Suppression of Global Protein Translation in SARS-CoV-2 Infection
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Abstract
The relationship of SARS-CoV-2 with the host translation remains largely unexplored. Using polysome profiling of SARS-CoV-2 infected Caco2 cells, we here demonstrate that the virus induces a strong suppression of global translation by 48 hours of infection. Heavy polysome fractions displayed substantial depletion in the infected cells, indicating the loss of major translational activities in them. Further assessment of the major pathways regulating translation in multiple permissive cell lines revealed strong eIF4E dephosphorylation accompanied by Mnk1 depletion and ERK1/2 dephosphorylations. p38MAPK showed consistent activation and its inhibition lowered viral titers, indicating its importance in viral survival. mTORC1 pathway showed the most profound inhibition, indicating its potential contribution to the suppression of global translation associated with the infection. Pharmacological activation of mTORC1 caused a drop in viral titers while inhibition resulted in higher viral RNA levels, confirming a critical role of mTORC1 in regulating viral replication. Surprisingly, the infection did not cause a general suppression of 5’-TOP translation, as evident from the continued expression of ribosomal proteins. Our results collectively indicate that the differential suppression of mTORC1 might allow SARS-CoV-2 to hijack translational machinery in its favor and specifically target a set of host mRNAs.
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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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