImager was used to determine the percent helix-destabilizing activity for each reaction. The values were normalized to that of 100% for the assay reaction wherein the substrate duplex was denatured by heating.

Article Snippet: Recombinant NSP2 was purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography following the protocol provided by the manufacturer (Qiagen).

Techniques: Labeling, Produced, Activity Assay

Journal: Antiviral research

Article Title: Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.

doi: 10.1016/j.antiviral.2019.104674

Figure Lengend Snippet: VEEV RNA synthesis peaks at 6–8 HPI. A) BHK 21 cells were infected with VEEV as described in the Materials and Methods and treated with act D and 3HU to selectively label newly synthesized viral RNA. Total RNA was isolated and subjected to liquid scintillation counting. The amount of label detected is reported as counts per minute (CPM) per microgram of isolated RNA. NC is an uninfected negative control. Bars represent three biological replicates from one representative experiment. Error bars indicate standard deviation. B) BHK 21 cells were infected with VEEV and total cell lysate was used for immunoblotting to detect nsP2. Beta-actin is included as a protein loading control. Image is from one representative experiment.

Article Snippet: Anti-nsP2 (Clone no. 8A4B3, available from Kerafast Cat. No EUL015) was generated as a custom mouse monoclonal antibody from GenScripts using bacterially expressed recombinant nsP2 protein.

Techniques: Infection, Synthesized, Isolation, Negative Control, Standard Deviation, Western Blot

Journal: Antiviral research

Article Title: Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.

doi: 10.1016/j.antiviral.2019.104674

Figure Lengend Snippet: Homology model of VEEV nsP2 N terminal region. A ribbon structure homology model of the first 465 amino acids of VEEV nsP2 was produced using I-TASSER. This model was made possible by the recent publication of the crystal structure of this same protein region from CHIKV by the Luo lab (Law et al., 2019) which was used as the basis for our model. Domains are color coded. N terminal domain (red), Stalk domain (orange), 1 B (yellow), Rec 1 (green), Rec 2 (light blue), Connector (dark blue). The region where our compounds are expected to bind is the stalk in the stalk domain. The location of one of the resistance residues, Y102, is indicated with the side chain. The ADP binding pocket is also marked.

Article Snippet: Anti-nsP2 (Clone no. 8A4B3, available from Kerafast Cat. No EUL015) was generated as a custom mouse monoclonal antibody from GenScripts using bacterially expressed recombinant nsP2 protein.

Techniques: Produced, Binding Assay