Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: CoV-2 nsp1 promotes translational suppression and mRNA decay in vitro and in cells (A) HBB-nLuc reporter RNA was incubated with HEK293T translation extracts alone or in the presence of increasing concentrations of purified WT, R124A/K125A, or K164A/H165A nsp1. Translation of the reporter was then evaluated by luciferase assay and normalized to a glutathione S-transferase (GST) protein control. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001; one-sample t test versus hypothetical value of 1. The bars represent the mean value of the replicates and error bars represent standard deviation. (B) A primer extension assay was used to measure cleavage of the HBB-nLuc RNA in the presence of purified WT or mutant nsp1. Lane 1 (no IVT) shows nsp1 and HBB-nLuc incubation in primer extension buffer only, whereas lanes 2–4 show reactions incubated in the presence of translation extracts. Hash marks denote cleavage intermediates. (C and D) HEK293T cells were transfected with a GFP reporter plasmid alone or together with the indicated nsp1-expressing plasmids and then harvested for protein or RNA. GFP and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (C). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (D). Each dot represents an independent experiment. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. For (D), the bars represent the mean value of the replicates and error bars represent standard deviation. (E) HEK293T cells transfected with a GFP reporter plasmid alone or together with the indicated nsp1-expressed plasmids were subsequently treated with 5-μg/mL actinomycin D (ActD) and harvested at the time points indicated after ActD treatment. GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, and the changes in GFP mRNA abundance are relative to the time point immediately before ActD treatment. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001; two-way ANOVA with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test versus “0”-h time point. See also and . The points represent the mean values of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: In Vitro, Incubation, Purification, Luciferase, Standard Deviation, Primer Extension Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: The N-terminal and central domains of nsp1 are required for translational suppression and mRNA depletion (A) Schematic of the N-terminal 3xFLAG-Halo-tagged versions of WT and mutant nsp1. Amino acids 122–130 encompass the RNA destabilization domain, which was either deleted (Δ122–130) or replaced with a size-matched glycine linker (G-linker). Mutant Δ118–180 lacks the central and C-terminal domains, whereas Δ1–117 lacks the N-terminal domain. (B and C) HEK293T cells were transfected with a GFP reporter plasmid alone or together with plasmids containing WT or the indicated mutant nsp1 and then harvested for protein or RNA. GFP, and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (B). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (C). Each dot represents an independent experiment. ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. See also Figure S2 . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Residue R99 located in the N-terminal domain plays key roles in nsp1-induced host shutoff (A and B) HEK293T cells were transfected with a GFP reporter plasmid alone or together with plasmids containing WT or the indicated mutant nsp1 and then harvested for protein or RNA. GFP and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (A). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (B). Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. For (B), the bars represent the mean value of the replicates and error bars represent standard deviation. (C) HBB-nLuc reporter RNA was incubated with HEK293T translation extracts in the presence of 80 nM of purified WT or the indicated mutant nsp1 protein. Translation of the reporter was then evaluated by luciferase assay and normalized to levels from lysates incubated with 80 nM of a control GST protein. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) Primer extension assay to measure degradation of the HBB-nLuc RNA in the presence and absence of purified WT or mutant nsp1. Lanes 1 and 2 are controls lacking translation extract (no IVT) or nsp1, respectively. See also Figure S3 .

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Quantitative RT-PCR, Standard Deviation, Incubation, Purification, Luciferase, Primer Extension Assay

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Nsp1 N-terminal and central domain mutants are defective for ribosome and mRNA binding (A) HEK293T cells were transfected with plasmids expressing WT or the indicated mutant 3xFLAG-Halo-tagged nsp1. Nsp1 was immunoprecipitated (IP) using ⍺-FLAG beads and coIP of ribosomal proteins RACK1, RPS2, RPS3, and RPS24 was monitored by western blotting, with vinculin serving as a loading control. Input lanes contain 1/10 of the amount of protein used for the IPs. (B) Equilibrium binding measurements of fluorescently labeled WT (blue), R124A,K125A (red), and R99A (green) nsp1 to purified ribosomes. Data represent a total of 3 biological replicates. (C) HEK293T cells were co-transfected with HBB-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 constructs. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were extracted and nLuc mRNA was quantified by qRT-PCR. The mRNA values were then normalized to the values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) HEK293T cells were co-transfected with a 3xFLAG-Halo-tagged nsp1 plasmid or empty vector control, together with a plasmid expressing either GFP with a 5′ stem loop (GFP+SL) or a control GFP lacking the stem loop (GFP). Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating GFP+SL or GFP mRNAs were quantified by qRT-PCR. The mRNA values were then normalized to those obtained from the empty vector control. The bars represent the mean value of the replicates and error bars represent standard deviation. (E) The levels of GFP+SL and GFP mRNA present in the input samples from (D) were quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 (empty vector control) set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; unpaired t test. See also , , and . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Labeling, Purification, Plasmid Preparation, Construct, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Protection from translational repression conferred by the CoV-2-leader sequence is selectively eliminated by nsp1 N-terminal and central domain mutants (A) HEK293T cells were co-transfected with a plasmid expressing CoV-2 leader-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 construct. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were quantified by qRT-PCR. The mRNA values were then normalized to the mRNA values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (B) HEK293T cells were transfected with either HBB-nLuc or CoV2L-nLuc together with control empty vector or the indicated nsp1 construct. Translation of HBB-nLuc or CoV2L-nLuc was measured by luciferase assay, and the fold change in luciferase activity was calculated relative to the empty vector control. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-sample t test versus hypothetical value of 1. The bars represent the mean value of the replicates and error bars represent standard deviation. (C) CoV2L-nLuc mRNA was quantified from the above experiment by qRT-PCR and normalized to 18S rRNA, with the level of CoV2L-nLuc mRNA in cells lacking nsp1 then set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. See also Figure S6 . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Luciferase, Activity Assay

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Model for how the N-terminal and central domains of nsp1 are critical for 40S ribosome association and preservation of leader-containing transcripts (A) All three nsp1 domains contribute to its interaction with the 40S ribosome. While the C-terminal domain interjects into the mRNA entry channel of the ribosome to block mRNA access, the N-terminal and central domains stabilize the interaction. When cellular mRNA encounters an nsp1-bound ribosome, it is translationally blocked and undergoes degradation. However, mRNA containing the CoV-2 leader sequence engages the N-terminal and central domains of 40S-bound nsp1 in a manner involving nsp1 residues R124, K125, and R99, leading to relief from translational repression. (B) Nsp1 mutants R124A/K125A and R99A have reduced affinity for the 40S ribosome, which alleviates the translational repression of cellular transcripts. However, CoV-2 leader-containing transcripts instead become translationally repressed, perhaps due to a “nonproductive” interaction with nsp1 in the absence of proper engagement with residues R124/K125 or R99.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Preserving, Blocking Assay, Sequencing

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet:

Article Snippet: pCDNA4TO-3XFLAG Halo delta 122-130 nsp1 , This paper , 175425 (Addgene).

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Transfection, Luciferase, SYBR Green Assay, Primer Extension Assay, Clone Assay, Software

Journal: STAR Protocols

Article Title: Protocol for quantitative evaluation of misfolded protein degradation using Agrobacterium -mediated expression system in Nicotiana benthamiana

doi: 10.1016/j.xpro.2024.103034

Figure Lengend Snippet:

Article Snippet: Plasmid: pBin-GFP2 , BioVector , http://www.biovector.net/product/425587.html.

Techniques: Virus, Recombinant, Protease Inhibitor, Cloning, DNA Extraction, Plasmid Preparation, Marker, Western Blot, Amplification, Software, Membrane, Electrophoresis, Fertilizer