SARS-CoV-2 Infection Causes Strong mTORC1 Inhibition and Massive Polysome Collapse
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Abstract
Viruses employ distinct strategies to ensure efficient translation of their mRNAs over the host transcripts. SARS-CoV-2 targets host mRNAs and ribosomes to favor its own protein synthesis. However, the modulation of the key signal pathways that control the host protein translation machinery during SARS-CoV-2 infection has not been sufficiently addressed. Here, by employing an early variant and a Delta variant isolate that evolved later in the pandemic demonstrates that SARS-CoV-2 infection results in massive polysome collapse starting from 24 hpi, a hallmark of global translation inhibition. Unexpectedly, eIF2α phosphorylation, commonly targeted by viruses to induce translation arrest, was not involved in the translation arrest, suggesting that SARS-CoV-2 countermeasures by the virus to suppress ISR. eIF4E phosphorylation remained unaltered during the infection, ruling out its involvement in the preferential translation of SARS-CoV-2 transcripts. We find that SARS-CoV-2 infection consistently causes mTORC1 inhibition in a comparable manner across both variants indicating that the virus likely targets mTORC1 pathway to suppress host translation. Interestingly, mTORC1 inhibition by SARS-CoV-2 did not impact the polysomal loading of ribosomal protein transcripts rpS3 and rpL26, suggesting that 5’TOP mRNAs are spared from the translation suppression and that ribosomal protein synthesis remains active during the infection. Pharmacological activation of mTORC1 did not significantly impact viral replication, suggesting that mTORC1 inhibition might be selectively restricting the host mRNAs from accessing the translation machinery, facilitating a more robust translation of viral transcripts. This study provides new insights into the molecular interactions by which SARS-CoV-2 variants, despite their different clinical outcomes, converge on a conserved mechanism to manipulate host translation regulatory pathways.
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SciScore for 10.1101/2021.05.08.443207: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells were continuously passaged at 70-80% confluency and mycoplasma contamination was monitored periodically. Table 2: Resources
Antibodies Sentences Resources Antibodies and inhibitors: All primary antibodies were purchased from Cell Signaling Technologies except the anti-SARS Spike antibody (Novus Biologicals; NB100-56578) and anti-SARS-CoV-2 Nucleocapsid (Thermo Fisher; anti-SARSsuggested: Noneanti-SARS-CoV-2suggested: NoneHRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch. HRP…SciScore for 10.1101/2021.05.08.443207: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells were continuously passaged at 70-80% confluency and mycoplasma contamination was monitored periodically. Table 2: Resources
Antibodies Sentences Resources Antibodies and inhibitors: All primary antibodies were purchased from Cell Signaling Technologies except the anti-SARS Spike antibody (Novus Biologicals; NB100-56578) and anti-SARS-CoV-2 Nucleocapsid (Thermo Fisher; anti-SARSsuggested: Noneanti-SARS-CoV-2suggested: NoneHRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch. HRP-conjugatedsuggested: Noneanti-rabbitsuggested: Noneanti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Colorectal adenocarcinoma Caco2 cells, were grown in DMEM supplemented with 20% FBS and 1× antibiotic. Caco2suggested: NoneAll the viral cultures were propagated in Vero (CCL-81) cells in serum and antibiotics free conditions. Verosuggested: NoneCaco2, Huh7 or Calu-3 cells were infected at 1 MOI for 2 hours in serum-free conditions after which the media was replaced with complete media and further incubated until the time of harvesting. Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)Software and Algorithms Sentences Resources Polysome profiles of mock and infected cells for each time point were digitized and overlaid on Inkscape. Inkscapesuggested: (Inkscape, RRID:SCR_014479)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.
Results from scite Reference Check: We found no unreliable references.
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