Journal: bioRxiv

Article Title: Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

doi: 10.1101/2021.11.03.467065

Figure Lengend Snippet: (A) Structural organization of SARS-CoV-2 Nsp1 protein showing the N-terminal and C-terminal domains (NTD and CTD) as well as the regions containing naturally occurring deletions ( , ). Also denoted are amino acids R124-K125 necessary for host mRNA cleavage and the R164-K165 pair reported to be necessary for the interaction with the 40S mRNA entry site . The secondary structure elements of wild-type Nsp1 are shown above the domain organization. (B) Schematic of the mRNA reporter construct containing the 5’ UTR of SARS-CoV-2 followed by a firefly luciferase open reading frame, with 5’ cap and 3’ poly A tail. (C) Results of the in vitro translation assay to assess the translation efficiency of the reporter mRNA. Graph showing luminescence signal in response to firefly luciferase activity in HEK293F translational lysate at the end of the reaction, in the absence and presence of increasing concentrations (0.1, 0.5, 1.0, 2.0, and 3.0 μM) of recombinant Nsp1 and deletion variants (Δ85; Δ82-83,85; Δ79-89; Δ141-143). The average luminescence (RLU) from three independent experiments are shown in the plot, error bars represent standard deviation.

Article Snippet: For expression of wild-type Nsp1, the nsp1 gene was amplified from the construct pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro (Addgene) ( ) and inserted into a 1B vector (Macrolab, UC Berkeley) using In-Fusion cloning kit (Takara Bio).

Techniques: Construct, Luciferase, In Vitro, Activity Assay, Recombinant, Standard Deviation

Journal: bioRxiv

Article Title: Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

doi: 10.1101/2021.11.03.467065

Figure Lengend Snippet: (A) Raw data of thermal shift assays with Nsp1 variants using SYPRO Orange. (B) Raw TSA data of the deletion variant Δ79-89. (C) Non-linear regression curve fit (Boltzman sigmoidal) of the unfolding curves of Nsp1 variants (except that of the Δ79-89 protein) to calculate the melting temperature. (D) Same as (C), for the Δ79-89 variant. (E) Tabular presentation of the result of the non-linear fitting of the unfolding curve of all the proteins.

Article Snippet: For expression of wild-type Nsp1, the nsp1 gene was amplified from the construct pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro (Addgene) ( ) and inserted into a 1B vector (Macrolab, UC Berkeley) using In-Fusion cloning kit (Takara Bio).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

doi: 10.1101/2021.11.03.467065

Figure Lengend Snippet: (A) Cartoon representation of the experimentally determined structure of the Nsp1 N-terminal β-barrel domain (PDBID: 7K7P, ). (B) Cartoon representation of the predicted wt Nsp1 structure (grey), truncated to the same boundaries as for (A). In red highlighted are residues 79-89. (C) Cartoon representation of the predicted Δ79-89 Nsp1 structure (orange), truncated to the same boundaries as for (A). In red highlighted is the disordered region, which in (B) forms the β-strands β3-β5. (D) Topological drawing of the wild-type Nsp1 fold with α-helices shown as black tubes and the β-strands as arrows. β-strands forming the β-barrel are shown in grey and non-β-barrel forming strands in light grey. Highlighted in red is the β-strand part deleted in Δ79-89 Nsp1. (E) Topological drawing of the Δ79-89 Nsp1 fold. α-helices are shown as black tubes and the β-strands as orange arrows. Highlighted in red is the disordered region as in (C). (D-E) Dashed-lined box highlights the location of change between the predicted wt and Δ79-89 folds.

Article Snippet: For expression of wild-type Nsp1, the nsp1 gene was amplified from the construct pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro (Addgene) ( ) and inserted into a 1B vector (Macrolab, UC Berkeley) using In-Fusion cloning kit (Takara Bio).

Techniques: