Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination
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Abstract
The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.
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SciScore for 10.1101/2020.11.11.377739: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pre-termination complex assembly: The 40S and 60S ribosomal subunits as well as eukaryotic translation factors eIF2, eIF3, eEF1H, and eEF2 were purified from RRL or HeLa lysate, as previously described (Alkalaeva et al., 2006). HeLasuggested: NoneRecombinant DNA Sentences Resources Nsp1 cloning and purification: Codon-optimized Nsp1 wt was amplified by PCR with Q5 polymerase (NEB) from the plasmid pDONR207 SARS-CoV-2 NSP1, which was a … SciScore for 10.1101/2020.11.11.377739: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pre-termination complex assembly: The 40S and 60S ribosomal subunits as well as eukaryotic translation factors eIF2, eIF3, eEF1H, and eEF2 were purified from RRL or HeLa lysate, as previously described (Alkalaeva et al., 2006). HeLasuggested: NoneRecombinant DNA Sentences Resources Nsp1 cloning and purification: Codon-optimized Nsp1 wt was amplified by PCR with Q5 polymerase (NEB) from the plasmid pDONR207 SARS-CoV-2 NSP1, which was a gift from Fritz Roth (Addgene plasmid # 141255; http://n2t.net/addgene:141255; RRID:Addgene_141255) (Kim et al., 2020a) using primers NSP1_F, NSP1_R. detected: RRID:Addgene_141255)Software and Algorithms Sentences Resources P-values were calculated in Microsoft Excel. Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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